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MindMap Gallery clinical immunology
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clinical immunology
Knowledge points for the intermediate professional title examination in medical testing include monoclonal antibodies, agglutination reactions, autoimmune diseases, immunoproliferative diseases, precipitation reactions, etc.
Edited at 2024-01-14 00:56:14
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clinical immunology
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clinical immunology
Introduction
Introduction
Immune Function
defense
Eliminate pathogenic microorganisms
Enhance
hypersensitivity reaction
weaken
immunodeficiency disease
foreign
self-stabilizing
Remove damaged or senescent cells
Enhance
autoimmune disease
Cannot be used by oneself
monitor
Eliminate mutated or aberrant malignant cells
weaken
malignant tumor
Anti-tumor
cell
cells derived from lymphoid precursor cells
NK cells
Cells that play a key role in acute inflammation
neutrophils
cells that can cause acute allergic reactions
basophils
Cells with the function of cooperatively clearing immune complexes from the blood circulation
red blood cells
immune response
A series of reactions that occur when stimulated by antigens
The purpose of excreting or breaking down the antigen
feature
Identify self and non-self
specificity
memory
Diversity, tolerance, self-regulation
Classification
innate immunity
Also known as innate immunity, non-specific immunity
first line of defense
tissue barrier
Skin and mucous membranes
Features
Early days
fast
innate
non-specific
adaptive immunity
Also known as acquired immunity, specific immunity
process
identify
Antigen presenting cells (APC)
Recognize, ingest, degrade
identify
ingest
swallow into stomach
degradation
broken down into antigenic peptides
Submit
released after decomposition
Present antigen information
To helper T cells and related lymphocytes
activation
T and B cells
The process of activation, proliferation, and differentiation after receiving antigen stimulation signals
direct stimulation
APC post-presentation stimulation
B cells
Plasma cell
Humoral immunity
Complete humoral immunity with the help of phagocytes
T cells
Effector cells (killer T cells)
cellular immunity
effect stage
Plasma cell
secrete antibodies
thymus-dependent cells
When activating B cells
Need helper T cell help
T cells
Helper T cells (Th cells)
secrete cytokines
Killer T cells (CTL)
Execute cytotoxic effects
target cells
tumor cells
cells infected by bacteria
transplanted organ
memory cells
After proliferation and differentiation, a small number of T cells and B cells
Does not directly perform effector functions
Acting on
immune response again
No activation stage required
Produce antibodies directly
primary immune response
Characteristics of producing antibodies
low affinity antibodies
Artificial active immunity and artificial passive immunity
artificial active immunity
Vaccination with vaccine (antigen)
artificial passive immunity
Immune serum (antibodies), lymphokines
such as tetanus
No need to stimulate
Immunity immediately
immune tissues and organs
system
immune organ
Immune Cells
immune molecules
immune organ
central immune organ
The place where immune cells are produced, differentiated and matured
marrow
important hematopoietic organ
The birthplace of blood cells and lymphocytes (T, B)
B cells
mature place
Body fluids also come from the bone marrow.
Thymus
T cells
mature place
peripheral immune organs
site of immune response
Places of identification and activation
lymphocyte recirculation
Starting point, intermediate station, and destination of homecoming
peripheral immune organs
blood
peripheral immune organs
organ
Lymph nodes
The biggest amount
spleen
maximum
mucosa-associated lymphoid tissue
tonsils, small intestinal lymph nodes, appendix
Immune Cells
Classification
Lymphocytes
T and B cells
NK cells
Mononuclear-phagocytic cells
Supporting role
late phase of humoral immunity
early stage of cellular immunity
antigen presenting cells
Monocytes enter tissues
It's phagocytes
Lymphocytes
T cells
Function
cellular immunity
of total peripheral blood lymphocytes
60%-80%
surface marking
CD28
expressed in
T cell surface
Can interact with CD80 on the surface of B cells
Stimulate T cell activation
CD45RO
memory T cell surface specific CD antigen
subpopulation
Th
Function
Promote and enhance immune response
Th1
Secrete IL-2, IFN-r (interferon-r), TNF-β (tumor necrosis factor β)
Promote cellular immunity
Th2
Secretes IL-4, 5, 6, 10
Promote humoral immunity
Negatively regulates cellular immune responses
TCF-β
Negative regulation of cellular immunity
CTL
specific recognition
Endogenous antigenic peptide-MHC class I molecule complex
Conditions under which tumor cells are killed
Expresses MHC class I factors
Specific killing of target cells
B cells
Function
Humoral immunity
of total peripheral blood lymphocytes
8%-15%
surface marking
mIgM=u chain Ig
CD80
expressed in
activated B cell surface
Interacts with CD28 on the surface of T cells
Stimulate T cell activation
CD21
CD molecules on the surface of mature B cells
Can bind to Epstein-Barr virus
B2 cells
Major B cells in peripheral blood
Primary cell that performs humoral immunity
B1 cells
Involved in the body's immune regulation, autoimmune diseases and B cell-derived tumors are closely related
Type of antigen recognized
polysaccharide
Natural killer cells (NK cells)
Features
non-specific
No antigen stimulation required
No need to activate
Directly kill target cells
Not restricted by MHC molecules
Cytoplasm contains azurophilic granules
surface marking
CD56, CD16
CD56 CD3-CD16
Two old men fuck directly without stimulation
Determination of active target cells
Human NK cell activity
K562 cell line
Mouse NK cell activity
YAC cell line
Antibody-dependent cell-mediated cytotoxicity
Cell-mediated: NK cells
ADCC
The killing function of NK cells also requires the help of antibodies
Antibodies needed
IgG
process
IgG binds to the corresponding antigenic determinant of the target cell
NK cells bind to the Fc segment of IgG
Activate NK cells
Release perforin, granzymes, etc. to kill target cells
target cell apoptosis
Detection
NK cell killing activity
Cytotoxicity test
immune helper cells
Glossary
Lymphocytes
Especially T cells
Activation requires the participation of non-lymphocytes
Classification
Mononuclear-phagocytic cells
Dendritic Cells
Mononuclear-phagocytic cells
large phagocytes
Monocytes in the blood
Macrophages in tissues
Surface contains scavenger receptors, TcrR, TLR, complement receptors
No antigen recognition receptor
Because macrophages are nonspecific
small phagocytes
neutrophils
surface markings
Mononuclear-phagocytic cells
CD14
neutrophils
CD33
Dendritic Cells
No phagocytic ability
Has strong antigen presentation ability
Antigen presenting cells (APC)
important surface markings
MHC class II antigen
After macrophage activation, high levels of
Expression levels are low in macrophages at rest
Full-time
Mononuclear-phagocytic cells
Dendritic Cells
Primary presenting cells of the primary immune response
B cells
No activation required
The main presenting cells of the immune response again
Part-time
Endothelial cells
Epithelial Cells
activated T cells
must be activated
Isolation and preservation of immune cells
separation
Mononuclear cell isolation
Ficoll separation liquid
Single Density Gradient Centrifugation Method
There is only one density, only one layer (middle layer) after centrifugation
Single core is also here
Isolate mononuclear cells
Lymphocytes
single core
layered
platelets, plasma layer
Mononuclear cell layer (Ficoll separation medium)
granulocyte, red blood cell layer
Specific gravity range
mononuclear cells
1.075-1.090
multinucleated granulocytes
Around 1.092
platelets
1.030-1.035
Lymphocyte isolation
Adhesion method, adsorption column filtration method
principle
Mononuclear cells can actively adhere to glass, plastic, nylon wool, cotton fiber, and dextran gel
Remove monocytes from suspension
Obtain pure lymphocyte population
magnet attraction method
Mononucleus can phagocytose iron particles
Percoll separation method
continuous density gradient centrifugation
layered
dead cells, platelets, plasma
Monocytes (upper layer)
Lymphocytes (lower layer)
granulocyte, red blood cell layer
Isolation of T and B cells
E rosette sedimentation method
T cells have sheep erythrocyte receptors (E receptor/CD2 receptor)
T cells
EA rosette sedimentation method
B cells have IgG Fc receptors
Can bind to the Fc segment of immunoglobulin IgG
B cells
Isolation of T cell subsets
principle
According to the characteristics of the corresponding cells and different surface markers
Th
CD4
CTL
CD8
Affinity plate binding and separation method
Magnetic microsphere separation method
Fluorescence activated cell separator separation method
save
Long-term preservation
Liquid nitrogen cryogenics
-196
Protective agent
dimethyl sulfoxide
Viability assay of isolated cells
See if it has any function
Check the functions before saving
trypan blue staining
Including checking whether the sperm is alive or dead
trypan blue
Intact cell membrane that is impermeable to living cells
Living cells do not stain
dead cell staining
blue
Detection of surface marks
Antibody-sensitized cell rosette method
E garland
EA Garland
Immunocytochemistry
Enzyme-labeled antibodies bind to specific receptors on cells
Percentage of stained cells observed under ordinary microscope
Immunofluorescence
Fluorescein-labeled antibodies bind to specific receptors on cells
Fluorescence microscope observation of the percentage of fluorescent cells
flow cytometry
Functional testing of lymphocytes
T cell function testing
T lymphocyte proliferation test/lymphoblast transformation (in vitro test) (PHA lymphocyte transformation test)
principle
After T cells are stimulated by mitogens or antigens in vitro
Increased intracellular protein and nucleic acid synthesis
Increased size, increased synthesis of DNA in the nucleus and RNA in the cytoplasm
Increased cytoplasm
Cavitation appears
Loose nuclear chromatin
Nucleoli are obvious
revert to lymphoblast
mother cellization
Transformed back into mother, in order to proliferate
irritants
non-specific mitogen
Phytohemagglutinin (PHA)
Concanavalin A (ConA)
Phytolacca americana (PWM)
specific antigen
Purified protein derivative (PPD) of Mycobacterium tuberculosis
Tetanus toxoid
Stimulate T cell proliferation
tumor antigen
allogeneic cells
Phytohemagglutinin (PHA)
Stimulate B cell proliferation
Epstein-Barr virus
Simultaneously stimulates T and B cell proliferation
Purified protein derivative (PPD) of Mycobacterium tuberculosis
Detection method
Morphological examination
3H-TdR incorporation method (radionuclide method)
Activated lymphocytes take up precursors for DNA synthesis
3H labeled thymidine
According to the amount of mixing
T cell-mediated cytotoxicity assay
principle
When the sensitized T cells encounter the corresponding target cell antigen again
Demonstrated destruction and lysis of target cells
Clinical application
Evaluate the immune level of body cells
In particular, measuring the ability of CTL in tumor patients to kill tumor cells
Determine prognosis and observe curative effect
In vivo testing
principle
After the normal body has established cellular immunity to a specific antigen,
Do a skin test with the same antigen
positive delayed hypersensitivity reaction
method
Tuberculin test (OT/PPD)
Subcutaneous vaccination
2-3 days later
redness, swelling, blisters
>20mm
Proof of tuberculosis infection
If the patient is diagnosed with tuberculosis
PPD negative
Demonstrates reduced cellular immune function
Clinical application
Whether it has specific cellular immune response ability to a certain antigen
Cellular immune status
Diagnosis of certain pathogenic microbial infections (tuberculosis, leprosy)
Diagnosing cellular immunodeficiency diseases
Determine disease prognosis
Functional testing of B cells
B cell proliferation assay
principle
Same as T cell proliferation assay
irritants
Mouse B cells
Bacterial lipopolysaccharide (LPS)
human B cells
Staphylococcus aureus and anti-IgM antibodies with SPA
SPA
Staphylococcal protein A
Hemolytic plaque test
The ability of B cells to produce antibodies
principle
Using sheep red blood cells (SRBC)
Immunized mice
Remove the mouse's spleen
Preparation of splenocyte suspension
Inside are plasma cells
Preparation of agar plates containing SRBC
Add spleen cell suspension to the plate
If anti-SRBC antibodies are produced, the surrounding area will be sensitized
With the participation of complement
Dissolve SRBC
Formation of hemolytic plaques visible to the naked eye
process
clinical significance
Each plaque contains an antibody-forming cell in the center
Number of plaques
Number of antibody-forming cells
plaque size
How many antibodies do each cell produce?
reverse hemolytic plaque test
Determine the number of antibody-producing cells
Enzyme-linked immunospot test (ELISPOT)
POT
spots meaning
application
In vitro testing
specific antibody B cells
Antibody secretion amount
Cytokine-secreting T cells
Can detect antibody-secreting cells
It can also detect the amount of antibody secreted
Phagocytic cell function test
Neutrophil function test
Chemotaxis function test
Filter membrane permeability method (Boyden chamber method)
skin window test
Intracellular bactericidal ability
Nitro blue tetrazolium (NBT) reduction test
Positive rate of normal people
5%-10%
The positivity rate increased significantly
systemic bacterial infection
Phagocytosis and metabolic activity
Chemiluminescence method
Substances that can enhance luminous efficiency
Luminol
Macrophage function test
Carbon particle clearance test
Phagocytes can swallow carbon particles
immune molecules
Immunoactive cells or related cells
Secreted to participate in the body's immune response or immune regulation
Protein and polypeptide substances
Classification
Immunoglobulin
Antibody
complement
Cytokines
cell adhesion molecules
human leukocyte differentiation antigen
Immunoglobulin
Immunoglobulin (Ig)
A globulin that has a similar chemical structure to an antibody molecule
Y shape
Antibodies (Ab)
Globulin produced by B cells that proliferate and differentiate into plasma cells after receiving antigen stimulation.
Exerting humoral immune function
Antibodies with minimal side effects
small molecule antibodies
The most effective carrier for therapeutic drugs
Monoclonal antibodies
All antibodies are immunoglobulins
Not all immunoglobulins are antibodies
such as multiple myeloma
Classification
Secreted (sIg)
Mainly found in body fluids
Have various functions of antibodies
Model(mIg)
Expressed on the surface of B cells as an antigen receptor
membrane surface immunoglobulin
chemical structure
Ig molecules are composed of 4 peptide chains
2 identical heavy chains (H)
γ, α, μ, δ, ε chain
Different heavy chains, different types of immunoglobulins
2 identical light chains (L)
K, L chain
K:L about 2:1
Two heavy and two light, upper change and lower constant, heavy and constant classification, light and constant classification, light and heavy variable knot antigen
Immunoglobulin classification
heavy chain constant region
Immunoglobulin typing
light chain constant region
Heavy chain constant region antigenicity (CH)
IgG (γ)>IgA (α)>IgM (μ)>IgD (δ)>IgE (ε)
Fc segment
Constant region (VH region)
Immunoglobulin isotype epitopes
Constant region (CH, CL)
immunoglobulin antigen binding site
Heavy chain (H) and light chain (L) variability (V region)
Hypervariable Region (HVR)
Within the antibody variable region (V region)
site that binds to an antigenic epitope
Also known as complementarity determining region (CDR)
There are 6 sites that actually bind to the antigen.
