Development and Applications of Novel Ultrasensitive Noncompetitive Immunoassay for Peptides

新型超灵敏多肽非竞争性免疫分析方法的开发及应用

• 批准号: 04808024
• 负责人: ISHIKAWA Eiji
• 金额: $ 1.15万
• 依托单位: Miyazaki Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04808024/
• 关键词: Peptide; Hapten; Noncompetitive essay; Enzyme immunoassay; Biotin; Biotinylation; Streptavidin; Peroxidase; アビジン

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.

尝试提高肽非竞争性酶联免疫分析(异位点酶联免疫分析)的适用性和灵敏度。1.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生物素化后的样品与过量的甘氨酰甘氨酸反应以消除未反应的试剂，随后与 2, 4-二硝基苯基-β-D-半乳糖苷酶-抗肽 Fab' 缀合物反应。将包含生物素化肽和缀合物的复合物捕获到涂有亲和纯化的（抗2，4-二硝基苯基）IgG的聚苯乙烯球上，洗涤后用过量的εN-2，4-二硝基苯基-L-赖氨酸洗脱，并转移到涂有链霉亲和素的聚苯乙烯球上。通过用甘氨酰甘氨酸进行生物素化后的样品孵育48-72 h，可充分降低β-D-半乳糖苷酶活性与链霉亲和素包被的聚苯乙烯球的非特异性结合。2。为了测量含有高浓度氨基酸的血浆等样品中不含有氨基但含有L-组氨酸和L-酪氨酸残基的肽， 证实在过量氨基和/或精氨酸存在下，优先与L-组氨酸和L-酪氨酸残基发生重氮偶联，并且通过重氮偶联反应证实生物素不会失去与链霉亲和素的反应性。 3.抗肽Fab'-过氧化物酶缀合物与链霉亲和素包被的聚苯乙烯球的非特异性结合 在某些情况下较高，限制了灵敏度。与γ-MSH 的情况一样，在聚苯乙烯球连续包被生物素化的非特异性兔IgG 和链霉亲和素后添加的牛血清白蛋白被明胶替代，从而降低了这一点。