Efficient use of monoclonal antibodies for immunofluorescence

Abstract

We describe here a simple and rapid small volume microplate-based immunofluorescence staining method in which fluorochrome-conjugated monoclonal antibodies (MAb) from three different manufacturers, used at a single standardized quantity (50 ng per test), resulted in optimal staining of human lymphocyte subsets. Staining reactions were robust, in that the number of lymphocytes used could be varied over a wide range (3 × 10⁴-1 × l0⁶ cells per microplate well) without significant effects on the fluorescence intensity of staining or nonspecific binding by MAb. A measure of the efficiency of MAb use was the number of tests theoretically possible to perform with nominal 100 test kits; this figure ranged from 400 to 20,000 tests, depending on the MAb in question. This method was readily adaptable to both single-and two-color immunofluorescence analysis.