Antigen antibody reactions
plasma• Albumin• Fibrinogen• Gobulins
– Alpha
– Beta
– Gammaglobulis
Gammaglobulis
• Five types– IgG– IgA– IgM– IgE– IgD
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
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)
Avidity• The overall strength of binding between an Ag
with many determinants and multivalent Abs
Keq = 104
Affinity106
Avidity1010
Avidity
Specificity
• The ability of an antibody combining site to react with only an antigenic determinant.
• The ability of antibody to combine with an antigen
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
Factors Affecting Happening of Ag/Ab Reactions
• Affinity
• Avidity
• Ag:Ab ratio
• Physical form of Ag
Ab excess Ag excess
Equivalence – Lattice formation
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
Antigen antibody tests
• Used in both directions– Qualitative– Quantitative
Uses of antigens/antibody reactions
• Diagnosis Infectious diseases
• Diagnosis of autoimmune diseases
• Determination of blood type and HLA types
• Determination of chemical levels
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
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
Antigen and antibody reactions in the lab
• Precipitation tests
• Agglutination
• Elisa
• Radioimmunoassay
• Immunofluorescence
• Complement Fixation
Precipitation tests
• The antigen and antibody are in, soluble form
• Combine to form a visible precipitate
• Presence of electrolytes
• Positive controls and negative controls
Precipitation tests
• Precipitation techniques– Tube precipitation test– Gel diffusion
• Double• Single radial
• precipitation in agar with an electric field– Immuno electrophoresis– Countercurrent electrophoresis (CEP),
Precipitation in a capillary tube
• Streptococcus grouping
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
Double diffusion method (Ouchterlony)
• indicates whether – antigens are identical, – Antigens not identical– Partially identical
Single immunodiffusion/Radial immunodiffusion
• Method
– Ab in gel
– Ag in a well
Radial Immunodiffusion (Mancini)
• Interpretation– Diameter of ring is
proportional to the concentration
• Quantitative– Ig levels
Ag Concentration
Dia
met
er2
AgAgAgAg
Ab in gel
Precipitation in agar with an electric field
Immunoelectrophoresis
• Application of electric current
• Separation of proteins
Interpertation
• serum proteins are characterized in terms of their
• presence,
• absence
• unusual pattern (e.g., human myeloma protein).
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- +
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.
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
Agglutination
• Used in both directions
• Antigen part of a particulate matter Salmonella
• Particulate matter– Latex– Carbon particles – cells– Bacteria
• Stablised staphylococcal cells
Agglutination
• Active agglutination test
• The antigen part of a particulate matter per se
• examples• Salmonella, vibrio,
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
coagglutination
• stabilized staphylococcal cells-protein affinity for FC fragment of antibody –protein A
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
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
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
Immunofluorescence• Direct
– Ab to tissue Ag is labeled with fluorochrome
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
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).
Immunofluorescence
• Flow Cytometry– Cells in suspension are labeld with fluorescent tag
•Cells analyzed on a flow cytometer
Assays Based on Complement
Lattice formation not required
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
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
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
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
Direct Elisa
• Ag detection– Add labeled antibody
– Amount of labeled Ab bound is proportional to the amount of Ag in the sample
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
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.
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).
Extras
Ag-Ab reactionsTests for Ag-Ab reactions
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
Coombs (Antiglobulin)Tests
• Incomplete Ab• Direct Coombs Test
– Detects antibodies on erythrocytes
+
Patient’s RBCs Coombs Reagent(Antiglobulin)
Coombs (Antiglobulin)Tests
• Indirect Coombs Test– Detects anti-erythrocyte antibodies in serum
Patient’s Serum
TargetRBCs
+ Step 1
+
Coombs Reagent(Antiglobulin)
Step 2
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
Agglutination Tests
Lattice Formation
Active Agglutination
• Definition - tests that have as their endpoint the agglutination of a particulate antigen– Agglutinin
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
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
Passive Agglutination/Hemagglutination
• Definition - agglutination test done with a soluble antigen coated onto a particle
• Applications– Measurement of antibodies to soluble antigens
Coombs (Antiglobulin)Tests
• Applications– Detection of anti-Rh Ab– Autoimmune hemolytic anemia
Precipitation Tests
Lattice Formation
Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent
Assays (ELISA)
Lattice formation not required
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
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
• Extras
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
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
Factors Affecting Measurement of Ag/Ab Reactions
• Affinity
• Avidity
• Ag:Ab ratio
• Physical form of Ag
Ab excess Ag excess
Equivalence – Lattice formation
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
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
Haemagglutination inhibiotion test
• Certain viruses measles and influenza arboviruses agglutinate RBC,s
• If specific antibodies are included in the system a virus is identified
Tests for Cell Associated Antigens
Lattice formation not required
Immunofluorescence
• Flow Cytometry– Cells in suspension are labeld with fluorescent tag
•Cells analyzed on a flow cytometer
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