Cellular enzyme-linked immunospecific assay (CELISA). I. A new micromethod that detects antibodies to cell-surface...

A novel, indirect immunoassay capable of detecting human anti-HLA antibody bound to lymphocytes has been developed. This cellular enzyme-linked immunospecific assay (CELISA) utilizes an antiglobulin covalently linked to alkaline phosphatase to quantitate the amount of anti-HLA antibody bound to cell-surface HLA antigens. During the CELISA, V-bottom polyvinyl microplates served as the receptacle in which as little as 5 microliters of sera and as few as 25,000 lymphocytes per well were incubated. We devised a rapid and simple technique to transfer the cells from the original V-bottom plate to a flat-bottom plate before adding the enzyme substrate. Using this strategy, background noise because of the nonspecific adsorption of the different protein immunoreactants to plastic was eliminated. This strategy, the identification of an optimal cell concentration and an optimal conjugate source and dilution, enabled us to detect the anti-HLA activity in sera diluted out 250-fold more than their maximum titer as determined by the microdroplet cytotoxicity test. Since this assay is capable of sensitively and objectively quantitating antibody bound to cell-surface antigens, it may be of value in the areas of transplantation, blood banking, autoimmune disease, tumor immunology, and the study of cell-surface differentiation and viral antigens.