Slide 1: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


Slide 2:Nature of Ag/Ab Reactions


Slide 3: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


Slide 5: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:


Slide 7: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.


Slide 9: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


Slide 10:Factors Affecting Measurement of Ag/Ab Reactions Affinity Avidity Ag:Ab ratio Physical form of Ag


Slide 11: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


Slide 14: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


Slide 16:Agglutination/Hemagglutination Definition - tests that have as their endpoint the agglutination of a particulate antigen Agglutinin/hemagglutinin


Slide 17: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.


Slide 18:RIA


Slide 19: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


Slide 21:ELISA


Slide 22: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.


Slide 23: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