anigen antiboy reactions
DESCRIPTION
immunologyTRANSCRIPT
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Antigen antibody reactions
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plasma• Albumin• Fibrinogen• Gobulins
– Alpha
– Beta
– Gammaglobulis
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Gammaglobulis
• Five types– IgG– IgA– IgM– IgE– IgD
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functions of antibodies• to neutralize
– toxins – viruses,
• to opsonize microbes – so they are more easily phagocytosed,
• to activate complement
• to prevent the attachment of microbes to mucosal surfaces.
• antibodies have a catalytic (enzymatic) capability
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Affinity = attractive and repulsive forces
Ab
Ag
High Affinity
Ab
Ag
Low Affinity
Affinity
• Strength of the reaction between an antigenic determinant (antigen) and an antigen combining site (antibody)
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Avidity• The overall strength of binding between an Ag
with many determinants and multivalent Abs
Keq = 104
Affinity106
Avidity1010
Avidity
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Specificity
• The ability of an antibody combining site to react with only an antigenic determinant.
• The ability of antibody to combine with an antigen
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Cross Reactivity• The ability of an individual Ab combining site to
react with more than one type of antigenic determinant.
• The ability of a Ab molecules to react with more than one Ag
Anti-A Ab
Ag A
Anti-A Ab
Ag C
Similar A.D
Cross reaction
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Factors Affecting Happening of Ag/Ab Reactions
• Affinity
• Avidity
• Ag:Ab ratio
• Physical form of Ag
Ab excess Ag excess
Equivalence – Lattice formation
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Tests Based on Ag/Ab Reactions
• All tests based on Ag/Ab reactions will have to depend on lattice formation itself or
• we will have to utilize ways to detect small immune complexes
• All tests based on Ag/Ab reactions can be used to detect either Ag or Ab
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Antigen antibody tests
• Used in both directions– Qualitative– Quantitative
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Uses of antigens/antibody reactions
• Diagnosis Infectious diseases
• Diagnosis of autoimmune diseases
• Determination of blood type and HLA types
• Determination of chemical levels
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Diagnosis of infectious diseases
• When the organism cannot be cultured-or difficult to culture hep A B C Syphilis
• When the organism is too dangerous to culture- rickettsial disease
• When the culture technique is not readily available-HIV,EBV
• When the organism takes too long to grow e.g Mycoplasma
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Qualitative /quantitative
• Qualitative– determines antigen or antibody is present or
absent
• Quantitative – determines the quantity of the antibody– Titer– The highest dilution of the specimen usually
serum which gives a positive reaction in the test
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Antigen and antibody reactions in the lab
• Precipitation tests
• Agglutination
• Elisa
• Radioimmunoassay
• Immunofluorescence
• Complement Fixation
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Precipitation tests
• The antigen and antibody are in, soluble form
• Combine to form a visible precipitate
• Presence of electrolytes
• Positive controls and negative controls
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Precipitation tests
• Precipitation techniques– Tube precipitation test– Gel diffusion
• Double• Single radial
• precipitation in agar with an electric field– Immuno electrophoresis– Countercurrent electrophoresis (CEP),
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Precipitation in a capillary tube
• Streptococcus grouping
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Double Diffusion
• antigen and antibody are placed in different wells in agar and
• allowed to diffuse and form concentration gradients.
• Where optimal proportions occur, lines of precipitate form
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Double diffusion method (Ouchterlony)
• indicates whether – antigens are identical, – Antigens not identical– Partially identical
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Single immunodiffusion/Radial immunodiffusion
• Method
– Ab in gel
– Ag in a well
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Radial Immunodiffusion (Mancini)
• Interpretation– Diameter of ring is
proportional to the concentration
• Quantitative– Ig levels
Ag Concentration
Dia
met
er2
AgAgAgAg
Ab in gel
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Precipitation in agar with an electric field
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Immunoelectrophoresis
• Application of electric current
• Separation of proteins
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Interpertation
• serum proteins are characterized in terms of their
• presence,
• absence
• unusual pattern (e.g., human myeloma protein).
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Counter-Immunoelectrophoresis
• Method– Ag and Ab migrate toward each other by
electrophoresis– Used only when Ag and Ab have opposite charges
• Qualitative–Rapid
Ag Ab- +
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uses
• The meeting of the antigen and antibody is greatly accelerated
• made visible in 30–60 minutes.
• detection of bacterial and fungal polysaccharide antigens in cerebrospinal fluid.
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Agglutination
• Visible clumping together of particulate matter by antigen combining with its specific antibody.
