Analysis of Monoclonal Antibody Binding Using Stepwise-Amplified Immunoperoxidase Staining

Abstract

Immunofluorescence techniques in combination with flow cytofluorometry are established tools for the analysis of monoclonal antibody binding. Drawbacks of the immunofluorescence techniques are that the cellular preparations are not permanent and are lacking in morphological detail. This is a particular problem when the cells under study have a low frequency and/or when morphological information is important (e. g., recognition of atypical cells) Immunoperoxidase techniques have been proposed as an alternative to immunofluorescence for these reasons [8, 9]. We have described immunoperoxidase procedures to detect monoclonal antibodies against cell-surface antigens [4], using a variant of the unlabeled antibody-enzyme method [12]. In this technique, binding of monoclonal mouse antibody to cells is detected using unlabeled antiserum to mouse immunoglobulin and peroxidase-antiperoxidase (PAP) complexes prepared with a monoclonal mouse antiperoxidase antibody. Adaptation of this technique for high-power microscopy of cytocentrifuged cells has now been introduced. Cells are labeled with monoclonal antibodies in suspension prior to cytocentrifugation and fixation. Sensitivity of immunoperoxidase staining is increased by repeating incubations with anti-mouse Ig and monoclonal PAP. Results obtained using such stepwise amplified immunoperoxidase staining with monoclonal antibodies which have not been previously described but were included in the workshop are given here.

The authors thank M.de Boer, M.Bos, and C.J. Joustra-Maas for technical assistance