Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes).

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Abstract
A murine monoclonal antibody specific for calf intestinal alkaline phosphatase was prepared and used in an unlabeled antibody bridge technique for labeling monoclonal antibodies. This procedure.sbd.the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) method.sbd.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 obtained which is very easily detected by the human eye. For this reason, the APAAP technique was particularly suitable for labeling cell smears (for both cytoplasmic and surface-membrane antigens) and for detecting low numbers of antigen-bearing cells in a specimen (e.g., carcinoma cells in a malignant effusion). It was possible to enhance the intensity of the APAAP labeling reaction substantially by repeating the 2nd and 3rd incubation steps (i.e., the unlabeled bridge antibody and APAAP complexes). The APAAP technique was superior to immunoperoxidase labeling for staining tissues rich in endogenous peroxidase, and could be used in conjunction with immunoperoxidase methods for double immunoenzymatic staining. The method was also applicable to the detection of antigenic molecules following their electrophoretic transfer from SDS[sodium dodecyl sulfate]-polyacrylamide gels to nitrocellulose sheets (immunoblotting).

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