An Enzyme-Linked Procedure for the Detection and Estimation of Surface Receptors on Cells

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Abstract
An enzyme immunoassay procedure has been developed for the visualization and estimation of lymphocyte surface receptors. β-Galactosidase (β-gal'ase) was covalently linked to sheep anti-rabbit immunoglobulin (SARIg), to ovalbumin (OA), and to adenosine (A). Exposure of rabbit peripheral lymphocytes to SARIg-β-gal'ase and subsequent incubation with the fluorogenic substrate fluorescein-β-digalactopyranoside allowed visualization of B cells by fluorescence microscopy. Treatment with each of the above β-gal'ase conjugates and incubation with another fluorogenic substrate, 4-methyl-β-D-umbelliferylgalactopyranoside, made possible the estimation of the various receptors by spectro fluorimetry. Procedures used for enrichment of T and B cell populations could be monitored. Among the findings was that immunoglobulin-bearing cells (presumably B cells) formed rosettes with sheep erythrocytes. The quantitative procedure was used to study lymphocyte population changes during immunization with an A-O A conjugate. An increase in the binding of A-β-gal'ase, SARIg-β-gal'ase, and OA-β-gal'ase by peripheral lymphocytes occurred repeatedly 6 to 8 days after immunization but lasted only a few days despite a continued high circulating antibody titer. These results are consistent with the possibility that immunization induces migration of lymphocytes into the circulation from bone marrow and/or thymus.

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