THE UNLABELED ANTIBODY ENZYME METHOD OF IMMUNOCYTOCHEMISTRY QUANTITATIVE COMPARISON OF SENSITIVITIES WITH AND WITHOUT PEROXIDASE-ANTIPEROXIDASE COMPLEX

JOHN P. PETRALI ¹, DENNIS M. HINTON ¹, GWEN C. MORIARTY ², and LUDWIG A. STERNBERGER ³

¹ Immunology Branch, Edgewood Arsenal, Maryland 21010
² Department of Anatomy, University of Nebraska School of Medicine, Omaha, Nebraska 63105
³ Immunology Branch, Edgewood Arsenal, Maryland 21010, and Department of Microbiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Araldite sections of rat pituitary intermediate lobe were used with anti-^17-39 adrenocorticotropin in the unlabeled antibody enzyme method to compare electron microscopic immunocytochemical staining by peroxidase-antiperoxidase complex (PAP) with antiserum or purified antibody to peroxidase followed by peroxidase (PO). Quantitation of normalized optical densities of secretory granules offered high significance comparison (P < 0.0001) of experimental with control values and of experimental values with each other. Use of purified antibody (prepared by a new density gradient method) yielded four times higher sensitivity than antiserum to PO, while a 6.5-fold increase would have been expected from the degree of contamination of anti-PO in the serum by nonanti-PO immunoglobulin. Use of PAP was four to five times more sensitive than purified anti-PO and 20 times more sensitive than antiserum to PO. The increased sensitivity of PAP is explained by the high over-all binding affinity of PO for anti-PO in the cyclic PAP molecule, thus preventing the losses of PO that occur during washing when anti-PO and PO have been applied in sequence. Identification of the characteristic, cyclic PAP molecules affords confirmation of specific staining at high resolution. In the sequential application of anti-PO and PO, no PAP molecules are formed, thus making distinction of specific from nonspecific deposition of enzyme reaction products ambiguous. With the use of anti-^17-39 ACTH and the intermediate lobe, the unlabeled antibody enzyme method was 16,000-100,000 times more sensitive than radioimmunoassay.

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