Classification
IgG
main force
The highest content
The only antibody that can cross the placenta
Autoantibodies, hypersensitive type II antibodies, secondary antibodies (secondary antibodies) in immunological testing
IgG1, IgG2, IgG3, IgG4
The highest content of IgG1
IgG4 cannot activate complement
Convective immunoelectrophoresis
IgG1 and IgG2
due to electroosmosis
Move towards the cathode
IgG3 and IgG4
due to current
move towards positivity
Long incubation period
High affinity
primary antibodies of secondary immune response
effect
IgM
March first
pentamer
Large molecular weight
Macroglobulin
The earliest antibodies synthesized and secreted during ontogeny
Increased IgM in cord blood of newborns
intrauterine infection
Antibodies first produced after antigen stimulation
Serum IgM levels increase during infection
recent infection
Can cause aggregation of red blood cells in saline medium
Blood type identification (slide method)
Low affinity
The strongest ability to activate complement
natural lectins
cold agglutinin
cold agglutinin syndrome
Autoimmune diseases caused by IgM immunoglobulins
IgA
border guard
Secreted (sIgA)
dimer structure
Contains a J chain and a secretory patch
Participate in local mucosal immunity
primary antibody
Presence of gastrointestinal tract, bronchial secretions, colostrum, saliva, tears
sIgA in breast milk improves local immune barrier in infants 4-6 months after birth
local antibodies
IgE
least content
Cytotropic antibodies
Mast cell and basophil affinity
Type I hypersensitivity reaction
increase
Type I hypersensitivity reaction
Bronchial asthma, allergic rhinitis
nonallergic diseases
IgE myeloma
parasitic infection
Preparation of immunoglobulin fragments
enzymatic method
Papain sensitivity
IgG is cleaved into 2 Fab fragments and an Fc fragment
Enzymes used to make Fc fragments of immunoglobulins
Pepsin
IgG is cleaved into F(ab)2 and several small fragments
subtopic
Body fluid immunoglobulin assay
Determination of IgG, IgA, IgM
Content g/L level
One-way circular immunodiffusion method
immunoturbidimetry
Most used
Enzyme-linked immunosorbent assay
Determination of IgG subclasses
lowest sensitivity
complement
Overview
Complement (C)
A group of glycoproteins in serum that have enzyme-like activity and are heat-labile (mostly β-globulin)
but not an enzyme
Synthesized by liver cells and macrophages
Antigens stimulate the body
will not produce
complement system
Composition
inherent ingredients
C1, C2, C3, C4, C5
complement regulatory protein
Complement receptor composition
C3
The highest content
C2
The lowest content
D factor (no C2)
The lowest content
Minimum molecular weight
Cq1
The largest molecular weight
A fragment of C1
chemical properties
Freeze drying
keep it active
complement inactivation
56℃
30min
Strong acids and bases
Strong shock
certain additives
Ether
ethanol
Protease
UV irradiation
activation pathway
C9 is N
identification stage
Requires the participation of immunoglobulins
IgM or IgG binds to antigen
Activate C1q and C1s
activation stage
C1s cleaves C4 and C2
C3 convertase (C4b2b) formation
C3 convertase cleaves C3
C5 convertase (C4bC2bC3b) forms
MAC film attack stage
C5 convertase cleaves C5
C5b67 complex formation
C5b678 embedded in cell membrane
N C9 enter
form transmembrane pores
External water can enter the cells
causing cell lysis
Complement is cleaved into multiple fragments during activation
smaller fragments
a
larger fragments
b
C2 exception
larger fragment
C2a
smaller fragment
C2b
The only small fragment in the classical path
effect
Helper antibodies exert cytolytic effect
Participate in the body’s defense effect and self-stability
Cause immunopathological reactions
C3a and C5a
Acts on the cell membrane of mast cell nuclei and basophils
Denucleates cells to release histamine and leukotrienes
Medical record changes for anaphylactoid reactions
anaphylatoxins
Raji cells
There are a large number of C3b, C1q and C3d receptors on the surface
no smig
Utilizes these receptors to bind to circulating immune complexes that bind complement
Then add radionuclide labeled anti-human IgG
Detectable circulating immune complexes
Total complement activity assay
Complement 50% hemolysis test (CH50 determination)
principle
SRBC (sheep red blood cells) antibodies bind to SRBC surface antigens
Activate complement (classical pathway)
Minimum serum volume for 50% hemolysis
Reflects total complement activity
Than 100% hemolysis
More sensitive and easier to observe
Dependence of hemolytic reaction on complement dosage
Special S-shaped curve
Hemolysin is mostly used
2 units (2U)
unit
U/ml
Influence
Decreased hemolytic activity
PH
Buffers and ionic strength
increase height
ion
Buffer calcium, magnesium ions
excess
Inhibit hemolytic activity
clinical significance
increase height
malignant tumor
C3 and C4 also increased
complement fixation test
principle
Antigen-antibody complexes bind complement
Free antigen or antibody cannot bind complement
dilution buffer
An appropriate amount of sodium chloride
Small amounts of calcium and magnesium ions
Have a certain pH value
Detection
Antigen or Antibody
indicating system
Hemolysin
Antibodies that lyse red blood cells
Sheep red blood cells
Antigen-antibody system
effect
Indicates whether complement is bound
Detect the presence of the antigen or antibody to be tested
Classification
reaction system
complement system
It's a reagent
General use
Fresh guinea pig serum
Not being tested
indicating system
Hemolysin
sheep red blood cells
process
Add reagent 1
Known Antigen/Known Antibody
Add serum (look at antibodies/look at antigens)
Add reagent 2
add complement
Add reagent 3
Join the instruction system
If there are antibodies in the serum, then complement has already combined with the first step, and reagent 3 can no longer combine.
Not hemolytic
Not hemolytic
Positive
Presence of antigen/antibody to be tested
Dosage unit
Utility unit
minimal complete hemolysis
Dosage
Antigen, antibody, hemolysin, sheep red blood cells
0.1ml each
complement
0.2ml
Total 0.6ml
Formal Trial Complement Dosage
formula
minimal amount of complete hemolysis
1:60 dilution
Y
Y*2：60=0.2：X
X: dilution factor
2: 2 complement units
0.2: Complement dosage
clinical significance
reduce
Increased consumption
RA (rheumatoid), SLE (systemic lupus erythematosus), active autoimmune hemolysis (type II hypersensitivity), transplant rejection
Bacterial infections
Especially Gram-negative cell infections
most sensitive microorganisms
Decreased serum complement levels often result from activation of the alternative complement pathway
Lipopolysaccharide (LPS) stimulation
Insufficient synthesis
severe liver disease
Malnutrition
massive loss
Extensive burns
Heavy bleeding
nephrotic syndrome
clinical significance
Genetic diseases of complement deficiency
C1 inhibitory molecule defect
It’s not a lack of C1
hereditary angioedema
C3 defect
severe infection
Cytokines
Activated immune cells and certain stromal cells
secreted active substances
chemical nature
Protein or small molecule peptide
Mostly glycoproteins
Mostly low molecular weight proteins
Mostly exist in single form
Not restricted by MHC
biological functions
Mediate and regulate immune responses and inflammatory responses
Regulate a variety of cell physiological functions
B cells alone cannot be stimulated to produce antibodies
B cell activation and proliferation
Requires dual signals, dual recognition and cytokines
Detection
most commonly used method
Immunological assay
ELISA
Preferred method
Measuring immunoreactivity only
Not tested for immune activity
flow cytometry
Enzyme-linked immunospot test (ELISAPOT)
Methods that cannot measure precursor molecules of cytokines
Biological activity assay
include
Interleukin (IL)
IL-2
T cell production
Promote and maintain long-term T cell culture
T cell growth factor
transplant rejection
After activation, IL-2 is secreted to promote effector T cells (CTL) to enter the graft and promote B cells to produce antibodies.
In delayed hypersensitivity (DTH)
not only cause antigen-activated T cell proliferation
It can also cause "bystander" T cells to proliferate
Can activate NK cells
Detection
Promoted cell proliferation assay
reduce
chronic hepatitis B
chronic hepatitis B
Mononuclear cells stimulated with LPS
The ability to secrete IL-1 and IL-2 is significantly lower than that of normal people.
IL-4
Th2 cell production
Promote immunoglobulin class switching
Peripheral blood mononuclear cells of tuberculosis patients stimulated with mycobacterial antigens
obviously increase
Start quiescent B cells to enter DNA synthesis phase
Type I hypersensitivity reaction
Stimulates the proliferation and differentiation of specific B cells into plasma cells
Produce characteristic IgE
Increase
active pulmonary tuberculosis
IL-3, IL-5
Type I hypersensitivity reaction
After binding to eosinophil surface receptors
stimulate its activation
Releases eosinophilic granules and bioactive substances
IL-5
Promote eosinophil proliferation, differentiation, and chemotaxis
IL-6
Multiple myeloma, myeloma patients
Important factors that increase the number of osteoclasts
Key factors that promote plasma cell proliferation and differentiation
Most commonly used to stimulate B cell hybridoma cell proliferation
Chronic rejection occurs after organ transplantation
Cytokine levels in patients
significantly increased
IL-6
IL-6, IL-1
Stimulates hepatocytes to secrete acute phase proteins
IL-8
Chemotactic effect on neutrophils
IL-10
negative immune regulator
Can inhibit the synthesis of IL-2 and IL-1
IL-18
Has chemotactic activity
method
Chemotactic activity assay
Interferon (IFN)
type I interferon
alpha interferon
human leukocyte production
beta interferon
Produced by human fibroblasts
Strong antiviral effect
type II interferon
rinterferon
T cell production
Th1 cell production
effect
Immunomodulatory effect
strongest
antiviral effect
Inhibit the transformation of Th0 cells into Th2 cells
Because this is produced by Th1, of course it is directed towards my mother
That is, inhibiting the activation and proliferation of Th2 cells
Can activate NK cells
IFN-α and IFN-r act on different receptors
Produced after stimulation by antigen
Detection method
Antiviral activity assay
growth factors
Transforming growth factor (TGF)
TCF-β
Negative regulation of cellular immunity
chemokine family
tumor necrosis factor (TNF)
TNF-α
cachexia
Major cytokines causing cachexia
stimulate target cells to produce
IL-1, IL-6
Can activate NK cells
Detection
Has cytotoxic activity
Available cytotoxic activity assays
TNF-β
by T cells
Th1 cell production
Colony stimulating factor (CSF)
Factors that activate NK cell function
IL-2, IFN-r, TNF-α
Cell adhesion factor (CAM)
Received from the mutual binding and adhesion between cells or between cells and extracellular matrix
Small molecule peptides or glycoproteins
form of functioning
Binding of receptor ligands
effect
Participate in immune response, inflammation, coagulation, wound healing and tumor metastasis, etc.
Molecular basis
Classification
Calcium ion dependent family/mucocadin family
integrin family
Immunoglobulin superfamily
mucin-like family
selectin family
Does not include inflammatory factor family
Main measurement methods
ELISA method
Features
Performance
Pleiotropy
overlap
Resistance effect
synergy
Need to specifically bind to high-affinity receptors on target cells
to exert biological effects
same as insulin
Has extremely strong biological effects
Only trace amounts of cytokines are needed
Can act in a paracrine, autocrine, or endocrine manner
same as hormones
clinical effects
Assessment of the body’s immune status and cellular immune function
Most used
Assisted diagnosis of specific diseases
specific
Just an auxiliary
Detection of disease treatment effects and guidance on medication use
disease prevention
disease treatment
Now many cytokines have been made into medicines
other
About cytokines produced by T cells
Types of antibodies that can affect B cells
Antigen-antibody reaction
principle
Structural complementarity and affinity of antigenic determinants (epitope) and antibody hypervariable regions
Antigen binding capacity
non-covalent binding
Does not form covalent bonds
electrostatic attraction
Van der Waals forces
weakest
hydrogen bonding force
hydrophobic force
strongest
This is because the hydration membrane of the antigen and antibody must be pushed aside to expose the binding site.
So it is the strongest
Affinity
The binding strength between an antigen-binding site on an antibody molecule and a responding antigen epitope
depending on
degree of spatial configuration complementarity
reflects
Antigen-antibody complementary relationship
express
Equilibrium constant K
The larger the K value, the stronger the affinity.
