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Journal of Histochemistry and Cytochemistry, Vol. 45, 1629-1642, Copyright © 1997 by The Histochemical Society, Inc.

ARTICLE

Enzyme-based Antigen Localization and Quantitation in Cell and Tissue Samples (Midwestern Assay)

Kevin A. Rotha,c, Jennifer W. Brennera, Lee A. Selznicka, Murat Gokdena, and Robin G. Lorenza,b
a Department of Pathology, Washington University School of Medicine, St. Louis, Missouri
b Department of Medicine, Washington University School of Medicine, St. Louis, Missouri
c Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri

Correspondence to: Kevin A. Roth, Dept. of Pathology (Box 8118), Washington Univ. School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110.

Quantitation of antigen concentration in cell and tissue samples typically requires antigen extraction, which precludes antigen localization in the same sample. Similarly, antigen immunolocalization in fixed cells or tissue sections provides limited information about antigen concentration. We have developed a rapid and sensitive assay for simultaneous antigen localization and quantitation in cell and tissue samples that does not involve antigen extraction, radioactive materials, or image analysis. Fixed cells and/or tissue sections are used with antigen-specific enzyme-linked probes to generate soluble reaction products that are spectrophotometrically quantifiable and deposited reaction products that are microscopically localizable. The amount of soluble reaction product is dependent on several variables, including antigen concentration, probe specificity and sensitivity, sample size, and enzyme reaction time. These variables can be experimentally controlled so that soluble reaction product is proportional to antigen concentration in the sample. This assay was used in multiple applications including detection of Ki-67 nuclear antigen immunoreactivity in human brain tumors, in which it showed a clear relationship with visually determined Ki-67 cell labeling indexes. This assay, termed the Midwestern assay, should be applicable to a wide variety of antigens in both clinical and research samples. (J Histochem Cytochem 45:1629-1641, 1997)

Key Words: enzyme-linked, immunosorbent assay (ELISA), chromogens, tyramide signal amplification, (TSA), anti-nuclear antibodies (ANA), progesterone receptors, Ki-67 nuclear antigen


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