• The clumps will be called agglutinates• Performed
– Slide
– Tube
– Tile
– Micrtitration plates
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Agglutination
• Used in both directions
• Antigen part of a particulate matter Salmonella
• Particulate matter– Latex– Carbon particles – cells– Bacteria
• Stablised staphylococcal cells
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Agglutination
• Active agglutination test
• The antigen part of a particulate matter per se
• examples• Salmonella, vibrio,
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Agglutination
• Passive agglutination test
• Antigen or antibody are not part of particulate matter but are attached (rided on inert particles like latex, carbon,)
+ Particulat matter
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coagglutination
• stabilized staphylococcal cells-protein affinity for FC fragment of antibody –protein A
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Prozone phenomenon
• Tube agglutination• The lower dilutions do not show agglutination • the tubes (prior/before the optimum zone )• The tubes in higher dilutions show agglutination• Reasons/factors
– Antibody excess:High level of antibody
– Non specific inhibitory factors
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Haemagglutination
• Active Haemagglutination test – (blood group)
• Passive haemagglutination (TPHA)– Known antigen coated on to treated RBC,s– Treated To remove their own antigens– Turkey,s RBC,s are used
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Immunofluorescence
• Fluorescent dyes illuminated by ultraviolet light are used to show combination of antigen antibody .
• The end point antigen antibody complexes are seen fluorescing against a dark background
• Direct• Indirect
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Immunofluorescence• Direct
– Ab to tissue Ag is labeled with fluorochrome
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Immunofluorescence
• Indirect– Ab to tissue Ag is
unlabeled– Fluorochrome-labeled
anti-Ig is used to detect binding of the first Ab.
• Qualitative to Semi-Quantitative
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Fluorescence-Activated Cell Sorting (Flow Cytometry)
• the patient's cells are labeled with monoclonal antibody to the protein specific to the cell of interest, e.g., CD4 protein
• The monoclonal antibody is tagged with a fluorescent dye, such as fluorescein or rhodamine.
• Single cells are passed through a laser light beam, • the number of cells that fluoresce is counted by
use of a machine called a fluorescence-activated cell sorter (FACS).
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Immunofluorescence
• Flow Cytometry– Cells in suspension are labeld with fluorescent tag
•Cells analyzed on a flow cytometer
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Assays Based on Complement
Lattice formation not required
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CFT
• The CFT is used to detect antibodies or antigens
• inference is made by the ability of the antibodies to fix the complement
• The fixation of the complement is measured by adding the indicator system
• Used in diagnosis of viral parasitic and rickettsial diseases
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Complement Fixation
– Antibody known mixed with test material to be assayed for Ag
– Standard amount of complement is added– Erythrocytes coated with Abs is added– Amount of erythrocyte lysis is determined
Ag
Ag NO Ag
Ag
• Methodology
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RadioImmunoassay
• The radioactivity of the specific labeled antibody or antigen is used to quantify the antigen or antibody in patient ,s serum
• HbsAg
• Hav IGM
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ELISA
• Uses an enzyme system to show the specific combination of antigen antibody
• An enzyme labeled or linked to a specific antigen • A substrate• A color reader• Double antibody technique to detect and assay
antigen• Indirect technique to Assay antibody
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Direct Elisa
• Ag detection– Add labeled antibody
– Amount of labeled Ab bound is proportional to the amount of Ag in the sample
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Indirect ELISA
• Ab detection– Immobilize Ag– Incubate with sample– Add labeled anti-Ig– Amount of labeled Ab
bound is proportional to amount of Ab in the sample
• Quantitative
SolidPhase
AgImmobilized
Ab in Patient’s
sample
LabeledAnti-Ig
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Western blot• HIV proteins are separated electrophoretically in a gel,
• discrete bands of viral protein.
• These proteins are then transferred from the gel, i.e., blotted, onto filter paper, and
• the person's serum is added.
• If antibodies are present, they bind to the viral proteins (primarily gp41 and p24) and can be detected by adding antibody to human IgG labeled an enzyme,
• produces a visible color change when the enzyme substrate is added.
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Neutralization Tests
• These use the ability of
• antibodies to block the effect of toxins or the infectivity of viruses.
• They can be used in cell culture ( inhibition of cytopathic effect
• in host animals ( mouse protection tests).
