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PAP soluble immune complexes are prepared by the method of Sternberger et al. (J Histochem. Cytochem. 1970. 12, 315). Theoretically, they consist predominantly of two anti-HRP antibodies in soluble complex with three molecules of HRP.
PAP soluble complexes are normally used at 25-50 μg/ml. Whole antisera against IgG (H+L) are recommended for bridging PAP to primary antibodies. Antisera against IgG (H+L) or against the F(ab')2 fragments of IgG will also bridge PAP to IgM primary antibodies (such as IgM monoclonal antibodies) by virtue of common light-chain recognition. Normal serum (5% v/v) from the same host species as the bridging antibody is suggested as a blocking solution to minimize non-specific binding. All PAP soluble complexes are freeze-dried at 20 mg/ml.