Stepwise amplified immunoperoxidase (PAP) staining. I. Cellular morphology in relation to membrane markers

A novel procedure for the assay of monoclonal antibodies is described. The technique is based on a combination of three principles. Unlabeled (sheep) antiserum to mouse immunoglobulin (Ig) and complexes of peroxidase with mouse monoclonal antiperoxidase (monoclonal PAP complexes) are used as reagents in a variant of the unlabeled antibody enzyme (PAP) method, described by Sternberger. The amount of peroxidase eventually bound to a monoclonal antibody can be varied over a wide range by repetition of incubation cycles with anti-mouse Ig and monoclonal PAP complexes. During the assay, incubations and wash steps are performed by immersion of whole slides. The influence of repetitive incubation cycles with anti-mouse Ig and monoclonal PAP complexes on background staining and detection of monoclonal antibodies at low concentrations was quantitated in a model system. At a given primary antibody concentration, a linear relationship was found between peroxidase activity and the number of incubation cycles. Application of the technique to the detection of monoclonal antibodies bound to cell-surface antigens is described. Peripheral blood cells were labeled in suspension with monoclonal antibodies. Cytocentrifuge preparations of labeled cells were prepared, and such preparations were fixed before stepwise-amplified PAP staining. Cells showed intense specific staining. Morphological detail of stained and unstained cells is preserved, allowing morphological analysis of labeled cells and rapid analysis of monoclonal antibody specificity. Because the reagents used in the assay can be produced in large quantities with uniform quality, the technique can be readily automated. This, together with the possibility to increase the sensitivity of antibody detection in a controlled, stepwise fashion to levels that cannot be reached with "single-step" techniques, may further expand the applications of monoclonal antibodies.