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Extras
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Ag-Ab reactionsTests for Ag-Ab reactions
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Nature of Ag/Ab Reactions
• Lock and Key Concept
• Non-covalent Bonds– Hydrogen bonds– Electrostatic bonds– Van der Waal forces– Hydrophobic bonds
• Reversible
• Multiple Bonds
Source: Li, Y., Li, H., Smith-Gill, S. J.,
Mariuzza, R. A., Biochemistry 39, 6296, 2000
http://www.med.sc.edu:85/chime2/lyso-abfr.htm
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Coombs (Antiglobulin)Tests
• Incomplete Ab• Direct Coombs Test
– Detects antibodies on erythrocytes
+
Patient’s RBCs Coombs Reagent(Antiglobulin)
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Coombs (Antiglobulin)Tests
• Indirect Coombs Test– Detects anti-erythrocyte antibodies in serum
Patient’s Serum
TargetRBCs
+ Step 1
+
Coombs Reagent(Antiglobulin)
Step 2
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Immunofluorescence
• Flow Cytometry cont.– Data displayed
Green Fluorescence Intensity
Nu
mb
er o
f C
ells
Unstained cells
FITC-labeled cells
One Parameter Histogram
Red Fluorescence Intensity
Gre
en F
luor
esce
nce
In
ten
sity
Two Parameter Histogram
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Agglutination Tests
Lattice Formation
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Active Agglutination
• Definition - tests that have as their endpoint the agglutination of a particulate antigen– Agglutinin
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Agglutination/Hemagglutination• Quantitative agglutination test
– Titer– Prozone
1/2
1/4
1/8
1/16
1/32
1/64
1/12
8
1/25
6
1/51
2
1/10
24
Pos
.
Neg
.
Titer
64
8
512
<2
32
128
32
4
Patient
1
2
3
4
5
6
7
8
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Agglutination/Hemagglutination
• Definition
• Qualitative test
• Quantitative test• Applications
– Blood typing– Bacterial infections
–Fourfold rise in titer
• Practical considerations– Easy– Semi-quantitative
1/2
1/4
1/8
1/16
1/32
1/64
1/12
8
1/25
6
1/51
2
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Passive Agglutination/Hemagglutination
• Definition - agglutination test done with a soluble antigen coated onto a particle
• Applications– Measurement of antibodies to soluble antigens
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Coombs (Antiglobulin)Tests
• Applications– Detection of anti-Rh Ab– Autoimmune hemolytic anemia
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Precipitation Tests
Lattice Formation
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Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent
Assays (ELISA)
Lattice formation not required
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Competitive ELISA for antigen
• Method– Determine amount
of Ab needed to bind to a known amount of labeled Ag
+
Prior to Test
Labeled Ag
+
Test
+Patient’ssample
LabeledAg
+
– Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor
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Competitive ELISA for Ag • Method cont.
– Determine amount of labeled Ag bound to Ab NH4SO4
anti-Ig • Immobilize the Ab
• Quantitative– Most sensitive test
+ Test
+Patient’ssample
LabeledAg
+
– Concentration determined from a standard curve using known amounts of unlabeled Ag
SolidPhase
SolidPhase
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• Extras
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Solid Phase RIA for antigen/direct
• Ag detection– Immobilize Ab– Incubate with sample– Add labeled antibody– Amount of labeled Ab
bound is proportional to the amount of Ag in the sample
• Quantitative
SolidPhase
Ag
Immobilized
Ag in Patient’s
sample
LabeledAb
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Solid Phase RIA for antibody/indirect
• Ab detection– Immobilize Ag– Incubate with sample– Add labeled anti-Ig– Amount of labeled Ab
bound is proportional to amount of Ab in the sample
• Quantitative
SolidPhase
AgImmobilized
Ab in Patient’s
sample
LabeledAnti-Ig
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Factors Affecting Measurement of Ag/Ab Reactions
• Affinity
• Avidity
• Ag:Ab ratio
• Physical form of Ag
Ab excess Ag excess
Equivalence – Lattice formation
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Cross Reactivity• The ability of an individual Ab combining site to
react with more than one type of antigenic determinant.
• The ability of a Ab molecules to react with more than one Ag
Anti-A Ab
Ag A
Anti-A Ab
Ag B
Shared A.D
Anti-A Ab
Ag C
Similar A.D
Cross reactions
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Immunoelectrophoresis• Method
– Ags are separated by electrophoresis
• Interpretation– Precipitin arc represent individual antigens
Ag-+
Ag
Ab
Ag
Ab
– Ab is placed in trough cut in the agar
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Haemagglutination inhibiotion test
• Certain viruses measles and influenza arboviruses agglutinate RBC,s
• If specific antibodies are included in the system a virus is identified
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Tests for Cell Associated Antigens
Lattice formation not required
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Immunofluorescence
• Flow Cytometry– Cells in suspension are labeld with fluorescent tag
•Cells analyzed on a flow cytometer
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Double Antibody ELISA
• Ag detection– Immobilize Ab– Incubate with sample– Add labeled antibody– Amount of labeled Ab
bound is proportional to the amount of Ag in the sample
• Quantitative
SolidPhase
Ag
Immobilized
Ag in Patient’s
sample
LabeledAb