Cell-ELISA using beta-galactosidase conjugated antibodies

Cell-enzyme-linked immunosorbent assay (cell-ELISA) is a technique for the rapid, convenient, and quantitative detection of molecules expressed on the cell surface. Here we present an evaluation of beta-galactosidase as an an tibody-tag for cell-ELISA. In contrast to substrates for horseradish peroxi dase (HRP) and alkaline phosphatase, murine splenocytes do not hydrolyze th e beta-galactosidase substrate chlorophenolred-beta-D-galactopyranoside (CP RG). beta-Galactosidase-antibody conjugates show much lower background bind ing to murine T cells than conjugates with HRP or alkaline phosphatase. We describe step-by-step procedures for direct and indirect beta-galactosidase based cell-ELISA to quantitate the expression of molecules on the surface of unfixed, live cells. Variations of the basic protocol are suitable for a dherent and non-adherent cells, large scale screening for expression of cel l surface molecules, and the screening of hybridomas for production of anti bodies to cell surface epitopes. Since relatively few beta-galactosidase co njugated antibodies are commercially available, we describe an efficient me thod to couple beta-galactosidase to antibodies using a novel water soluble heterobifunctional crosslinker, sulfosuccinimidyl 4-[N-maleimidomethyl]-cy clohexane-1-carboxylate (sulfo-SMCC). We demonstrate the utility of this me thod by conjugating F(ab')(2) fragments of an anti-B7-2 antibody, and using this conjugate to assay B7-2 on Fc-receptor bearing cells. (C) 2000 Elsevi er Science B.V. All rights reserved.