A murine monoclonal antibody specific for calf intestinal alkaline phosphatase has been prepared and used in an unlabeled antibody bridge technique for labeling monoclonal antibodies. This procedure-the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) method-gives excellent immunocytochemical labeling of tissue sections and cell smears, comparable in clarity and intensity to that achieved with immunoperoxidase labeling. If the enzyme label is developed with a naphthol salt as a coupling agent and Fast Red or hexazotized new fuchsin as a capture agent, a vivid red reaction product is obtamed which is very easily detected by the human eye. For this reason the APAAP technique was found particularly suitable for labeling cell smears (for both cytoplasmic and surface-membrane antigens) and for detecting low num-APAAP 220 CORDELL ET AL APAAP STAINING OF MONOCLONAL ANTIBODIES

SUMMARY This paper describes the use of a panel of seven monoclonal antibodies (selected so as to include reagents reactive with both epithelial and lymphoid cells) for distinguishing between anaplastic carcinoma and high grade lymphoma. Details are given of the immunohistological reactions of these antibodies against a wide range of both normal and malignant tissues and of a number of practical instances in which use of the antibody panel enabled a diagnosis to be made when routine histological examination had been inconclusive.

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The production of mouse peroxidase:antiperoxidase (PAP) complexes suitable for immunohistological use in conjunction with monoclonal antibodies is described. Three approaches were explored: 1) production of conventional polyclonal PAP complexes; 2) conversion of rabbit PAP to "pseudo-mouse PAP" by incubation with monoclonal mouse anti-rabbit immunoglobulin; 3) formation of PAP complexes from monoclonal mouse antiperoxidase. PAP complexes prepared by the latter technique gave the best PAP LABELING OF MONOCLONAL ANTIBODIES

The increasing number of antigens detectable in human lymphoid tissue (particularly since the advent of monoclonal antibodies) makes it necessary to have techniques available for studying the relative distribution patterns of pairs of antigens in tissue sections. Double immunoenzymatic labelling (using peroxidase and alkaline phosphatase) offers a number of advantages over double immunofluorescence, including the fact that the two antigens can be visualised simultaneously (rather than sequentially) and that the labels are permanent. In studying paraffin-embedded human lymphoid tissue an important application of the double immunoenzymatic technique lies in distinguishing Ig-positive cells containing exogenous Ig (which stain for only a single light chain class). In addition double staining of paraffin sections for IgG and IgM has been used to show that "switch" cells containing both these classes of heavy chain are rare in reactive lymphoid tissue. The potential scope of the double immunoenzymatic technique has been extended by showing that the procedure is applicable to cryostat sections (in which antigenic reactivity is better preserved than in paraffin sections) and by adapting it for use with monoclonal antibodies (by preparing "monoclonal PAP" complexes).

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