Antigen-AntibodyAntigen-AntibodyInteractions:Interactions:Principles & ApplicationsPrinciples & Applications --by K.R.Deepthi

- A bimolecular association - A bimolecular association

involving various noncovalent interactionsinvolving various noncovalent interactions- Is similar to an enzyme-substrate interactions, Is similar to an enzyme-substrate interactions,

but not lead to an irreversible chemical alterationbut not lead to an irreversible chemical alteration

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

Nature of Ag/Ab Reactions

1.1. Strength of Antigen-Antibody InteractionsStrength of Antigen-Antibody Interactions

2.2. Precipitation ReactionsPrecipitation Reactions

3.3. Agglutination ReactionsAgglutination Reactions

4.4. ImmunodiffusionImmunodiffusion

5.5. RadioimmunoassayRadioimmunoassay

6.6. Enzyme-Linked Immunosorbent AssayEnzyme-Linked Immunosorbent Assay

7.7. Western Blotting/immunoblottingWestern Blotting/immunoblotting

8.8. ImmunofluorescenceImmunofluorescence

9.9. ImmunohistocompatibilityImmunohistocompatibility

10.10.Localization of cells in tissue immunoblotting.Localization of cells in tissue immunoblotting.

contents:

Structure of an antibody

Affinity = attractive and repulsive forces

Ab

Ag

High Affinity

Ab

Ag

Low Affinity

Affinity

• Strength of the reaction between a single antigenic determinant and a single Ab combining site

Calculation of Affinity

Ag + Ab Ag-Ab

Keq = [Ag-Ab]

[Ag] x [Ab]

Applying the Law of Mass Action:

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 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

Anti-A Ab

Ag A

Anti-A Ab

Ag B

Shared epitope

Anti-A Ab

Ag C

Similar epitope

Cross reactions

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 in fluids yield a precipitin curve. FIGURE 6-4

( Lattices or large aggregates )

( no precipitate is formed if an Ag contains only a single copy of each epitope )

Precipitation ReactionsPrecipitation Reactions

Radial Immunodiffusion (Mancini)

• Interpretation– Diameter of ring is

proportional to the concentration

• Quantitative– Ig levels

• Method– Ab in gel– Ag in a well

Ag Concentration

Dia

met

er2

AgAgAgAg

Ab in gel

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

in the Ab-containing semisolid medium

The region of equivalence

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

Precipitation Reactions(immunoelectrophorosis)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

• Qualitative–Rapid

Ag Ab- +

Agglutination/Hemagglutination

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

+

• Qualitative agglutination test– Ag or Ab

FIGURE 6-7

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

Agglutination ReactionsAgglutination Reactions

+ + + (control)

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

RIA

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

RIA

RIA

• Radioimmunoassay is widely-used because of its great sensitivity.

• Using antibodies of high affinity, it is possible to detect a few picograms (10−12 g) of antigen in the tube.

• The greater the specificity of the antiserum, the greater the specificity of the assay

FIGURE 6-10Variations in the enzyme-linked immunosorbent assay (ELISA) technique, similar to RIA except using an Enzyme (alkaline , ⓟ horseradish peroxidase, & β-galactosidase) : safer & less costly.

to detect Ab (HIV, HCV)

to detect Ag

to detect Ag

ELISA ELISA

FIGURE 6-12

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.

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

Immunofluorescence

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

FIGURE 6-14

Fluorochromes-Fluorescein (490→517nm)-Rhodamine (515→546nm)-Phycoerythrin : absorb light of one wavelength & emit fluorescence at a longer wavelength than fluorescein.

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.

• It is similar to grafting

Immunohistocompatibility