The stronger the binding to the antigen
Affinity
The adaptive binding strength between an antigen-binding site on an antibody molecule and the corresponding antigenic determinant
The inherent binding force between antigen and antibody
reflects
The binding strength between antigen and antibody
Features
specificity
Reversibility
Proportionality
stage
specificity
Complementarity between antigenic determinants and antibody hypervariable regions
cross-reactivity
two different antigen molecules
Antigenic epitopes with partially identical or similar structures
May cross-react with each other's corresponding antisera
Waifei reaction
Mutans bacteria and Klebsiella have the same antigenic epitope
Replacement of Chrysotitis sp. with mutans
Diagnosing Typhus (Crittsiosis)
I'm off from get off work outside, come back immediately
Outside: Epiphysis Side: Metamorphosis Class: Macula Immediately: Rickettsia
Reversibility
Dissociation can occur under certain conditions
affinity chromatography
Changing the pH and ionic strength of the solution promotes the dissociation of the antigen-antibody complex
thereby purifying the antigen or antibody
Remains biologically active after dissociation
Proportionality
The amount of conjugates formed is related to the concentration of the reactants
Optimum ratio (equivalence point)
The concentration ratio of the antigen to antibody that produces the most rapid visible reaction
Equivalence zone
The appropriate range for the ratio of antigen-antibody molecules
The maximum precipitation reaction also occurs at
band phenomenon
Excess antigen and antibody without precipitation
front strap
Antibody overdose
back strap
Antigen excess
Plain before and plain behind
Antibodies are relatively flat
stage
The first stage
Antigen and antibody specific binding
Fast, invisible
seconds to minutes
second stage
Agglutination, precipitation, cell lysis
Slow response, visible
minutes to hours
Influencing factors
electrolyte
Make the antigen and antibody lose part of their charge
prone to agglutination or precipitation
0.85% sodium chloride
increase the rate of immune complex formation
from slow to fast
SCN-, CLO4-, NO3-, SO4-, H2PO4-
The fastest is
CL-
pH
Antigen-antibody reactions must be carried out in a suitable pH environment
Optimum pH6-9
Because human pH is 7.35-7.45
temperature
temperature rise
Can accelerate molecular motion
Increased chances of antigen-antibody collision
Reaction acceleration
Temperature: 37℃
Preparation of immunogens and antisera
Preparation of immunogen
Immunogen
Can induce the body to produce antibodies or sensitized lymphocytes
Substances that cause specific reactions
characteristic
Immunogenicity
immune response occurs
The ability to induce the development of antibodies or sensitized lymphocytes
Immunoreactivity (antigenicity)
Characteristics of specific binding ability
Classification
complete antigen
immunoreactive
immunogenic
hapten
immunoreactive
Non-immunogenic
No immune response
Does not produce antibodies or sensitize lymphocytes
Peptides, steroid hormones, nucleosides, certain drugs (penicillin)
Non-immunogenic
Only combined with a carrier (bovine serum albumin is most commonly used)
to be immunogenic
thymus-dependent antigen
I-worker-cannot be relied upon
preparation
Fresh or stored at -40℃
particulate antigen
big
Cellular antigens, bacterial antigens, parasite antigens
intravenous immunization
soluble antigen
Small molecule
Proteins, glycoproteins, lipoproteins, enzymes, complement, bacterial toxins, immunoglobulin fragments, nucleic acids
rough extraction
Acquisition of single cells
Mechanical mashing
Enzyme treatment
Cell disruption method
repeated freeze-thaw method
Ultrasonic disruption
Autolysis method
Enzyme treatment
Surfactant treatment method
Extraction and purification
Salting out method
Ammonium sulfate salting out method
Ammonium octanoic acid sulfate extraction method
Sodium sulfate salting out method
Neutral salt (33-50% saturated ammonium sulfate)
Can destroy hydration membrane
neutralize charge
aggregate and precipitate proteins
The most classic
roughest
Protein separation and purification technology
Does not affect activity
Polymer precipitation method (CIC)
Circulating immune complex detection
PEG precipitation method
Cannot reflect the situation of small molecule circulating immune complexes
Determination of antigen-nonspecific immune complexes
Features
Simple
Poor specificity
susceptible to temperature effects
Polyethylene glycol (PEG)
molecular weight
6000
The greater the protein molecular weight
The lower the PEG concentration required to be precipitated
3%-4%PEG
Precipitable immune complexes
>5%
The feature of selecting precipitated CIC disappears
8%-12%
Precipitates IgG
Complement participation technology detects CIC
method
C1q solid phase method
Antibody C3-CIC-ELISA method
Unable to detect circulating immune complexes formed by IgA class antibodies
Because IgA cannot bind complement
Formalized red blood cells
Features
Ability to withstand heating at 60°C
Can be frozen and thawed repeatedly and not easily broken
The blood coagulation reaction is stronger than that of fresh red blood cells
Long storage time
Multiple viruses can be purified simultaneously
Strong applicability to viruses
immune adjuvant
Helps generate immune response
Injected into the body before or at the same time as the antigen
Can be non-specific
Enhance immune response
Change the type of immune response
type
immunogenic
Cytokines, lipopolysaccharide, Bacillus pertussis, BCG vaccine
cells and bacteria
Non-immunogenic
Aluminum hydroxide, liquid paraffin, lanolin, alum
Adjuvants for immunizing animals
Freund's adjuvant
Freund's complete adjuvant
Liquid paraffin, lanolin, BCG vaccine
One more BCG vaccine
Freund's incomplete adjuvant
Liquid paraffin, lanolin
Mechanism
Change the physical properties of the antigen
Enhance immunogenicity
Delay antigen degradation and elimination
Effectively stimulates the immune system
Stimulates the monocyte-phagocytic cell system
Enhance its ability to process and present antigens
Stimulate lymphocyte proliferation and differentiation
Increase the body's immune response antibody titer
Change antibody type
Produce more IgG
Induction of delayed-type hypersensitivity reactions
Type IV
Cannot change antigen specificity
If the specificity is changed, this vaccine will no longer be the same.
Preparation of antiserum (preparation of antibodies)
antiserum: serum containing antibodies
Multiple B cells in the body activate and produce antibodies against a certain antigen
polyclonal antibodies
Each B cell produces an antibody
Multiple B cells are polyclonal antibodies
type
Type R
Rabbits and other small animals
Strong affinity and difficult to dissociate
Suitable for wide range
for diagnostic reagents
H type
Horses and other large animals
Weak affinity, easy to dissociate
Suitable for narrow range
for immunotherapy
Tetanus vaccine
choose
program
time
1st and 2nd time
10-20 days
3rd time and later
7-10 days
Blood collection after immunization
5-7 days
5.7.10.20
Blood collection site
Carotid artery blood collection
Large and medium animals
Eye removal or tail docking
mouse
Purification of Antiserum
Removal of heteroantibody adsorbates
Antigen solution without specific antigen
affinity chromatography
Specific affinity (specificity)
Purify antibodies, enzymes and receptor proteins
best, most effective
Such as extracting high-purity specific IgG
affinity binding Dedicated enough
gel filtration
Separate proteins according to their molecular weight
Large molecules pass through first
gel via hole Look at the size
Salting out method
The most classic and rough
Does not affect protein activity
Caprylic acid extraction method
Salting out crude extraction very classic
Ion exchange chromatography
According to the charge carried by the protein
Ions are charged differently
affinity chromatography
process
As the antiserum passes through the column
The specific affinity of the solid phase carrier for the separated substance
IgG is adsorbed by the chromatography column
Then change the mobile phase to make the specific adsorption of the target substance disappear.
IgG dissociates from the column
To achieve the purpose of purification
gel filtration
Gel chromatography/molecular sieve filtration
According to protein molecular weight
large protein
Directly through the gaps between gels
washed off first
small protein
will enter the gel
was eluted later
Salting out method
Ammonium sulfate salting out method
Ammonium octanoic acid sulfate extraction method
Sodium sulfate salting out method
Neutral salt (33-50% saturated ammonium sulfate)
Can destroy hydration membrane
neutralize charge
aggregate and precipitate proteins
The most classic
roughest
Protein separation and purification technology
Does not affect activity
Ion exchange chromatography
Different charges
positively charged
anion exchange resin
Because what I want to exchange is anion
negatively charged
Cation exchange resin
Because what I want to exchange is cations
Identification and preservation of antisera
identification
Antibody specificity
two-way immunodiffusion
Determination of antibody titer
Checkerboard titration (square titration method)
two-way immunodiffusion
Optimum working concentration
Judgment criteria
Antigen and antibody
Strong positive
highest dilution
lowest concentration
save
short term storage
2-8℃
Keep for 5 years
Common methods
Avoid repeated freezing and thawing
Cryopreservation
Keep for 5-10 years
vacuum drying
Monoclonal antibodies
Basic principles of hybridoma technology
Making monoclonal antibodies
Protective agent
dimethyl sulfoxide
Monoclonal antibodies
Single B cells isolated
A colony of clones formed after proliferation
single epitope
Same structure
Functionally homogeneous antibodies
Dilute the fused cells
Each culture well
Actually
0-number of cells
The theory is
1 cell
Antigen molecules produced by hybridoma cells
a single antigenic determinant
of antibodies
specificity
depending on
Screening of clonal cells
Clonal culture of positive hybridoma cells
limiting dilution method
Most used
microscopy
Intuitive and reliable
Long operating time and easy to contaminate
fluorescence activated cell sorter
High efficiency and expensive
soft agar plate method
Difficult to master
Fundamental
Splenocytes
Sensitized B cells
Antibody secretion function
myeloma cells
non-secretory
Does not produce immunoglobulins
Can be divided infinitely and has immortality
Prevent rentback
8-azaguanine
Drug that can breed myeloma cell lines lacking hypoxanthine-guanine phosphoribose convertase (HGPRT)
save
-196℃ liquid nitrogen
cell fusion agent
Polyethylene glycol (PEG)
HAT medium
Hypoxanthine (H), aminopterin (A), thymidine (T)
Aminopterin (A)
Folic acid antagonist that blocks the main pathway of DNA synthesis in cells
Unfused cell death
Hybridoma cell survival
type
B lymphocyte hybridoma technology
Prepare monoclonal antibodies
T lymphocyte hybridoma technology
Preparation of various lymphokines
preparation
In vivo induction method
Take BALB/c mice
Intraperitoneal injection of 0.5 ml liquid paraffin followed by phytane
inducer
induce ascites
1 week later
Intraperitoneal inoculation of hybridoma cells
Proliferate in the abdominal cavity of mice
Production and secretion of monoclonal antibodies
10-14 days after vaccination
Aspirate ascites
peritoneal cells
feeder cells
Obtain large quantities of monoclonal antibodies
characteristic
Highly specific
Only binds to the corresponding epitope
Target only one epitope
high degree of uniformity
Repeatability
weak agglutination reaction
Does not precipitate
After precipitation, there is no way to perform immunoturbidimetry.
Highly sensitive to the environment
Purification method
Salting out method
Caprylic acid extraction method
gel filtration
Ion exchange chromatography
affinity chromatography
The most effective
Same as purification of antiserum
save
vacuum drying
Genetic Engineering
principle
DNA recombinant and protein engineering technology
Modify and assemble antibody-coding genes according to different needs
Import appropriate recipient cells
re-expressed antibody
advantage
Reduce or even eliminate the body’s rejection of antibodies
humanized antibodies
Retains the affinity and specificity of the parent antibody
Reduce antibody heterogeneity
Conducive to application in human body
type
chimeric antibodies
Functional variable region gene human Ig approved degene
Mouse-human chimeric antibodies
Features
Reduce heterogeneity
Retains the specificity and affinity of the parent antibody
Reduce rejection
modified antibody
Complementarity determinants (CDRs) in human antibody variable regions (hypervariable regions)
Change to mouse source sequence
Features
Has the specificity of mouse-derived monoclonal antibodies
Maintain antibody avidity
human antibodies
Fully human antibodies
The most ideal form of therapeutic antibodies
Bispecific antibodies/bifunctional antibodies
Two antigen binding sites have different specificities
combines two different antigen molecules
Combinatorial Antibody Library Technology
Light and heavy chain variable regions
random combination
Produce new light-heavy chain pairing method technology
agglutination reaction
Overview
particulate antigen
Bacteria, red blood cells
or surface covered with soluble antigen or antibody
particulate matter
Soluble antigen must coat the particulate matter
Otherwise invisible
Binds to corresponding antibody or antigen
Visible clots
Classification
direct agglutination reaction
particulate antigen
indirect agglutination reaction
soluble antigen
Need carrier help
Features
Qualitative
Semi-quantitative
After dilution
Use the highest dilution factor as the potency or titer
Convenient and simple
No special equipment required
two stages
specific binding of antigen and antibody
Visible agglomeration of particles occurs
Influence
Promote agglutination
Increase electrolytes or protein
Increase the viscosity of the test solution
cystinase treatment
Neuraminidase treatment
direct agglutination reaction
particulate antigen
agglutinogen
Antigen (particulate)
Lectin
Antibody
method
slide method
Known antibodies
test antigen
In blood type identification, blood is the antigen, so known antibodies are used
Can detect both antigens and antibodies
application
Strain identification
Can detect both antigens
Antibodies can also be tested
Serological typing
ABO blood group identification
test tube method
Semi-quantitative
known antigen
Test for antibodies
Waifei test, cross match, Feida test
Tip: Diplomats are so fat that they bleed.
Waifei test
Using the same epitope of Proteobacteria and Klebsiella
test for typhus
Feida test
Typhoid agglutination test
So fat, so sad
Determine antibodies
heterophile agglutination test
Epstein-Barr virus
IgM antibodies appear
Can non-specifically agglutinate sheep red blood cells and horse red blood cells
heterophile antibody
surface receptors
C3d receptor
Diagnosing infectious mononucleosis
indirect agglutination test
Soluble antigen or antibody
Unable to detect
AFP antigen
forward indirect agglutination test
known antigen
Test for antibodies
Agglutination is positive
Forward, there is an antigen first, so it is a known antigen
Antigen sensitizing carrier
reverse indirect agglutination test
Known antibodies
test antigen
Agglutination is positive
Antibody sensitizing carrier
application
latex pregnancy diagnosis
Motto: Make things right
Antibodies are relatively flat
Difference: Antigen or antibody sensitizing carrier
forward indirect inhibition agglutination test
in turn
Known antibodies
test antigen
Agglutination is negative
The interpretation of the results is also the other way around.
However, it is known that antibodies do not sensitize the carrier
One more allergenic agent 2
process
application
Ascoli test for anthrax
reverse indirect inhibition agglutination test
in turn
known antigen
Test for antibodies
Agglutination is negative
The interpretation of the results is also the other way around.
