Antigen-Antibody Interactions:

Principles & Applications

--by

K.R.Deepthi

- A bimolecular association involving various noncovalent interactions - Is similar to an enzyme-substrate interactions, but not lead to an irreversible chemical alteration

Nature of Ag/Ab Reactions

- Four types of non-covalent forces operates over a very short distance ( generally 1 angstrom )

contents:
1. Strength of Antigen-Antibody Interactions 2. Precipitation Reactions 3. Agglutination Reactions 4. Immunodiffusion 5. Radioimmunoassay 6. Enzyme-Linked Immunosorbent Assay 7. Western Blotting/immunoblotting 8. Immunofluorescence 9. Immunohistocompatibility 10.Localization of cells in tissue immunoblotting.

Structure of an antibody

Affinity
Strength of the reaction between a single antigenic determinant and a single Ab combining site High Affinity Ab Low Affinity Ab

Ag

Ag

Affinity = attractive and repulsive forces

Calculation of Affinity

Ag + Ab Ag-Ab Applying the Law of Mass Action: [Ag-Ab] [Ag] x [Ab]

Keq =

Avidity
The overall strength of binding between an Ag with many determinants and multivalent Abs

Keq =

104 Affinity

106 Avidity

1010 Avidity

Specificity
The ability of an individual antibody combining site to react with only one antigenic determinant.

Cross Reactivity
The ability of an individual Ab combining site to react with more than one antigenic determinant. The ability of a population of Ab molecules to react with more than one Ag

Cross reactions
Anti-A Ab Anti-A Ab Anti-A Ab

Ag A

Ag B

Ag C

Shared epitope

Similar epitope

Factors Affecting Measurement of Ag/Ab Reactions

Affinity Avidity Ag:Ab ratio Physical form of Ag
Ab excess
Ag excess

Equivalence Lattice formation

Precipitation Reactions

( no precipitate is formed ( Lattices or if an Ag contains only a large aggregates ) single copy of each epitope ) FIGURE 6-4

Precipitation reactions in fluids yield a precipitin curve.

Radial Immunodiffusion (Mancini)

Method
Ab in gel Ag in a well
Ab in gel
Ag Ag Ag Ag

Interpretation
Diameter2

Diameter of ring is proportional to the concentration

Quantitative
Ig levels

Ag Concentration

in the Ab-containing semisolid medium

-> The area is proportional to the conc. of Ag.

The region of equivalence

FIGURE 6-5
Diagrammatic representation of radial & double immunodiffusion. : precipitation reactions in gels yield visible precipitin lines; no visible precipitate forms in regions of Ab or Ag excess.

Precipitation Reactions(immunoelectrophorosis)

FIGURE 6-6 (a) Immunoelectrophoresis. - an antigen mixture is first electrophoresed to separate its components by charge - diffusion & producing lines of precipitation.

Countercurrent electrophoresis
Method
Ag and Ab migrate toward each other by electrophoresis Used only when Ag and Ab have opposite charges

Ag Qualitative
Rapid

+ Ab

Agglutination/Hemagglutination
Definition - tests that have as their endpoint the agglutination of a particulate antigen
Agglutinin/hemagglutinin

Qualitative agglutination test

Ag or Ab

Agglutination Reactions
-visible clumping by interaction between Ab & a particulate antigen such as RBC, latex beads. -routinely performed to type RBCs for blood transfusion.

+
FIGURE 6-7

(control)

Demonstration of hemagglutination using Ab against sheep red blood cells (SRBCs).

RIA

RIA

From these data, a standard binding curve, like the one shown in red, can be drawn.

RIA
Radioimmunoassay is widely-used because of its great sensitivity. Using antibodies of high affinity, it is possible to detect a few picograms (1012 g) of antigen in the tube. The greater the specificity of the antiserum, the greater the specificity of the assay

to detect Ab (HIV, HCV)

ELISA

to detect Ag

to detect Ag

FIGURE 6-10

Variations in the enzyme-linked immunosorbent assay (ELISA) technique, similar to RIA except using an Enzyme (alkaline , horseradish peroxidase, & -galactosidase) : safer & less costly.

Western blotting
: separates the components according to their molecular weight.

: the proteins in the gel are transferred to the sheet of nitrocellulose or nylon by the passage of an electric current.

: probed with Ab & then radiolabeled or enzyme-linked 2nd Ab.

FIGURE 6-12

: a position is visualized by means of an ELISA reaction.

Immunofluorescence

Fluorochromes
-Fluorescein (490517nm) -Rhodamine (515546nm) -Phycoerythrin : absorb light of one wavelength & emit fluorescence at a longer wavelength than fluorescein.
FIGURE 6-14

mIgM-producing B cells indirectly stained with rhodamine-conjurated secondary Ab under a fluorescence microscope.

Localization of cells in tissue immunoblotting

Nonsymbiotic hemoglobins (ns-Hbs) previously have been found in monocots and dicots. however, very little is known about the tissue and cell type localization as well as the physiological function(s) of these oxygen-binding proteins. The immunodetection and immunolocalization of ns-Hbs in rice (Oryza sativa L.) by Western blotting and in situ confocal laser scanning techniques. Ns-Hbs were detected in soluble extracts of different tissues from the developing rice seedling by immunoblotting. Levels of ns-Hbs increased in the germinating seed for the first six days following imbibition and remained relatively constant thereafter.

Immunohistocompatibility
It is similar to grafting