ENZYME-BASED ANTIGEN LOCALIZATION AND QUANTITATION IN CELL AND TISSUESAMPLES (MIDWESTERN ASSAY)

Quantitation of antigen concentration in cell and tissue samples typic ally requires antigen extraction, which precludes antigen localization in the same sample. Similarly, antigen immunolocalization in fixed ce lls or tissue sections provides limited information about antigen conc entration. We have developed a rapid and sensitive assay for simultane ous antigen localization and quantitation in cell and tissue samples t hat does not involve antigen extraction, radioactive materials, or ima ge analysis. Fixed cells and/or tissue sections are used with antigen- specific enzyme-linked probes to generate soluble reaction products th at are spectrophotometrically quantifiable and deposited reaction prod ucts that are microscopically localizable. The amount of soluble react ion product is dependent on several variables, including antigen conce ntration, probe specificity and sensitivity, sample size, and enzyme r eaction 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 det ection 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, sho uld be applicable to a wide variety of antigens in both clinical and r esearch samples.