But the known antigen is not a sensitizing carrier
One more allergenic agent 2
synergistic agglutination test
Staphylococcus aureus protein A (SPA) as carrier
Non-specific binding to the Fc segment of IgG (sensitized particle carrier)
Test antigen (unknown)
for soluble antigens
Direct detection of bacterial viruses
indirect hemagglutination test
carrier
red blood cells
Commonly used
Sheep, rabbits, chickens
Type O human red blood cells
Because there is no antigen on the O-type surface
Coating antigen or antibody on red blood cells
form sensitization vector
Simple red blood cells are not sensitizing carriers
See if red blood cells are passively agglutinated together
result
Positive
agglutination
around the bottom of the hole
Negative
Bottom of sedimentation hole
a small dot
Like the TPPA test for syphilis
latex agglutination test
indirect agglutination test
latex particles
adsorbable protein
principle
adsorbed antigen
Detect antibodies
antigen
Denatured IgG
Rheumatoid factor (RF)
Also known as rheumatoid factor
Hemolysin "O"
Antihemolysin "O" (ASO)
vector used
polystyrene latex
diameter
0.8um
Features
test tube method
slide method
Bonding firmness
Difference
Less sensitive than coagulation tests
Gelatin agglutination test
indirect agglutination test
Antigen adsorbed on pink gelatin
Detect antibodies
Testable viruses
Antiglobulin test (Coombs test)
Coombs test
Detecting autoimmune hemolysis
Methods to detect incomplete antibodies to red blood cells
Antibody classification
complete antibody
IgM
Because it is a pentamer
Can cause red blood cells to stick together and then cross-link together
visible to naked eye
incomplete antibodies
Features
Can bind to antigens (sensitize)
No visible agglutination reaction should occur
a fragment of an IgG antibody
Immune antibodies are mostly incomplete antibodies
type
direct antiglobulin test
Determination of surface bound
It has diagnostic value for neonatal Rh hemolytic disease
Because this is to detect whether cells are sensitized
indirect antiglobulin test
Determine free
Detection of maternal Rh(D) antibodies (anti-D antibodies)
application
blood matching test
anti-D antibody
Diagnosing neonatal hemolytic anemic disease
Detection of incomplete antibodies to bacteria
Cold condensation test
cold agglutinin syndrome
Detecting autoimmune hemolysis
Caused by IgM/macroglobulin/cold agglutinin
divided into
Single plant type
Hybrid
process
at lower temperatures
Antibodies and autologous red blood cell agglutination
Symptoms of cyanosis of hands and feet
The temperature rises to 20-25℃
Complement is activated and hemolysis occurs
The temperature rises by 37℃
Completely reversible separation
Symptoms disappear
Cold condensation test
in vitro
Optimum temperature 0-4℃
Dissociate at 37°C
Judgment of results
Negative
Cold agglutinin titer <1:32
Positive
Cold agglutinin titer＞1:64
diagnosis
primary atypical pneumonia
precipitation reaction
circulating immune complexes
optimal precipitation temperature
4℃
precipitation reaction in liquid
immunoturbidimetry
definition
Buffer
Phosphate solution
Turbidity increasing agent
Polyethylene glycol (PEG)
Can rapidly form immune complex (IC) particles
In the case of a slight excess of antibody and immobilization
IC is proportional to the amount of antigen to be measured
principle
Optical measurement instruments combined with automated analysis systems
Immunoturbidity classification
immunoturbidimetry
Transmitted light
No light hits the complex (IC)
The light hitting the complex (IC) is absorbed
Absorbance value (amount of light absorbed) and IC amount
Positive correlation
The more IC, the higher the absorbance
immunoturbidimetry
Scattered light
Light that is refracted after hitting the complex (IC)
When the particle diameter is much smaller than the wavelength of the incident light
The scattered light distribution is relatively uniform
Become a Rayleigh Scatter
Detect scattered light intensity
Positive correlation
The more ICs, the stronger the scattered light
Classification
end-point scattering turbidimetry
After the antigen-antibody reaches equilibrium
Determine the amount of complex
rate scattering turbidimetry
Immunoglobulin assay
most sensitive
The most appropriate approach
For enzyme activity determination
More accurate
Kinetic determination method
application
The second peak signal appears
The first peak signal is generated by all antigens to be tested
No second peak signal appears
Tested antigen amount is too high
premise
Antibody overdose
Antibody overdose detection system
Detection system developed to ensure excess antibody
Prevent hook effect
Prevent antigen overdose (afterzone phenomenon)
Threshold limits for antigen excess
Influencing factors
Antibody quality
Strong specificity, high potency and strong affinity
Use R-type antibodies
detection reagent
Clinical application
Test Ig content
most commonly used method
Test serum apolipoprotein
most commonly used method
Detect immunoglobulins
IgG, IgA, IgM
High content
Detection of immunoglobulin subclasses
Complement C3, C4
Urinary trace protein
therapeutic drug concentration
In-gel sedimentation test
One-way agar diffusion test
Can be done manually
Can only test for antigen
principle
Mix a quantitative amount of antibody into the agar gel
agar plate
Drill holes in the plastic board
Inject serum (antigen) into the well
plate method
In 0.9% agar gel
37℃, free expansion
ring diffusion
test tube method
0.7% agarose solution
Diffusion at 50℃
Formation of visible precipitation rings
Positive correlation
The higher the antigen content
The larger the precipitation ring
A standard curve must be produced
Add 5-7 antigen standards of different concentrations at the same time
Determine the sedimentation ring diameter
abscissa
Antigen standard concentration
Y-axis
Sedimentation ring diameter
Application of monoclonal antibodies to measure polymorphic antigens
measured value
On the low side
Single and more low
Measurement of M protein using polyclonal antibodies
measured value
On the high side
How high
method
test tube method
plate method
Calculation method
Mancini curve
Suitable for large molecular antigens and long-term diffusion (>48 hours)
The square of the precipitation ring diameter d2 has a linear relationship with the antigen concentration c
c/d2=k
k: constant
Fahey curve
Suitable for small molecule antigens and shorter diffusion times
logc/d=k
c: Antigen concentration
d: Sedimentation ring diameter
k: constant
clinical significance
Used by primary hospitals
Determination of IgG, IgA, IgM and complement C3 and C4 contents
When measuring M protein using polyclonal antibodies
The sedimentation circle expands
Methodological evaluation
Not very sensitive
Detection time
48-72 hours
2-3 days
A standard curve must be produced at the same time
for quantitative determination
Two-way agar diffusion test
form
test tube method
First add agar containing antibodies to the test tube
After solidification, add a layer of ordinary agar in the middle
After cooling, add the antigen solution to the upper layer
After placement, the antigen and antibody diffuse freely into the middle agar layer.
plate method
two holes
an antigen
Ag
an antibody
Ab
Judgment of results
close to antigen well
High levels of antibodies
Close to the antibody well
High antigen content
Whoever stays away from is more likely to be
An arc appears
Because the molecular weight is small
Spread quickly
Due to the small slow diffusion circle, the local concentration is large
The precipitation line will bend toward the side with the larger molecular weight.
Whose head is smaller?
Antibody titer determination
Checkerboard titration (Founder titration method)
A biphasic immunodiffusion test
Purpose
Choose the optimal working concentration
Highest dilution that was strongly positive
The lowest concentration that is strongly positive
immunoelectrophoresis
principle
DC electric field
Combined product of electrophoretic analysis and gel diffusion (precipitation reaction)
Most commonly used gels
agarose
concentration
0.8%-1.0%
Generally, the electrophoresis of protein molecules in gel is almost the same as that of free electrophoresis.
Because the ratio of buffer in the gel
99%
principle
Antigens and antibodies are negatively charged
Because the antigen is more negatively charged
put on cathode
swimming towards the anode
Because antibodies are less negatively charged
Put on the anode
Under the action of electroosmotic force
Electroosmotic force: water swims from anode to cathode
swimming towards the cathode
Convective immunoelectrophoresis
IgG1 and IgG2
due to electroosmosis
Move towards the cathode
IgG3 and IgG4
Because it is more negatively charged
move towards positivity
Classification
Convection immunoelectrophoresis (CIEP)
Dual-phase immunodiffusion combined with electrophoresis
antigen cathode
Antibody anode
IgG antibodies
due to electroosmosis
A milky white precipitation line forms at the optimum ratio of antigen to antibody.
phenomenon called reverse negative pole regression
Influence
electroosmosis
Antibody electrophoretic power is lower than electroosmotic power
So swim towards the cathode
pH
How much charge does a molecule have?
Depends on buffer pH
The farther the buffer pH value is from the isoelectric point
The more charged
swim faster
Isoelectric point
The pH value of a solution when proteins dissociate into equal positive and negative charges
Rocket immunoelectrophoresis (RIE)
Measuring range
ug/ml or above
Single-phase immunodiffusion combined with electrophoresis
The height of the peak is proportional to the amount of antigen
The top of the peak is in the shape of unclear clouds
Electrophoresis has not reached the end point
such as combined with autoradiography
Increased sensitivity
1000 times
Immunoelectrophoresis (IEP)
Dual-phase immunodiffusion combined with zone electrophoresis
process
Zone technology first
Detect unused zones
After electrophoresis stops
Then put serum (antibody) into the parallel antibody tank.
Only make one hole and put the serum in
Look at the arc-shaped sedimentation line
result
measurable
M protein
clinical significance
Qualitative test
Serum protein analysis of multiple myeloma and macroglobulinemia
M protein
between γ and β
Antigen component identification
Monoclonal gamma antibody identification
Can roughly reflect the immunoglobulin type
can only roughly reflect
Not accurate
IgG
Between α and slow γ
IgA
β and γ1
IgM
β2 or γ
IgD
β or γ
IgE
β to slow γ
Immunofixation electrophoresis (IFE)
first person to report
Alfonso
Immunochemical methods (antigen-antibody binding reaction) combined with zone electrophoresis
principle
Zone technology first
detect different zones
Then cover the kappa and lambda light chains with antibodies (antibody against kappa and lambda light chains) or heavy chain antiserum (antibody against heavy chain)
Formation of complex precipitate
Rinse and dye
renders colored zones
clinical significance
M protein
most commonly used method
Monoclonal antibodies
Qualitative and typing (type of light and heavy chain) identification
Preferred method
Identification of immunoglobulin light chains and detection of weekly proteins in urine and κ and λ typing
Result graph
G, A, M, kappa and lambda chains
SP stands for total serum protein
Polyacrylamide gel electrophoresis (SDS-PAGE)
principle
zone electrophoresis
The gel has a network structure
Has molecular sieve effect
The smaller the molecular weight
move faster
Several bands can be separated
shortcoming
Proteins with similar molecular weight cannot be separated
process
separate bands
The separation effect is invisible to the naked eye
Electrophoretic bands appear only after staining
Add stacking gel to the upper layer of the gel to improve separation effect
Compact protein to promote swimming
Separating serum proteins can give
20+ ingredients
isoelectric focusing electrophoresis
principle
Special buffer creates a pH gradient in the gel
Commonly used gels
polyacrylamide gel
Same as SDS
Features
Isolated molecular weights are similar
Proteins with different isoelectric points
radioimmunoassay
radioimmunoassay
Commonly used radionuclides
125 I (iodine 125)
125 I (iodine 125) labeling method
direct notation
Iodination labeling of peptides, proteins and enzymes
method
Chloramine T (ch-T) method
Lactoperoxidase labeling method
indirect labeling
Applicable to
Small molecule compounds lacking iodine labeling groups
method
Connection notation method (Bolton-Hunter method)
Require
Radiochemical Purity
＞95%
Immune activity requirements
＞80%
Stabilizer added to the eluate of radionuclide markers
The same amount of 1% egg white
specific radioactivity
The intensity of radioactivity contained in a unit chemical quantity of a marker
Radiochemical Purity
test methods
Use trichloroacetic acid to precipitate all proteins in the sample to be tested
Insulin standard
Reduces the maximum binding of 125I insulin to antiserum by 50%
DosageX
Proinsulin dosage Y
Cross-reactivity rate=X/Y
Titer
After serial dilution of an antiserum
react with labeled antigen
Methodology
high sensitivity
Samples and reagents
Less dosage
Strong specificity
Good repeatability
easy to use
easy to standardize
shortcoming
radioactive contamination
Clinical application
Hormones (such as hCG, insulin, etc.), drugs (digoxin, morphine, barbiturates), tumor markers (AFP, CEA), trace proteins
They are all trace substances
Radioimmunoassay (RIA)
RIA
principle
competitive inhibition response
There is competition
So the speed becomes slower
Can only measure
antigen
unit
cpm or cps
Inversely proportional to
The more antigens to be tested
The less radioactive material
process
Labeled antigens and known antibodies
All are limited
Separate labeled antigen-antibody complex (B) and unbound labeled antigen (F)
method
Second antibody precipitation method
Polyethylene glycol (PEG) precipitation method
PEG can non-specifically precipitate macromolecular proteins such as antigen-antibody complexes.
Does not precipitate small molecule antigens
shortcoming
More non-specific precipitation
When the temperature is higher than 30°C, the precipitate is easy to redissolve
PR reagent method
Retains the advantages of secondary antibodies and PEG
Save dosage, quick and easy separation
Activated carbon adsorption method
Immunoradiometric Analysis (IRMA)
IRMA
principle
noncompetitive binding reaction
Can detect antigen
Antibodies can also be tested
Single point IRMA
reaction system
labeled antibody
Antigen or antibody to be tested
Antigen coated on solid phase
the difference
principle
First use excess labeled antibody to react with the antigen to be tested
form complex
in the supernatant
Measure the amount of radiation in the supernatant
Solid-phase antigen is then used to bind unbound labeled antibodies.
and separate it
Can detect antigen
Antibodies can also be tested
Two-site IRMA
double antibody sandwich method
reaction system
labeled antibody
Antigen to be tested
Antibodies coated on solid phase
the difference
principle
double antibody sandwich method
Need to wash away remaining labeled antibodies
Measure solid phase radioactivity
Can only test for antigen
Compared
application
Raji cells
There are a large number of C3b, C1q and C3d receptors on the surface
no smig
Utilizes these receptors to bind to circulating immune complexes that bind complement
Then add radionuclide labeled anti-human IgG
Detectable circulating immune complexes
Fluorescence Immunotechnique
Overview
The earliest established marking technology
Cannot be used for quantitative determination
concept
Fluorescein
Markers directly involved in luminescence
fluorescence
Ground state molecules (stable, low energy)
After absorbing light of a certain wavelength
Excited singlet state (higher energy)
When quickly returning to the ground state
Emits light from a specific excitation luminescence plant
Fluorescence efficiency
The percentage of fluorescein that converts absorbed light energy into fluorescence
quenching of fluorescence
Under certain physical and chemical factors, fluorescence can weaken or even disappear.
UV irradiation
high temperature
Use fluorescence quenchers to eliminate non-specific fluorescence
methylene blue
Evans Blue
Basic fuchsin
iodine solution
fluorescent substance
Fluorescent pigments
Fluorescein isothiocyanate (FITC)
yellow-green
molecular weight
389.4kDa
Maximum absorbed light (excitation wavelength)
490-495nm
Maximum emitted light
520-530nm
The most widely used
advantage
Human eyes are sensitive to yellow-green
Usually there is less green fluorescence in sectioned specimens than red fluorescence
Tetraethyl Rhodamine (RB200)
Orange
Maximum absorbed light (excitation wavelength)
570nm
Maximum emitted light
595-600nm
Insoluble in water
Soluble in ethanol and acetone
Can be stored for a long time
Tetramethylrhodamine isothiocyanate
orange red
Maximum absorbed light (excitation wavelength)
550nm
Maximum emitted light
620nm
Can be dual labeled with FITC
Phycoerythrin (R-RE)/(PE)
orange color
Maximum absorption (excitation wavelength)
565nm
488nm
Maximum emitted light
620nm
575nm
Insoluble in water
Soluble in ethanol and acetone
Can be dual labeled with FITC
widely used
Shared 488nm excitation light
Other fluorescent substances
Lanthanide chelates
Europium (Eu3)
The most widely used
Applicable to
time-resolved fluorescence immunoassay
Generates fluorescence after enzyme action
No fluorescent effect itself
Fluorescence can be produced under the action of enzymes
Preparation of fluorescent antibodies
Fluorescein labeling of antibodies
method
Stirring method
Dialysis
Purification of labeled antibodies
method
Dialysis
chromatographic separation
Identification of antibodies
Antibody titer
biphasic immunodiffusion test
When the antigen content is 1g/L
Titer>1:16
Influencing factors
temperature
The lower the temperature
Higher efficiency and higher strength
pH
Depends on fluorescent pigments
For example, FITC decreases in acidic
Lanthanide chelates strengthen in acidity
concentration
Concentration too high
self-extinguishing phenomenon
Because it is too high, the molecules should collide violently
lose energy
Solvent
Fluorescent impurities in ethanol solutions
Need processing
enhancer
Speed up dyeing
Has no effect on coloring effects
Fluorescence microscope
light source
excitation light source
High pressure mercury lamp
xenon lamp
Halogen lamp
filter
Thermal filter
In front of the condenser of the light room
Blocks the passage of infrared rays and insulates heat
excitation filter
between light source and objective lens
Selectively transmits light in the ultraviolet visible wavelength range
Classification
UV filter (UG)
275-400nm UV light passes through
Maximum transmittance
365nm
Blue UV filter (BG)
325-500nm blue ultraviolet light passes through
Maximum transmittance
410nm
Absorption filter
between objective lens and eyepiece
Blocks the excitation light and allows the emitted fluorescence to pass through
dark background
Show fluorescence
easy to observe
Eyes protected from strong stimulating light irritation
Transmittance range
410-650nm
Classification
OG
orange yellow
GG
light greenish yellow
other
Watch FITC
excitation filter
BG12
Absorption filter
OG4 or GG9
Watch RB200
excitation filter
BG12
Absorption filter
OG5
Fluorescent Antibody Technology
Also known as
Fluorescence immunohistochemistry
Required for tissue or cell response
Fluorescence microscopy
Need to look at it under a microscope
process
labeled antibody
Only antibodies can be labeled
React with tissue or cell antigens in the section
After washing and separation
Observe complexes and parts under a microscope
clinical significance
Characterize or localize tissue cell antigens
position
Which part emits light (nucleus, cytoplasm, envelope, etc.)
