Development and Applications of Novel Ultrasensitive Noncompetitive Immunoassay for Peptides

为提高非竞争酶免疫分析法（杂二位点酶免疫分析法）对多肽的适用性和敏感性进行了尝试。试验了一种去除生物素化后不需要的生物素化物质的新方法。用n -羟基琥珀酰亚胺生物素化后的样品与过量的甘氨酸反应以消除未反应的试剂，随后与2,4 -二硝基苯- β -d -半乳糖苷酶抗肽Fab'偶联物反应。由生物素化肽和偶联物组成的配合物被捕获在涂有亲和纯化的（抗2,4 -二硝基苯基）IgG的聚苯乙烯球上，洗涤后用过量的epsilonn - 2,4 -二硝基苯基赖氨酸洗脱，转移到涂有链霉亲和素的聚苯乙烯球上。经甘氨酸生物素化处理48-72小时后，充分降低β - d -半乳糖苷酶与链霉亲和素包被的聚苯乙烯球的非特异性结合活性2。为了在含有高浓度氨基酸的血浆等样品中测量不含氨基但含有l -组氨酸和l -酪氨酸残基的肽，在存在过量氨基和/或精氨酸的情况下，证实了重氮偶联反应优先与l -组氨酸和l -酪氨酸残基发生，并且证实了生物素不会通过重氮偶联反应失去与链霉亲和素的反应活性。抗肽Fab'-过氧化物酶偶联物与链霉亲和素包被的聚苯乙烯球的非特异性结合在某些情况下很高，限制了灵敏度。在聚苯乙烯球被生物素化的非特异性兔IgG和链亲和素依次包被后，用明胶代替牛血清白蛋白降低了这一比例，就像γ - msh的情况一样。

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.