Antigen binding sites blocking

Enzyme-multiplied immunoassay technique Perhaps the best known homogeneous assay format is the enzyme-multiplied immunoassay technique (EMIT), in which the analyte is covalently attached to an enzyme, and the formation of an analyte-antibody complex blocks the active site and inhibits enzyme activity. When this blocked enzyme is mixed with the experimental sample, there is competition between the enzyme-linked analyte and the sample analyte for occupation of the antibody s antigen-binding site. The more of the analyte present in the sample, the more of the enzyme is released from inhibition, and the level of enzyme activity can thus be used to determine the quantity of the analyte. 

All passages are carried out at room temperature unless otherwise stated. After slides are dewaxed and brought to water, endogenous peroxidases are suppressed by the incubation of sections with 3 % H2O2 in 10 % methanol in PBS for 7 min. Subsequently, sections are incubated for 20 min in normal serum to block nonspecific antigen-binding sites. 

For indirect immunoassay methods, the antigen (analyte) is bound to support materials and excess binding sites are blocked. Analyte and primary antibody are then added simultaneously, followed by the addition of enzyme-labeled secondary antibody and color reagent. The bound analyte (coating antigen) and free analyte (in... 

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