Antigen localization tests can be done
Fluorescent Antibody Technology
enzyme tissue immunochemistry
preparation of specimens
Must have cells
Serum and plasma are not acceptable
source
Tissue sections, tissue prints, cell smears, microbial smears, exfoliated cell smears, concentrated body fluid specimens (sediments of pleural effusion and ascites)
Types
direct method
React directly with antigen
labeled on the antibody
Only the antigen can be checked
Features
Highest specificity
Method with the lowest non-specific staining
High direct specificity
poor sensitivity
Few non-specific fluorescent staining factors
indirect method
Test unknown antibodies using known antigens
primary antibody
humanIg
secondary antibody
Firefly or enzyme-labeled sheep/rabbit Ig
The same goes for ELISA testing for antibodies.
There is secondary antibody
Labeled on secondary antibody
Fluorescein-labeled secondary antibody conjugated to the primary antibody
Can detect antigen
Antibodies can also be tested
Advantages compared to direct method
Detect different antigens
Just prepare a fluorescent antibody
Because fluorescein is labeled on the secondary antibody
Detection
antinuclear antibodies
The most effective
double labeling method
Label different antibodies with FITC and rhodamine respectively
Stain the same specimen
Clinical application
Not a substitute for other routine testing
Autoantibody testing
Antinuclear antibodies (ANA) in serum
Pathogen detection
Identify pathogens
Specific antibody levels in serum
Immunopathological testing
Surface antigen and receptor detection
Identification of lymphocytes and subpopulations
Flow Cytometry
Fluorescence immunoassay type
Time-resolved fluorescence immunoassay (TRFIA)
principle
With lanthanide elements
Label antigen or antibody
Determined using time-resolved techniques
time resolution
non-specific fluorescence
Fluorescence lifetime is short
Lanthanide chelates
Long fluorescence lifetime
After non-specific fluorescence decay
Detect the fluorescent signal again
Stokes displacement
Wavelength difference between excitation spectrum and emission spectrum
If the Stokes displacement is small
Excitation and emission spectra often overlap
interfere with each other
Lanthanide chelates have large displacements
Easily eliminates scattering interference of excitation light
signal strength
Fluorescence can be enhanced in acidic enhancement solution
Clinical application
similar to radioimmunoassay
Detect trace substances
One more protein to detect
Fluorescence polarization immunoassay (FPIA)
principle
Using antigen-antibody competition reactions
Difference in fluorescence polarization degree of labeled antigen and complex
application
in blood or urine
Determination of small molecules
drug concentration
First method
Fluorescent enzyme immunoassay
principle
Utilize the reaction of antigen and antibody, with the help of enzyme reaction fluorescent substrate
The previous one is the double-antibody sandwich method.
Add substrate
process
Substrate 4-MUP has no fluorescence
Emit fluorescence under the stimulation of ALP
Flow Cytometry Analyzer (FCM)
Overview
Utilizing the characteristics of integrated optics, fluid mechanics, electronic computer technology, and fluorescein to produce fluorescence
Technology for multi-parameter quantitative measurement and sorting of each cell
Commonly used light sources
light emitted
Light absorbed by fluorescein
laser
structure
Liquid flow system
Optical and signal conversion test system
Computer systems for signal processing and amplification
principle
Single-cell suspensions and sheath fluids labeled with specific fluorescent fuels
Enter the flow room
Forming a single-cell fluid column that encapsulates the cell suspension in sheath fluid
Liquid column and horizontal laser beam (excitation light source)
vertical intersection
data
Forward scattered light (FS)
particle size
Side scattered light (SS)
The complexity inside the particle
surface smoothness
It is the same as the five categories
Fluorescence (FL)
The number of fluorescent parts stained by particles
PI staining
cell cycle
Commonly used DNA markers
PI (propidium iodide)
Commonly used excitation wavelengths
488nm
Commonly used emission wavelengths
620nm
Sorting harvest rate
There is a corresponding relationship between harvest rate and purity
High purity of sorted cells
The harvest rate is relatively low
Sheath fluid
effect
Wrap around the sample stream
Center the sample flow in the nozzle
Guaranteed accuracy
Prevent cells from getting close to the nozzle wall and blocking the hole
application
T cell subsets
Highest detection sensitivity
B cells stained with anti-Ig fluorescent antibodies
The surface shows fluorescence changes with time
ring, spot, cap, disappear
enzyme immunoassay
enzyme immunoassay
Utilizing the efficient and specific catalytic activity of enzymes
enzyme conjugate
Enzyme labeled antigen or antibody
Biomagnification of catalytic substrates
A small amount of enzyme can reflect a large amount of substances
Marking technology
Antigen or antibody
Positioning, qualitative, quantitative
Features
enzymes and substrates
enzyme requirements
Strong activity
High purity
Easy to couple with antigen and antibody
Activity is stable after labeling
Does not affect the immune reactivity of antigens and antibodies
specificity
No endogenous enzyme or inhibitor identical to the labeled enzyme is present in the sample being tested
products produced by enzyme catalysis
easy to judge or measure
Commonly used enzymes
Horseradish peroxidase (HRP)
substrate
Tetramethylbenzidine (TMB)
most commonly used
blue
good stability
No need to avoid light, no mutagenic effect
shortcoming
Poor water solubility
O-phenylenediamine (OPD)
One of the most sensitive chromogen substrates
orange yellow
unstable
Available as tablets or powder
Reconstitute before use
concept
Derived from vegetables
a glycoprotein
Sugar content 18%
composition
colorless glycoprotein
main enzyme
Independent of enzyme activity
Maximum absorption peak
275nm
Ferrous blood red
coenzyme
active group of enzyme
Maximum absorption peak
403nm
most commonly used
Alkaline phosphatase (AP/ALP)
substrate
p-Nitrophenylphosphate (p-NPP)
color
yellow
concept
Extracted from E. coli or calf intestinal mucosa
Classification
Bacteriogenic
intestinal mucosa
High activity
β-Galactosidase (β-Gal)
substrate
4-methylumbelanoyl-R-D galactopyranoside (4-MUU)
Features
Higher sensitivity than HRP
30-50 times
But when measuring, it is necessary
Fluorometer
concept
Escherichia coli extraction
Human blood lacks this enzyme
No interference from endogenous enzymes during measurement
Enzyme markers
Marking method
Glutaraldehyde cross-linking method
Modified sodium periodate method
HRP (horseradish peroxidase) labeled antigen and antibody
most commonly used method
Solid phase carrier
The most commonly used solid phase carrier for ELISA
polystyrene
covered
The process of binding an antigen or antibody to a solid support
Buffer
PH9.6
carbonate buffer
closed
1%-5% bovine serum albumin or 5%-20% calf serum
Covered again
Eliminate non-specific color development
Prevent the entry of impurity proteins in serum and interfere with the results
Classification
enzyme immunohistochemistry
used in pathology
Localization of antigens in tissue sections or other specimens
only antigen
Just positioning
Enzyme immunoassay (EIA)
Qualification or quantification of antigens or antibodies in liquids
Homogeneous enzyme immunoassay
Enzyme Amplified Immunoassay Technology (EMIT)
Clonase Donor Immunoassay (CEDIA)
The enzyme labeled on the antigen is inactive Only active after specific binding
heterogeneous enzyme immunoassay
solid phase enzyme immunoassay
liquid phase enzyme immunoassay
Separate and distinguish bound and free enzyme markers according to whether it is necessary
separated into heterogeneous phases
Divorce requires separation
Homogeneous enzyme immunoassay
Homogeneous without separation
No need for carrier
enzyme immunoassay
competition law
principle
The combined enzyme loses its activity
Test enzyme activity
What is missing is the binding enzyme
advantage
For measurement of small molecule antigens (hormones) or haptens (drugs)
heterogeneous enzyme immunoassay
heterogeneous phase separation
principle
The combined enzyme remains active
Just need to wash the board
It's a different phase
Classification
liquid phase enzyme immunoassay
Determination of extremely small amounts of short peptide hormones
Drugs and other small molecule haptens
Sensitivity
ng to pg
solid phase enzyme immunoassay
Use solid supports as carriers
common
Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay (ELISA)
How to detect antigens
Double-antibody sandwich method (two-step method)
process
solid phase antibody
First connect the specific antibody to the solid phase carrier
Antigen-antibody reaction
Add the antigen to be tested
Formation of solid-phase antibody-antigen complex
first wash
Enzyme labeled antibodies
Add enzyme-labeled antibodies
Formation of solid-phase antibody-antigen-enzyme-labeled antibody complex (on two different epitopes of the antigen to be tested)
second wash
Color development
Add substrate to develop color
The amount of colored product is proportional to the antigen to be tested
Applicable to
most commonly used method
Multivalent antigens with at least two epitopes
macromolecular antigen
Such as hepatitis B surface antigen, alpha-fetoprotein, hCG
extracellular soluble adhesion molecule
soluble antigen
Two-site (one-step method)
process
Combine the antigen to be tested and the enzyme-labeled antibody
Join the reaction at the same time
The two antibodies do not interfere with each other
After one wash
Add substrate to develop color
shortcoming
When the concentration of the antigen to be tested is too high
hook effect (backband phenomenon)
Reduced color rendering
false negative
Samples can be diluted and retested
competition law
Inversely proportional to
The higher the amount of antigen to be measured
The less colored products
process
solid phase antibody
First connect the specific antibody to the solid phase carrier (limited amount)
Antigen-antibody reaction
Add the antigen to be tested and enzyme-labeled antigen (limited quantity)
Competing binding specific antibodies
washing
Color development
Add substrate to develop color
Applicable to
Small molecule antigens with only a single epitope
Drugs, hormones, etc.
How to detect antibodies
indirect method
Hepatitis C virus antibodies
process
solid phase antigen
Coating the antigen on a solid phase carrier
Antigen-antibody reaction
Add the antibody to be tested
Formation of solid-phase antigen-antibody complex to be tested
first wash
Enzyme-labeled secondary antibody
Add enzyme-labeled secondary antibody (goat anti-human IgG)
Formation of solid-phase antigen-antibody-to-be-detected-enzyme-labeled secondary antibody complex
second wash
One marker detects multiple analytes
Enzyme-labeled secondary antibodies can detect any antigen-antibody complex
Color development
Directly proportional
competition law
Hepatitis B virus core antibody (HBcAb), hepatitis B virus e antibody (HBeAb)
Inversely proportional to
The higher the amount of antibody to be measured
The less colored products
process
solid phase antigen
Coating the antigen on a solid phase carrier
limited edition
Antigen-antibody reaction
Add the antibody to be tested and enzyme-labeled antibody (limited quantity)
Competing binding specific antibodies
washing
Color development
Add substrate to develop color
Influencing factors
Insufficient centrifugation of specimen
presence of fibrin
Enzyme labels will stick to fibrin
Resulting in being unable to be washed off
dark color
false negative
Hemolysis
Because hemolysis is dark in color
false negative
double antigen sandwich method
sensitivity and specificity
higher than indirect method
application
Detection of hepatitis B surface antibodies
capture method
also known as reverse indirect method
process
Secondary antibody against IgM coated on solid phase carrier
Add specimen to be tested
IgM can be captured by solid phase carriers
Add specific antigen
Add enzyme-labeled antibodies to specific antigens
Formation of solid-phase secondary antibody-IgM-antigen-enzyme labeled antibody complex
application
IgM type antibody
application
extracellular soluble adhesion factor
ELISA
Main measurement methods
Chemiluminescence immunoassay technology
principle
Combining the high sensitivity of luminescence analysis with the high specificity of antigen and antibody
Labeled immunoassay technology for detecting trace amounts of antigens or antibodies
Same as radioimmunoassay
Marking method
carbodiimide condensation method
condensing agent
carbodiimide
Sodium periodate oxidation method
Labeled glycoproteins have good stability
Not easy to fall off
Diazonium salt coupling method
Simple, low cost and good repeatability
N-hydroxysuccinimide activation method
glow
Ground state
absorb light
jump to excited state
return to ground state
The process of releasing photons
Same principle as fluorescence
chemiluminescent agent
type
direct chemiluminescence immunoassay
Use acridinium ester
in alkaline environment
Direct labeling of antigens (antibodies)
Antigen-antibody reaction occurs
Chemiluminescent enzyme immunoassay
Enzyme markers involved in luminescence through catalytic reactions or energy transfer
Use horseradish peroxidase or alkaline phosphatase
Label antigen or antibody
Antigen-antibody reaction occurs
Microparticle chemiluminescence immunoassay
Mark
alkaline phosphatase
Electrochemiluminescence immunoassay
Participate in oxidation reactions through energy transfer
Ruthenium terpyridine labeled antibody (antigen)
tripropylamine
Electron donor (electron acceptor)
In the electric field, a specific chemiluminescent reaction occurs on the electrode surface due to electron transfer.
