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 antibody-tag for cell-ELISA. In contrast to substrates for horseradish peroxidase (HRP) and alkaline phosphatase, murine splenocytes do not hydrolyze the beta-galactosidase substrate chlorophenolred-beta-D-galactopyranoside (CPRG). beta-Galactosidase-antibody conjugates show much lower background binding 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 adherent and non-adherent cells, large scale screening for expression of cell surface molecules, and the screening of hybridomas for production of antibodies to cell surface epitopes. Since relatively few beta-galactosidase conjugated antibodies are commercially available, we describe an efficient method to couple beta-galactosidase to antibodies using a novel water soluble heterobifunctional crosslinker, sulfosuccinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (sulfo-SMCC). We demonstrate the utility of this method 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.
Liu
Z
Z
School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA.
Gurlo
T
T
von Grafenstein
H
H
D000818:Animals; D015703:Antigens, CD / Q000276:immunology; D051940:B7-2 Antigen; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D008562:Membrane Glycoproteins / Q000276:immunology; D051379:Mice; D001616:beta-Galactosidase / Q000235:genetics / Q000276:immunology*
D015703:Antigens, CD; D051940:B7-2 Antigen; C497965:Cd86 protein, mouse; D007140:Immunoglobulin Fab Fragments; D008562:Membrane Glycoproteins; D001616:beta-Galactosidase