Development and Applications of Novel Ultrasensitive Noncompetitive Immunoassay for Peptides

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Attempts were made to improve applicability and sensitivity of noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for peptides.1.A new method was tested to remove unwanted biotinylated substances after biotinylation. Samples after biotinylation with N-hydroxysuccinimidobiotin were reacted with excess of glycylglycine to eliminate unreacted reagent and subsequently with 2, 4-dinitrophenyl-beta-D-galactosidase-anti-peptide Fab' conjugate. The complex comprising biotinylated peptides and the conjugate was trapped onto polystirene balls coated with affinity-purified (anti-2, 4-dinitrophenyl group) IgG, eluted with excess of epsilonN-2, 4-dinitrophenyl-L-lysine after washing and transferred to polystirene balls coated with streptavidin. The nonspecific binding of beta-D-galactosidase activity to streptavidin - coated polystirene balls was sufficiently lowered by incubating samples after biotinylation with glycylglycine for 48-72 h.2.In order to measure peptides containing no amino groups but L-histidine and L-tyrosine residues in samples such as plasma containing amino acids at high concentrations, diazo coupling was confirmed to take place preferentially with L-histidine and L-tyrosine residues in the presence of excess of amino groups and/or arginine, and biotin was confirmed not to lose its reactivity with streptavidin by diazo coupling reaction.3.The nonspecific binding of anti-peptide Fab'-peroxidase conjugate to streptavidin-coated polystirene balls was high in some cases, limiting the sensivity. This was lowered by substitution of gelatin for bovine serum albumin added after polystirene balls were coated successively with biotinylated nonspecific rabbit IgG and streptavidin, as was the case for gamma-MSH.