Including two processes: electrochemistry and chemiluminescence
advantage
Similar to radioimmunoassay
But no pollution
High sensitivity and strong specificity
ng or even pg
Wide linear range
Marker is stable
The reagent has a long validity period and is non-toxic
high degree of automation
application
Hormones, tumor markers, drug concentrations, myocardial markers, viral antibodies and other trace biological activities
Detection
small amount of
Biotin-avidin amplification technology
activated biotin
Utilizing the carboxyl group of biotin
Use chemical modification to make derivatives of various reactive groups
After activation
Easily coupled to corresponding groups in various antigens, antibodies, enzymes and nucleic acid molecules
biotinylated markers
structure
I ring
imidazole copper ring
Avidin: tryptophan
II ring
Thiophene ring
Binding antibodies and other large molecules
Biotin (B)
Avidin (A)
high affinity
Combining speed, specificity and stability
Avidin
cross shape
Biotin
triangle
Avidin has four sites and can bind 4 biotins
So there is an amplification reaction
Label different types of activated biotin
activated biotin
Cannot label carboxyl groups
Because it was chemical modification made by him
Applications of Biotin-Avidin System (BAS)
B: biotin, A: avidin, S: amplification
Avidin labeling method
sodium periodate method
ABC method
principle
C: The meaning of enzyme
biotin labeling enzyme
biotin-binding avidin
Biotinylated antibody conjugated avidin
labeled enzymes and antibodies
It's all biotin
Avidin is only used to bind to biotin
coupled with ELISA
Greatly improve the sensitivity of ELISA
The enzyme is labeled in
on biotin
solid phase membrane immunoassay
Commonly used solid phase membranes
Nitrocellulose membrane (NC)
Colloidal gold
Antigen-antibody complexes are labeled with gold particles
under microscope
Dark brown particles
naked eye
red or pink spots
hCG
Detect hCG
Monoclonal Antibody Colloidal Gold Test
Highest sensitivity
hCG test during ovulation
To avoid cross-reaction
Monoclonal two-point enzyme immunoassay
8-10 weeks
reach peak
hCG in serum is slightly higher than in urine
The only hormone that does not increase in secretion as the weight of the placenta increases
Differentiating molar pregnancy from normal pregnancy
hCG concentration quantitative test
Classification
Dotted gold immunofiltration test
on NC film
Liquid flows vertically downward
Colloidal gold like lung clothing
Dot gold immunochromatographic assay
on NC film
liquid horizontal flow
like pregnancy test strips
Enzyme-linked immunospot test (ELISPOT)
Measurable B cells
secrete specific antibodies
Antibody secretion can also be measured
Measurable T cells
Cytokine-secreting T cells
Western-blot/wb
process
three phases
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
zone electrophoresis
electrotransfer
under transfer electrophoresis conditions
Transfer to nitrocellulose membrane (NC membrane)
Blot protein onto solid phase membrane
Still invisible to the naked eye
Enzyme reaction detection
Blotted protein (equivalent to a solid-phase carrier coated with antigen)
React with specific antibodies and enzyme-labeled secondary antibodies
Add substrate to develop color
Comprehensive SDS-PAGE and ELISA
Clinical application
HIV
Confirmatory test
Detection of antibody components
gp41/gp120/gp160 appears
two strips in
If he dies, call 120 and it won’t cure him. He’s going to run away.
gp120 binds to CD4
Destroy CD4 T cells
CD4/CD8
Ratio decreases
Anti-extractive antigen antibody assay (ENA antibody)
Southern blot
Blotting methods for detecting nucleic acids
immunohistochemistry
Overview
immunohistochemistry
labeled specific antibodies
Tests for locating, quantifying, and qualitatively identifying antigens
Can only test for antigen
Specimen source
living tissue
Tissue sections
Various body fluids and puncture fluids (serum and plasma are not acceptable because they have no cells)
Smears and prints
Cultured cells
Live cell slide
The purpose of specimen fixation
coagulation protein
Terminate the action of intracellular enzymes
Prevent autolysis
Maintain cell shape and structure
Maintain cell antigenicity
Prevent cell layers from falling off
Remove lipids that prevent antibody binding
Inhibit bacterial growth
Anticorrosion
Immunohistochemistry process
Antigen extraction and purification
Antigen to be tested
Immunizing animals and cell fusion
Preparation of specific antibodies
hybridoma technology
Antibody purification
Conjugate markers to antibodies
Form labeled antibodies
Specimen handling and preparation
primary reagent
Antibody
Monoclonal antibodies
Because it can only detect tissue, it must be an antigen, so it can only detect antigens
key step
Immunostaining
immunofluorescence histochemistry
Fluorescein labeled antibodies
Can only test for antigen
Features
Can detect multiple antigens in specimens simultaneously
Advantages compared to enzyme immunohistochemistry techniques
During fluorescence microscopy observation
Precautions
Observe specimens immediately after staining
It is not advisable to observe one field of view for a long time
Use non-fluorescent lens oil
Dark room observation
mistake
Observation is best at 37℃
enzyme immunohistochemistry
Use enzyme-labeled antibodies (antigens)
Antibodies and antigens can be measured
Classification
Enzyme labeled antibody immunohistochemistry technology
Same as enzyme immunoassay
Non-labeled antibody immunoenzyme histochemical staining-enzyme bridge method
The enzyme is not labeled on the antibody
Anti-enzyme antibodies as third antibodies
Have a bridging antibody as a secondary antibody
Formation of test antigen-specific antibody-bridge antibody-anti-enzyme antibody complex
Non-labeled antibody enzymatic staining-PAP method (peroxidase-anti-peroxidase method)
The third antibody is different
The third antibody (anti-enzyme antibody) and enzyme form a soluble complex (PAP complex)
This complex consists of 2 anti-enzyme antibodies and 3 peroxidase enzymes
pentagonal structure
very stable
transplant immunity
Classification
autotransplantation
The graft is derived from the host itself
No rejection reaction
syngeneic transplant
Transplantation between genetically identical individuals
Identical twins
Rejection reactions generally do not occur
allogeneic transplant
Transplantation between individuals of the same species but with different genotypes
target antigen causing rejection
major histocompatibility antigen (MHA)
Major histocompatibility antigen (MHA) of the major histocompatibility complex (MHC) on chromosome 6
Also known as human leukocyte antigen (HLA)
Human MHC=HLA
Classification
HLA I
HLA-A, HLA-B, HLA-C
All nucleated cell surfaces
Most lymphocytes
nerve cells
no nucleus
HLA-C
Has no significant effect on the transplantation immune process
HLA II
HLA-DR, HLA-DQ, HLA-DP
Distributed in professional APC cells, activated T cells and thymic epithelial cells
immunoglobulin-like domain
α2 and β2 functional domains
Antigen polypeptide binding region
α1 and β1 functional domains
CD4 binding region
β2 functional area
HLA-DR
The most immunogenic
Most important for transplant rejection
CDC method to detect HLA-DR antigen
Cells to be tested
purified B cells
Because class II proteins are present on the surface of B cells and macrophages (professional APCs)
HLA-I class interference has not been ruled out
with purified B cells
biological typing
PCR-RFLP
PCR-SSO
The most widely used method for class II HLA typing
PCR/SSP
principle
Design a set of sequence-specific primers (SSP) for HLA alleles
PCR amplification of DNA to be tested
get
allele-specific amplification products
PCR-SSCP
SBT
The most accurate and intuitive method
must
DNA isolation of cells to be tested
PCR amplification using locus-, group- or allele-specific primers
Purification and sequencing of amplified products
Comparison of the measured gene sequence with the known DNA sequence of the gene bank
HLA typing cytology
negative typing
standard cells
Homozygous typed cells with only one LD antigen on their surface
positive typing
standard cells
presensitized lymphocytes
Structure and function of MHC molecules
MHC
A group of closely linked genes on a chromosome encoding major histocompatibility antigens
MHC-I
Tumor cells can only be killed by Tc cells if they express MHC-I.
Platelets express only MHC-1
MHC-II
Important surface markers of presenting cells (APCs)
B cells can express MHC class I and class II
Recognized by helper CD4 T cells
Ribbon
Allotypic epitopes (polymorphic portion of HLA class II molecules)
Antigenic peptide binding region
Recognize CD4 and CD8 molecules
Ig sample area
Immunoglobulin-like region: β2 domain
Anchoring HLA molecules to the cell membrane
transmembrane region
and intracellular signaling
Intracellular region (cytoplasmic region)
HLA-II
Antigen polypeptide binding region
α1 and β1 functional domains
immunoglobulin-like domain
α2 and β2 functional domains
CD4 binding region
β2 functional area
Factors that determine MHC polymorphism
MHC genes are multiple alleles
are co-dominant
identification pathway
direct identification pathway
Donor APC
Donor transplant antigen (MHC)
presented to recipient T cells
indirect identification pathway
RecipientAPC
Donor transplant antigen (MHC)
presented to recipient T cells
Recognition of donor MHC by T cells
recognized molecule
are peptides derived from processed allotype MHC molecules
As long as there is one amino acid difference between the donor and the recipient
rejection reaction can occur
type
Host versus graft reaction (HVGR)
Rejection of the Donor by the Recipient
Graft versus host reaction (GVHR)
Donor rejection of recipient
Host versus graft reaction (HVGR)
Classification
hyperacute rejection
Within minutes to 1-2 days
Irreversible humoral rejection occurs
Quick, strong, irreversible
humoral immune response
main substance mediating
Cytotoxic antibodies (cytotoxic T cells/killer T cells/Tc cells)
reason
Those with incompatible ABO and other blood types, incompatible Rh blood types, multiple pregnancies, repeated blood transfusions, or those who have received organ transplants
Poor perfusion of the transplanted organ or excessive ischemia time
Improper preservation and handling of grafts
acute rejection
weeks to months
most common type
diagnosis
sIL-2R and serum creatinine values increased simultaneously
3H-TdR 4-hour incorporation method
3H-TdR 4-hour cell transformation assay
Methods for detecting sensitized T cells in recipients
A satisfactory test for predicting acute rejection crisis
Differentiation between acute rejection and viral infection
1-5 days before clinical symptoms appear
Increased CD4/CD8 ratio
>1.2
Indicates that acute rejection is about to occur
cytomegalovirus infection
CD4/CD8 decrease
<1.08
High possibility of infection
chronic rejection
months or even years
The disease progresses slowly
Graft versus host reaction (GVHR)
bone marrow transplant
If you do not receive immunosuppressive treatment
Depends on HLA-DR
Before treatment, high-dose radiotherapy and chemotherapy are required to kill all cells in the bone marrow.
exhibit immune incompetence
Show this
It only requires ABO blood type matching.
The bone marrow that enters the recipient's body has a large number of immune cells
Can carry out immune response to recipient's organs and tissues
HLA serological typing
Microcomplement-dependent cytotoxicity test (CDC)/microlymphocytotoxicity test
HLA-I and HLA-II typing methods
Complement-dependent cytotoxicity test
genetically predisposed
Unidentifiable
HLA-DP
Cytological HLA typing is required for identification
Identification of presensitized cells by cell typing
Known specific anti-HLA standard typing sera
Known antibodies
If characterized as anti-HLA-DR or DQ
Using platelets to adsorb HLA-I antibodies
Combine with lymphocytes to be tested
Antigen to be tested
add complement
Cytological HLA typing
Identifiable HLA-DP
Standard cells used
negative typing
Homozygous typed cells with only one LD antigen on their surface
positive typing
presensitized lymphocytes
Two-way mixed lymphocyte test/mixed lymphocyte test
Determine whether donor and recipient HLA antigens are compatible
If the amount of infiltration and transformed cells is the largest
Indicates strong MLC positivity
Indicates complete mismatch of D antigen
Unable to determine HLA type
Prevention and treatment
HLA crossmatch
Lymphocyte cross-match test (lymphocyte cross-toxicity test)
lymphocyte cross-toxicity test
Purpose
Whether there are antibodies against donor lymphocytes in the recipient's serum
Donor lymphocytes
Antibodies in recipient serum
Monophasic mixed lymphocyte reaction between cells from the recipient and cells from the donor treated with rhomycetin C
and cross-matching
Same as main side
treat
Allogeneic anti-CD3 antibody therapy
Most important complications
serum sickness
tumor immunity
Macrophages
Infiltrating macrophages in tumor lesions
and tumor spread and metastasis
negative correlation
Anti-tumor mechanism of action
Kill tumor cells through ADCC pathway
tumor antigen
Newly emerged or overexpressed during tumor occurrence and development
Antigenic substances
specific classification
Tumor Specific Antigen (TSA)
neoantigens specific to tumor cells
Only expressed in tumor cells
Absent and normal tissue cells
melanoma associated rejection antigen
MARA
prostate specific antigen
PSAs
tumor associated antigen
Not specific to tumor cells
Normal tissues and cells also express
When cancer occurs
The content increased significantly
alpha-fetoprotein
AFP
Anti-tumor effect mechanism
cellular immunity
The most important role
T cells, NK cells, macrophages
Humoral immune mechanism
Cytolytic effect of complement
Antibody effect
Antibody-dependent cell-mediated cytotoxicity (ADCC)
Immunomodulatory phagocytosis of antibodies
Antibodies block certain receptors on the surface of tumor cells
Antibodies bind to receptors and block signaling in tumor cells
Antibodies interfere with tumor cell adhesion
Common tumor markers
embryonic antigens
Alpha-fetoprotein (AFP)
primary liver cancer
AFP＞300ug/L
rise
pregnant women
newborn
acute hepatitis
primary liver cancer
AFP and ALT dynamic curves
Differentiation between liver cancer and benign liver disease (active hepatitis)
dynamic curve follower
benign liver disease
As high as you want
dynamic curve separator
Elevated AFP
ALT decrease
Consider liver cancer
Carcinoembryonic Antigen (CEA)
Has multiple isoforms
CEA-A, CEA-P, CEA-M, etc.
