Highly sensitive enzyme immunoassay system: determination of nervous system-related proteins at the level of single neurons.
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 substrate, 4-methylumbelliferyl-beta-D-galactoside. (2) The nonspecific binding of labeled antibodies to the polystyrene ball solid-phase (blank value of the assay) was low. (3) Antibodies could be coupled selectively with galactosidase, which brings about an increase in the efficiency of the immunoreaction of the labeled antibodies and a decrease in the nonspecific binding of the label on the solid-phase in the assay.(ABSTRACT TRUNCATED AT 250 WORDS)
Kato
K
K
Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Japan.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D003402:Creatine Kinase / Q000032:analysis; D019204:GTP-Binding Proteins / Q000032:analysis; D006801:Humans; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000302:isolation & purification; D007527:Isoenzymes / Q000032:analysis; D008832:Microchemistry; D009414:Nerve Growth Factors / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000032:analysis*; D009474:Neurons / Q000032:analysis*; D010751:Phosphopyruvate Hydratase / Q000032:analysis
D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007527:Isoenzymes; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D003402:Creatine Kinase; D019204:GTP-Binding Proteins; D010751:Phosphopyruvate Hydratase