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A new amplification technique is described for the detection of small amounts of antigen in ethanol-fixed cytologic specimens and formalin-fixed tissues. By adding a third antibody layer to the already sensitive avidin-biotin immunoperoxidase system, we have demonstrated a considerable increase in both the quantitative and qualitative aspects of this immunostaining method. In evaluating this technique, we employed a previously defined small cell lung carcinoma cell surface antigen system and monoclonal antibodies.
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