Contains complex antigenic determinants and multiple isomeric glycoproteins
smoker
Mild increase
Intestinal tumors
Colon cancer, rectal cancer, pancreatic cancer
Sugar chain antigens
CA153
breast cancer
Want me to touch
Breasts to touch
CA125
ovarian cancer
Want me
A son is about to be born, so in the ovary
Human epididymis protein 4 (HE4)
New tumor markers
ovarian cancer
CA199
gastrointestinal tumors
Want 99
I can’t digest it, it costs 99
pancreatic cancer
9 When viewed sideways, it looks like a pancreas
Pancreatic cancer, gallbladder cancer, colon cancer, stomach cancer
Squamous cell carcinoma antigen (SCCA)
Squamous cell carcinoma
Esophageal squamous cell carcinoma
cervical cancer
Enzymes and isoenzymes
Prostate-specific antigen (PSA)
prostate cancer
organ-specific tumor markers
f-PSA and f-PSA/t-PSA ratio
Differentiation between prostate cancer and benign prostate diseases
Both t-PSA and f-PSA are elevated
f-PSA/t-PSA ratio
<10%
Prostate cancer is likely
Prostatic acid phosphatase (PAP)
prostate cancer
Neuron-specific enolase (NSE)
Small cell lung cancer (oat cell carcinoma)
a-L-fucosidase (AFU)
primary liver cancer
Not related to AFP
complementary effects
Elevated AFU can also be seen in negative AFP
Hormones
Vanillyl Mandelic Acid (VMA)
Pheochromocytoma
human chorionic gonadotropin
Trophoblast tumors and germ cell tumors
Hydatidiform mole, erosive mole, choriocarcinoma
calcitonin
medullary thyroid cancer
adrenocorticotropic hormone (ACTH)
Small cell lung cancer (oat cell carcinoma)
protein
β2-Microglobulin (β2-MG)
Malignancy, myeloma, lymphoma, kidney disease
Because β2-microglobulin
Found on the surface of all nucleated cells
Ferritin
malignant tumors, inflammation
Protein of the Week (BJP)
multiple myeloma
Expresses ABO blood group antigen
gastric cancer cells
Not possible to be
Monosaccharide
immunodeficiency disease
primary immunodeficiency disease
primary B cell deficiency
X-linked agammaglobulinemia
Gamma globulin = immune globulin
Bruton syndrome
Absence of tonsils
Common clinical manifestations
concept
Congenital agenesis of B cells
Reduced or defective B cells
Reduced or deficient antibodies
Hard to find in bone marrow
Plasma cell
Normal number of T cells
Recurrent infections occurred in the child 6 months after birth (preschooler)
Current IgG and secretory IgA resistance within 6 months
selective IgA deficiency
Most common humoral immunodeficiencies
Both serum IgA and sIgA decreased
Often accompanied by recurrent infections of the digestive tract and respiratory tract
in the mucous membrane
Gamma globulin replacement therapy
invalid
common variable immunodeficiency disease
B cell immunodeficiency disease
Due to defective B cell function and abnormal signaling
humoral immunodeficiency disease
Bruton syndrome
Primary T cell deficiency
congenital thymic hypoplasia syndrome
cellular immunodeficiency disease
DiGeorge syndrome
Often accompanied by parathyroid insufficiency
Susceptibility to viral and fungal infections
Because neither cellular nor humoral immunity works
Cellular immune function deficiency
Humoral immune deficiency
Because CD4 T cells (Th cells) are needed
Embryonic thymus transplantation is effective
primary phagocytic deficiency disease
chronic granuloma
Abnormalities in the number, movement or adhesion function, and bactericidal activity of phagocytes
Recurrent infections with purulent bacteria or fungi
G-6-PD deficiency can cause
Due to the lack of G-6-PD, macrophages have insufficient energy supply and reduced phagocytosis and killing capabilities.
neutrophils
Chemotaxis ability significantly decreased
Chediak-Higashi syndrome
Lasy leukocyte syndrome
Chronic cutaneous and mucosal Candida albicans infection
Candida albicans skin test
diabetes
burn
normal newborn
Phagocytic ability is significantly reduced
Complement or antibody deficiency
Decreased enzyme metabolism
chronic granuloma
The positive rate of NBT test increased significantly
septicemia
complement deficiency
C1 inhibitory molecule defect
hereditary angioedema
recurring bacterial infections
not a virus
With systemic lupus erythematosus and chronic nephritis
Not necessarily
Severe combined immunodeficiency (SCID)
The patient developed developmental disabilities 6 months after birth
Susceptible to severe infection and death
sex-linked severe combined immunodeficiency disease
X-SCID
X-linked recessive inheritance
T cells
lack or significantly reduced
B cells
Normal but dysfunctional
Causes reduced Ig production and type conversion disorder
adenosine deaminase deficiency
Adenosine deaminase (ADA) deficiency
autosomal recessive inheritance
principle
Enzyme deficiency impairs the decomposition of adenosine and deoxyadenosine
Causes large amounts of storage of nucleotide metabolites dATP and dGTP
Toxic effects on early T and B cells
Causes T cell and B cell defects
clinical significance
After infant vaccination with BCG vaccine
Fatal and disseminated infection occurs
secondary immunodeficiency disease
Acquired immunodeficiency syndrome (AIDS)
AIDS
Infect
CD4 T cells
main
Macrophages
Dendritic Cells
B cells
Microglia in brain tissue
index
HIV antibody positive
Susceptibility to Pneumocystis carinii pneumonia
CD4/CD8 ratio inversion
Kaposi's sarcoma
Increased B cells and immunoglobulins
preliminary screening test
ELISA method
Confirmation test
immunoblotting
test
Humoral immunoassay
B cell defect detection
Cellular immunoassay
T cell defect detection
skin test
antigen
Substances that are easily sensitized by contact in the natural environment
tuberculin
Candida albicans
trichomycin
Streptokinase-Streptokinase (SK-SD)
mumps virus
test
Simultaneous testing of several antigens
Positive
Positive skin test for more than 3 antigens
Children under 2 years old
an antigen positive
Negative
Positive for less than 2 antigens
48 hour reaction diameter
<10mm
hint
Immunodeficiency or reduced responsiveness
PHA stimulation test
non-specific cellular immune function
PHA-L: Phytohemagglutinin
PHA stimulates lymphocyte proliferation and transformation test
Determine T cell function
Lymphocyte proliferation and transformation test
Newborn one week later
PHA irritation reaction occurs
Rule out the possibility of severe cellular immune deficiency
AIDS
Mainly invades CD4 cells (helper T cells Th)
gp120 binds to CD4 receptor protein
Decreased CD4/CD8 ratio
Cellular immune function deficiency
Phagocytosis Deficiency Disease
Intracellular bactericidal ability of neutrophils
Nitro blue tetrazolium (NBT) reduction test
Positive significantly increased
septicemia
Chemotaxis function test
Boyden Chamber Method
Chemotaxis in a small room
macrophage phagocytosis
Carbon particle clearance test
immunoproliferative disease
Diseases caused by abnormal proliferation of lymphocytes
plasma cell disease
Caused by excessive proliferation of plasma cells or Ig-producing B lymphocytes
Excessive amounts in serum or urine
monoclonal immunoglobulin
or light or heavy chain fragments
plasmacytoma
Reduced IL-4 synthesis, inhibition of B cell activation, and inhibition of APC antigen presentation ability
heavy chain disease
Mutations and abnormal proliferation of plasma cells
can only produce
Immunoglobulin heavy chain or defective heavy chain
Large amounts in serum or urine
Diseases caused by free immunoglobulin heavy chains
malignant plasma cell disease
light chain disease
Mutated plasma cells produce large amounts of abnormal light chains
Excess light chain deposits in the kidneys and other visceral tissues
Causes kidney damage and amyloidosis
Light chain protein types are divided into lambda type and k type
Lambda type nephrotoxicity is stronger
K-type M proteinemia
K/λ type ratio in serum
>4:1
Classification
malignant plasma cell disease
Multiple myeloma (MM)
A malignant tumor characterized by abnormal proliferation of a single plasma cell line in the bone marrow
mature B cell tumors
primary macroglobulinemia
heavy chain disease
benign plasma cell disease
Primary benign monoclonal Igemia
Secondary monoclonal hyperIgemia
clinical manifestations
Overflow proteinuria/prerenal proteinuria
=immunoglobulin light chain
=urinary B-J protein/BJP/week protein/agglutinin/immunoglobulin light chain
Monoclonal plasma cell hyperplasia
Monoclonal immunoglobulin (M protein)
hyperviscosity syndrome
Rising erythrocyte sedimentation rate
Urinary protein
blockage of renal tubules
leading to renal insufficiency
Plasma cells invade bone marrow
Causes bone marrow destruction, bone pain (most commonly), or fractures
hypercalcemia
Anemia, infection, bleeding, immune dysfunction
Because normal bone marrow cells cannot produce
laboratory tests
Quantitative detection of serum immunoglobulin (M protein)
M protein
monoclonal proliferation of plasma cells
Class, subclass, genotypic and idiotype homogeneous
Immunoglobulin
No antibody activity
No other immunological activity
More common in
Multiple myeloma, hypergammaglobulinemia, malignant lymphoma, heavy chain disease, light chain disease
Increased total protein concentration and increased M protein
IgG is the most common
Increased IgA
preliminary screening test
one-way agar immunodiffusion
immunoturbidimetry
Detection of urine light chain protein
Protein of the Week (BJP)
Immunoglobulin (M protein) light chain
Condensin
preliminary screening test
Thermal precipitation-dissolution method
Under the condition of PH: 4.9±0.1
Heat to 40-60℃
coagulation occurs
Raise to 90-100℃
redissolve
Reduce to 56℃
Resolidify
Can be negative in blood
Because this week’s protein has a small molecular weight
Easily excreted from the kidneys
Serum zone electrophoresis
use
Cellulose acetate membrane and agarose electrophoresis
M protein band
dense narrow protein band
in the r zone (sometimes in the beta or a zone)
The most basic, preferred, preliminary screening and qualitative test methods
Unable to correctly determine the type of immunoglobulin
immunoelectrophoresis
We can only estimate which type of Ig it is.
Unable to confirm
immunofixation electrophoresis
Confirmatory test for M protein
most commonly used method
Qualitative and typing identification of monoclonal antibodies
Preferred method
Identification of M protein light chain types
most commonly used method
subtopic
autoimmune disease
basic concept
Immune tolerance
to one's own tissue and cellular components
No immune response
generate a minimal immune response
Has immune stabilizing effect
Trace amounts of autoantibodies and sensitized lymphocytes may exist in normal human serum
self-immune
Immune tolerance is disrupted
Generate an immune response to self-components
The presence of autoantibodies or sensitized T lymphocytes
autoimmune disease
Autoantibodies or sensitized T lymphocytes can produce immune responses with corresponding self-components
Damage to tissues and organs or cause dysfunction due to immune response
Classification
organ-specific autoimmune diseases
limited to a certain organ or tissue
Chronic thyroiditis, Hashimoto's thyroiditis
Addison's disease (primary chronic adrenal insufficiency)
Myasthenia gravis, atrophic gastritis
Idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia
Specific platelets and red blood cells
non-organ-specific autoimmune diseases
A group of diseases that invade multiple tissues, organs or systems
Systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjogren's disease (SS)
Common feature
Most causes are unknown
There is no incentive to become more
spontaneity
Idiopathic
Mostly women
More women than men
More old people than young people
genetically predisposed
Sensitized T lymphocytes with autoantibodies or autologous tissue cells
Diseases can overlap
incurable
Alternating attacks and remissions
autoantigen
Release of cryptic antigens
Sperm, eye contents, brain, etc.
Those who have not been in contact with the immune system, the immune system does not know
Sympathetic ophthalmia
One eyeball injured, contents released
The body will also attack the other good eye
changes in one's own composition
Denatured IgG
Stimulates the body to produce anti-denatured IgG antibodies (rheumatoid factor RF)
rheumatoid arthritis
Cross-reactivity caused by common antigens
rheumatic fever
M protein in group A hemolytic streptococci
Same as endocardial, joint and glomerular basement membrane antigens
cause cross-reaction
acute glomerulonephritis
rheumatic fever
Common autoimmune diseases
Type I hypersensitivity reaction
II hypersensitivity reaction
myasthenia gravis
autoantigen
acetylcholine receptor
mechanism
Autoantibodies bind to acetylcholine receptors at the neuromuscular junction
Internalized and degraded
Causes decreased responsiveness of muscle cells to acetylcholine released by motor neurons
Causes skeletal muscle weakness
Hyperthyroidism (Graves' disease)
autoantigen
Thyroid Stimulating Hormone Receptor (TSHR)
Antibodies against TSH receptors
mechanism
diffuse toxic goiter
Type III hypersensitivity reaction
Systemic Lupus Erythematosus (SLE)
autoantigen
nuclear antigen
Nucleic acids, nucleoproteins, histones
autoantibodies
antinuclear antibodies (ANA)
mechanism
Immune complexes are deposited in
Cardiovascular connective tissue, glomerular basement membrane, serosa, joint synovium and small blood vessel walls of organs
activate complement
Attract neutrophil infiltration
Features
More common in young women
Cumulative multi-organ, multi-system
face
Butterfly erythema
skin
annular erythema
Photosensitivity, fever, rash
Kidney damage (glomerulonephritis), cardiovascular disease
anemia, psychiatric symptoms
Arthralgia, serositis
complement
active period
due to overactivation
level drops
After stabilization
increased reactivity
autoantibodies
antinuclear antibodies (ANA)
Most autoimmune diseases are positive
Mainly found in untreated SLE
Mainly IgG type
Not organ or species specific
Reacts with cell nuclei of all animals
Classification
anti-DNA antibodies
anti-histone antibodies
Anti-non-histone antibodies
anti-nucleolar antibodies
Detection
indirect immunofluorescence
anti-dsDNA antibodies
Anti-double-stranded DNA antibodies
Belongs to antinuclear antibodies (ANA)
Specific, characteristic antibodies
Frequent occurrence during active periods
method
Trypanosoma equi or Greenfly
antigen matrix
indirect immunofluorescence
The most specific, sensitive and commonly used method
anti-Sm antibodies
signature autoantibodies
Rheumatoid arthritis (RA)
autoantibodies
type
Rheumatoid factor (RF)
Denatured IgG Fc segment
autoantibodies to target antigens
Mainly IgM type
IgA, IgG, IgE
A negative result does not rule out RA
Antikeratin Antibodies (AKA)
Signs of prognosis
high titer
The condition is more serious
Anti-cyclic citrullinated antibodies (anti-CCP antibodies)
Highly specific
Early diagnosis may result in positive
RF detection method
Latex particle agglutination test
indirect method
Can only check IgM
ELISA
RF for different Ig types
Rabbit IgG is used as the antigen coated on the solid phase carrier
Combined with the sample to be tested
Add enzyme-labeled anti-human IgG, IgM, IgA, and IgE respectively
RF as bridging antigen
Sjogren's syndrome (SS)
also known as
Sjogren's syndrome
feature
Secretory gland dysfunction
Dryness of skin and mucous membranes
Tear and salivary glands are most commonly affected
Dry eyes and mouth
divided into
primary
Cumulative tissues other than exocrine glands
Secondary
Flag antibody
anti-SSA/Ro antibodies
anti-SSB/La antibody
Polymyositis (PM) and dermatomyositis (DM)
polymyositis
Mainly muscle damage
Dermatomyositis
Muscle damage and skin damage
Flag antibody
Jo-1
I kicked my skin with my feet.
