Immunological Techniques

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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

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Nature of Ag/Ab Reactions

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Strength of Antigen-Antibody Interactions Precipitation Reactions Agglutination Reactions Immunodiffusion Radioimmunoassay Enzyme-Linked Immunosorbent Assay Western Blotting/immunoblotting Immunofluorescence Immunohistocompatibility Localization of cells in tissue immunoblotting. contents:

Structure of an antibody : 

Structure of an antibody

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Affinity = ? attractive and repulsive forces Affinity Strength of the reaction between a single antigenic determinant and a single Ab combining site

Calculation of Affinity : 

Calculation of Affinity Ag + Ab ? Ag-Ab Applying the Law of Mass Action:

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

Specificity : 

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

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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

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Factors Affecting Measurement of Ag/Ab Reactions Affinity Avidity Ag:Ab ratio Physical form of Ag

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Precipitation Reactions

Radial Immunodiffusion (Mancini) : 

Radial Immunodiffusion (Mancini) Interpretation Diameter of ring is proportional to the concentration Quantitative Ig levels Method Ab in gel Ag in a well

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Precipitation Reactions(immunoelectrophorosis)

Countercurrent electrophoresis : 

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

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

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FIGURE 6-7 Demonstration of hemagglutination using Ab against sheep red blood cells (SRBCs). Agglutination Reactions visible clumping by interaction between Ab & a particulate antigen such as RBC, latex beads. -routinely performed to type RBCs for blood transfusion.

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RIA

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

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

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ELISA

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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.

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Immunofluorescence mIgM-producing B cells indirectly stained with rhodamine-conjurated secondary Ab under a fluorescence microscope. FIGURE 6-14

Localization of cells in tissue immunoblotting : 

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 Immunohistocompatibility