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