PMID: 2499320Jan 1, 1989Paper

Highly sensitive enzyme immunoassay system: determination of nervous system-related proteins at the level of single neurons

Biomedica Biochimica Acta
K Kato

Abstract

We established a highly sensitive sandwich-type enzyme immunoassay system which was applicable to the measurement of macromolecular antigens at the level of single cells. The assay system was composed of a solid-phase (polystyrene ball, 3.2 mm in diameter) with immobilized monospecific antibodies and the same antibodies labeled with beta-D-galactosidase from Escherichia coli. The antibody IgG was purified from antisera by means of immunoaffinity chromatography. It was then digested with pepsin to obtain the F(ab')2 fragments of antibodies. The antibody F(ab')2 fragments were immobilized noncovalently on polystyrene balls. Other aliquots of antibody F(ab')2 fragments were reduced with 2-mercaptoethylamine, and the resulting Fab' fragments (which contained one thiol group in each molecule) were coupled with beta-D-galactosidase in a two-step procedure by use of N,N'-o-phenylenedimaleimide. The assay system was highly sensitive, and amol (1 x 10(-18) mol) levels of antigens were detected. The high sensitivity of the present assay was the result of the following factors. (1) A very small amount (1 amol) of the galactosidase was determined using conventional laboratory instruments by measuring its activity with a fluorogenic substra...Continue Reading

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