PM-1
Scleroderma (Scl)
Systemic disease
Progressive systemic sclerosis (PSS)
Performance
Skin becomes tighter and harder
Flag antibody
anti-Scl-70 antibody
Be tough on your wife (7)
Serum total antinuclear antibodies (total ANA) detection method
Indirect immunofluorescence assay (IIF)
antigen
HEp2 cells
Human head cancer cells, rich in nuclear components
Reacts with tested serum
Then add FITC-labeled anti-human IgG (secondary antibody)
The most effective
Fluorescence patterns and clinical significance
Homogeneous type
High potency
Seen in systemic lupus erythematosus (SLE)
Peripheral/nuclear membrane type
High potency
Only found in SLE
Especially during the active period SEL
Prompt disease activity
Most common
spotted type
Mixed Connective Tissue Disease (MCTD)
mixed spots
nucleolar type
Scleroderma (SCL)
Hard-hearted people
Type IV hypersensitivity reaction
T cell responses to self-antigens
autoreactive T cell mediated
type 1 diabetes
The presence of autoreactive T cells in patients
CD8 CTL
Th1 cells
Common autoantibody tests
Anti-ENA antibody profile
ENA
General name for extractable zygogenic antigens
Available in saline or phosphate buffer
extract it
ANA cannot
ENA antibodies
Mainly IgG
Detection method
immunoblotting
clinical significance
Does not include anti-double-stranded DNA (anti-dsDNA antibodies)
Because it belongs to ANA
Detection of other autoantibodies
Antineutrophil cytoplasmic antibodies (ANCA)
Vasculitis
Target antigen of perinuclear (pANCA) antibodies
Myeloperoxidase (MPO)
Myeloid-specific
So around the core
Target antigen of cytoplasmic (cANCA) antibodies
Protease-3 (PR3)
Antimitochondrial antibodies (AMA)
Primary biliary cirrhosis (PBC)
Antiphospholipid antibody (APLA)
Includes anticardiolipin antibodies (ACLA)
Hypercoagulable state of blood
thrombosis
ACLA
Women with SLE are more likely to develop blood clots
Miscarriage is prone to occur during pregnancy
Anti-smooth muscle antibody (ASMA)
Autoimmune hepatitis (AIH)
Indirect immunofluorescence (IIF) assay
as a substrate
Monkey liver cells
Rat kidney, stomach or liver tissue
Antithyroglobulin Antibodies (ATGA)
Hashimoto's Thyroiditis (HT)
Anti-parietal cell antibody (PCA), anti-intrinsic factor antibody
atrophic gastritis
pernicious anemia
hypersensitivity disease
hypersensitivity reaction
allergy
After initial response to sensitization
answer again
physiological dysfunction
tissue cell loss
Abnormal immune response
immune defense
too high
Type I hypersensitivity reaction
IgE antibody mediated
Cytotropism
binds to mast cells
Immediate hypersensitivity/anaphylaxis
few minutes
principle
Production of allergen-specific IgE after primary response
Binds to IgE Fc receptors on mast cells
sensitization
Sensitized mast cell degranulation during re-response
Release bioactive mediators
Histamine, leukotrienes
cause
smooth muscle spasm
Mainly caused by leukotrienes
Main substances causing bronchial asthma
Increased small blood vessel permeability
Increased secretion of mucosal glands
Sensitive nerve endings
eosinophil activation
The main components involved in the reaction
allergens
certain medications
penicillin
inhalant allergens
pollen, mites
food allergens
milk, shrimp
Requires the participation of platelet activating factor
Antibody
IgE antibodies
IgG subclasses may also occur
IgG4
Because this does not activate complement
Type I hypersensitivity without complement involvement
participating cells
Mast cells
eosinophils
parasitic infection
Can be increased
basophils
Clinical symptoms
formula
Type I rapid onset, I billion acid-base, type I shock asthma and allergies
Billion:E:IgE
Systemic
anaphylactic shock
Caused by medication or injection of heterogeneous serum
Such as tetanus antitoxin, diphtheria antitoxin
penicillin anaphylactic shock
blood pressure drops
Caused by vasoactive substances
telangiectasia
parasitic infection
locality
respiratory allergic reaction
Allergic rhinitis and allergic asthma
Gastrointestinal allergic reaction
allergic gastroenteritis
allergic skin reaction
Urticaria, eczema, angioedema
Cause functional damage to effector organs
but no substantial pathological damage
causing type I hypersensitivity reactions
IL3/IL5, IL4
Testing method
Specificity test
Allergen intradermal test
Picking, scratching, intradermal injection
intradermal injection
Skin test such as penicillin
positive control
Histamine hydrochloride
false positive
A large number of hormonal drugs
false negative
Effects of antihistamines
process
A syringe injects the allergen extract into the skin
Antigen should be appropriately diluted
Injection volume
0.01-0.02ml
injection site
inner forearm
Cannot bleed or inject subcutaneously
multiple allergens simultaneously
spacing
2.5-5.0cm
Highly suspicious and sensitive
5.0cm
20-30 minutes to observe the results
Blushing (prick test)
Wheals (intradermal test)
Specific IgE (sigE) detection
Radioallergen adsorption test (RAST)
process
Adsorb purified allergens onto solid carriers
Add serum to be tested
Put in radioactively labeled anti-IgE antibody (secondary antibody)
Quantitative detection of specific IgE levels in serum
Such as serum allergy
Tetanus antitoxin injection required
Small and multiple injections
Type II hypersensitivity reaction
Cytotoxic or cytolytic
Type II poison, 23mygod
Target cell surface antigens bind to IgG or IgM class antigens
With the participation of complement, macrophages, and NK cells
Main ingredients
Antibody
IgG or IgM
Complement involvement
dissolution
participating cells
Macrophages
Opsonophagocytosis
NK cells
ADCC effect
clinical disease
formula
Hyperthyroidism, muscle strength, heat and hemorrhage
transfusion reaction
hemolytic disease of newborn
Rh for pregnant women (-), Rh for first child ( )
Prevent fetal hemolysis in another pregnancy
Within 72 hours after delivery
Injection of anti-Rh antibodies
No matter how fast the speed is
Autoimmune hemolytic disease (AIHA)
Drug-allergic cytopenia
Idiopathic thrombocytopenic purpura (ITP)
Pulmonary hemorrhage nephritis syndrome (Goodpasture syndrome)
Hyperthyroidism
(Graves' disease)
myasthenia gravis
(MG)
acute rheumatic fever
(ARF)
Detection
Anti-blood cell antibody test
Can be caused by ABO antigen
So here’s everything about blood
clinical significance
Helps in the diagnosis of autoimmune hemolytic anemia
anti-Rh antibodies
Helps with hemolytic disease in newborns caused by Rh blood group incompatibility
Streptolysin O (ASO)
Streptococcal cell wall M protein
Consistent with the heart and glomerulus
Therefore, these will be combined to cause damage.
Streptococcus-related diseases
pharyngitis
tonsillitis
otitis media
scarlet fever
acute glomerulonephritis
acute rheumatic fever
Type III hypersensitivity reaction
immune complex
Medium size soluble immune complex (IC: IgG, IgM)
Deposited in capillary basement membrane locally or in multiple places throughout the body
activate complement
Complement causes damage to blood vessel walls
And then induce the participation of platelets, basophils, and neutrophils
Congestion, edema, local necrosis
Neutrophil infiltration
The main characteristic of inflammatory reaction
memory method
Type III Compound, 23mygod, Third Plenary Session
Main ingredients
Antibody
IgG, IgM
participating cells
neutrophils
The most potent cause of tissue damage
basophils
Mast cells
platelets
Immune complex (IC), circulating immune complex (CIC)
Medium size soluble antigen-antibody immune complex
disease
local immune complex disease
Arthus reaction/Arthur reaction
Experimental local allergic reaction
Multiple subcutaneous injections of horse serum into rabbits
After 4-6 injections
Local edema, hemorrhage and necrosis
Arthus-like reaction
insulin
Therefore, when injecting insulin, you need to inject it at the patient’s location
vaccine
antiserum injection
systemic immune complex disease
formula
Fenglang kidney refreshing A
A: Arthus-like reaction
serum sickness
Onset occurs 1-2 weeks after injection of xenogeneic animal serum
Fever, rash, lymphadenopathy, joint swelling and pain, and transient proteinuria
Immediate shock
Type I hypersensitivity reaction
Poststreptococcal glomerulonephritis
Group A hemolytic streptococcus infection
2-3 weeks
Deposited on the glomerular basement membrane
immune complex nephritis
Rheumatoid Arthritis
Denatured IgG
Anti-IgG autoantibodies = rheumatoid factor (RF)
Anti-IgG autoantibodies
Mainly IgM antibodies
Occurs in small joints
rheumatoid arthritis
Occurs in large joints
systemic lupus erythematosus
DNA and anti-DNA antibody complexes
basement membrane of blood vessels deposited in the kidneys, joints, and other parts of the body
Causes glomerulonephritis, arthritis and other multiple organ damage
Detection
Detection in the organization
immunohistochemistry
Detection of immune complexes (CIC)
(CIC)
Classification
Antigen-specific methods
non-antigen specific methods
Often used clinically
Type IV hypersensitivity reaction
Delayed type hypersensitivity (DTH)
Effector T cells (CD4 Th1) (CD8 CTL)
After binding to specific antigen
An inflammatory response characterized by mononuclear-macrophage infiltration and tissue damage.
Main ingredients
antigen
intracellular parasites
Brucella, Mycobacterium tuberculosis, Salmonella typhi
It’s so sad not to get married
Viruses, parasites
Chemical material
Dyes, paints, pesticides, cosmetics
drug
penicillin
Type I hypersensitivity can also occur
target cells
cells harboring Mycobacterium tuberculosis
Transplanted cells or organs
tumor cells
protein to which hapten is attached
participating cells
effector T cells
CD4
Th1
CD8
CTL
Mononuclear-macrophages
disease
infectious delayed-type hypersensitivity reaction
Infection with intracellular parasitic pathogens
Mycobacterium tuberculosis
cavitary tuberculosis
Virus
protozoan
contact dermatitis
Exposure to small molecule hapten substances
transplant rejection
host versus graft reaction
HVGR
graft versus host reaction
GHR
Detection
Skin test principle
Intradermal injection, skin patch
24-48h
Local redness, swelling, induration, blisters, etc.
Purpose
Detecting Type IV Hypersensitivity Reactions
Cellular immune function status
Tuberculin test (OT)
method
4-8 weeks after infection
can appear
Old tuberculin (OT) or purified protein derivative (PPD) of Mycobacterium tuberculosis
48-72h observation
redness, swelling or hardness
>5mm
Positive
Judgment of results
<5mm
Negative
It does not mean that the patient is infected with tuberculosis
Because Mycobacterium tuberculosis is late-onset
5-9/10-19mm
Positive
Have been infected with Mycobacterium tuberculosis or vaccinated with Bacillus Calmette-Guérin (BCG)
≥20mm or blisters or necrosis
Strong positive
High risk of contracting tuberculosis
Purpose
Choose BCG vaccine recipients
Rule out tuberculosis infection
Understand the function of cellular immunity
skin patch test
Gauze soaked in allergen solution and placed on the inside of the forearm or back
Cover with cellophane or wax paper
24-72h
redness, swelling, blisters
Skin test difference
infiltrative pneumonia
Lung patchy shadow
Fever, cough, blood in sputum
diagnosis
Sputum acid-fast bacilli culture
Diagnose tuberculosis
Summarize
formula
Type I immediate onset, type II poison, type III complex, IV delayed onset
Antibodies and cells
100 million fertilizer acid-base, 23mygod, the Third Plenary Session of the CPC Central Committee, 4T cells
disease
Type I
Shock Asthma and Allergies
Type II
Hyperthyroidism, muscle strength, heat and hemorrhage
Type III
Fenglang kidney refreshing
Type IV
Transplant tuberculosis contact dermatitis
Infectious diseases
bacterial infection
strep infection
Gram-positive streptococci
Streptococcus pyogenes
Group A-beta-hemolytic Streptococcus
Testing method
latex agglutination test
immunoturbidimetry
Commonly used immunological markers
Antistreptolysin "O"
ASO
increase height
Acute pharyngitis and other upper respiratory tract infections
rheumatic fever
Type III hypersensitivity reaction
rheumatic myocarditis
rheumatoid arthritis
pericarditis
acute glomerulonephritis
Salmonella typhi
Clinical symptoms
self-healing gastroenteritis
Fatal typhoid fever (enteric fever)
Testing method
hypertrophic response
So fat, so sad
Enzyme-linked immunosorbent assay
ELISA
Mycobacterium tuberculosis
TB
Testing method
tuberculin test
Old Tuberculin (OT)
Tuberculin purified protein derivative (PPD)
Symbols of tuberculosis activity
anti-PPD antibodies
Positive
viral infection
influenza virus
structure
Hemagglutinin (HA)
Neuraminidase (NA)
method
Hemagglutination inhibition test
principle
The virus suspension is mixed with the serum (antibody) to be tested
Have corresponding antibodies
Can inhibit the binding of hemagglutinin on the surface of the virus to red blood cells
If you add red blood cells, it will no longer combine.
No agglutination
Positive
Coxsackie virus
Can infect the body and stimulate the body's immune system
Attack pancreatic beta cells
cause diabetes
Hepatitis A
Detection method
ELISA and chemiluminescence technology
Detection of HAV-IgM and IgG
IgM
Diagnose current infection
early infection
IgG
Late stage of infection
past infection
Hepatitis B
HBsAg (surface antigen)
appeared earlier
HBsAb (anti-HBs antibody)
protective antibodies
HBeAg (e antigen)
Indicates that HBV replicates actively in vivo
Very contagious
HBeAb (anti-HBe antibody)
Reduced ability to replicate
Reduced infectivity
HBcAg (core antigen)
HBV is present and actively replicating
direct indicator
Hard to detect
HBcAb (anti-HBc)
IgM
Hepatitis B acute phase and early infection
IgG
past infection
HBVDNA
most sensitive
most direct
chronic active hepatitis
clinical manifestations
Have a history of chronic hepatitis
HBc IgM positive
abnormal liver function
damage gene
extrahepatic injury
Caused by immune complex deposition
destruction mechanism
CD8 cells cause cell lysis by recognizing HBcAg and HLA-I expressed on the liver cell membrane
Ask for a double serum
Purpose
Observe whether the antibody titer in the recovery period is higher than that in the initial stage
congenital infection
Infections received by the fetus in the mother's body
collectively called TORCH
4 items of eugenics and education
Toxoplasma gondii (TOX)
Rubella virus (RUV)
Cytomegalovirus (HCMV)
Herpes simplex virus (HSV)
Clinical symptoms
abortion
deformity
Mental retardation, hearing impairment
parasitic infection
Schistosoma infection
ring egg precipitation reaction
Antigen-antibody reaction
Secretion of mature miracidia within the eggs (around the eggs)
Binds to antibodies within human serum
Formation of bubble-shaped, finger-shaped, strip-shaped specific precipitates with obvious refractive properties
Positive
polyethylene glycol
hybridoma technology
fusion agent
immune turbidity
Turbidity increasing agent
Antigen purification
polymer precipitation method