Analysis of Factors Influencing Sensitivity in Enzyme Immunoassay

酶联免疫分析灵敏度影响因素分析

• 批准号: 62570960
• 负责人: HOSODA Hiroshi
• 金额: $ 1.28万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1988
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62570960/
• 关键词: Enzyme Immunoassay; Label Enzyme; Enzyme Labeling Method; Assay Sensitivity; Steroid Hormone; Monoclonal Antibody; Enzyme Activity Measurement Method; ブリッジ現象; エンザイムノアッセイ; 酵素活性測定法; グルコースオキシダーゼ; β-ガラクトシダーゼ; モノクローン抗体

Numerous enzyme immunoassays (EIAs) have been developed using various enzymes as labels. In general, however, development of a specific and sensitive EIA for haptens, such as steroids and drugs, is not always easy owing to various factors. In order to obtain a practical basis for selecting the enzyme, enzyme labeling method, steroid/enzyme molar ratio in the label, method of determining enzyme activity, and bridging phenomenon were investigated in a testosterone or 11-deoxycortisol EIA system; alkaline phosphatase (AP), -galactosidase ( -GAL), horseradish peroxidase (HRP), glucose oxidase (GOD) were compared with regard to the sensitivity. There is the possibility that the heterogeneity of polyclonal antibodies influencing the results on assay sensitivity. The use of monoclonal antibody eliminates this problem, simplifying the analysis of immune reactions. Thus, the production of monoclonal antibodies to steroid hormones was also carried out.The following results were obtained: 1) The … More N-succinimidyl ester method is useful for these four enzymes. The labeled antigens prepared at molar ratios of 4-8, 20-40, 10-60, and 10-20 in the GOD, AP, HRP, and -GAL labelings gave high sensitivities, respectively.2) The use of 3,3',5,5'-tetramethylbenzidine, a safe substrate for HRP, as a chromogen in an EIA system with a GOD label gave satisfactory results.3) In the testosterone EIA, the highest sensitivity was obtained by the use of HRP, with GOD next, AP third and -gal fourth. The amount of testosterone needed to displace 50% of the bound label in the colorimetric EIAs ranged from 9 to 90 pg.4) Previously, using -GAL as a label in bridge heterologous assay systems for cortisol, we showed that the bridge length is an important factor influencing the sensitivity, and the use of a shorter bridge for enzyme labeling results in an increase in sensitivity. The present findings suggest that the bridge length effect may depend on the label enzyme.5) EIA systems using a monoclonal antibody were also developed for the determination of 11-deoxycortisol or 17 -hydroxyprogesterone. Less

许多酶免疫分析（EIA）已开发使用各种酶作为标记。然而，总的来说，由于各种因素，针对半抗原（例如类固醇和药物）开发特异性和敏感的EIA并不总是那么容易。为了获得一个实际的基础上选择酶，酶标记方法，类固醇/酶的摩尔比的标签，测定酶活性的方法，和桥接现象进行了研究，在睾酮或11-脱氧皮质醇EIA系统;碱性磷酸酶（AP），-半乳糖苷酶（GAL），辣根过氧化物酶（HRP），葡萄糖氧化酶（GOD）进行了比较的灵敏度。多克隆抗体的异质性可能会影响检测灵敏度的结果。单克隆抗体的使用消除了这个问题，简化了免疫反应的分析。因此，我们也进行了抗甾体激素单克隆抗体的制备，获得了以下结果：1） ...更多信息 N-琥珀酰亚胺酯法对这四种酶都是有效的。在GOD、AP、HRP和-GAL标记中，以4-8、20-40、10-60和10-20的摩尔比制备的标记抗原分别具有高的灵敏度。2）在具有GOD标记的EIA系统中，使用HRP的安全底物3，3 '，5，5'-四甲基联苯胺作为色原获得了令人满意的结果。HRP法的敏感性最高，GOD法次之，AP法次之，-gal法最低。在比色EIA中取代50%结合标记所需的睾酮量范围为9至90 pg。4）以前，在用于皮质醇的桥异源测定系统中使用-GAL作为标记，我们表明桥长度是影响灵敏度的重要因素，并且使用较短的桥用于酶标记导致灵敏度增加。目前的研究结果表明，桥长效应可能取决于标记酶。5）EIA系统使用的单克隆抗体也被开发用于测定11-脱氧皮质醇或17 -羟孕酮。少

项目成果

HOSODA,Hiroshi: "Sensitivity of steroid enzyme immunoassays. Comparison of alkaline phosphatase, -galactosidase and horseradish peroxidase as labels in a colorimetric assay system" Chem. Pharm. Bull.35. 3336-3342 (1987)

HOSODA，Hiroshi：“类固醇酶免疫测定的灵敏度。比色测定系统中碱性磷酸酶、β-半乳糖苷酶和辣根过氧化物酶作为标记的比较”Chem。

HOSODA,Hiroshi: "Enzyme labeling of steroids by the N-succinimidyl ester method. Preparation of glucose oxidase-labeled antigen for use in enzyme immunoassay" Chem. Pharm. Bull.35. 4856-4861 (1987)

HOSODA，Hiroshi：“通过 N-琥珀酰亚胺酯法对类固醇进行酶标记。用于酶免疫测定的葡萄糖氧化酶标记抗原的制备” Chem。

HOSODA,Hiroshi: Chem.Pharm.Bull.35. 3336-3342 (1987)

细田浩：Chem.Pharm.Bull.35。

TSUJI,Akio: "Eluorescence and chemiluminescence enzyme immunoassays of 17 -hydroxy-progesterone in dried blood spotted on filter paper" J. Steroid Biochem.27. 33-40 (1987)

TSUJI，Akio：“滤纸上干血中 17-羟基孕酮的荧光和化学发光酶免疫分析”J. Steroid Biochem.27。

TSUJI,Akio: J.Steroid Biochem.27. 33-40 (1987)

TSUJI，Akio：J.Steroid Biochem.27。