Abstract：
In this study, we have developed bridge heterologous ELISA for the detection of 17α- Methyltestosterone by incorporating aromatic spacers between 17α-Methyltestosterone-3-Carboxymethyloxime and Horseradish peroxidase label through N-hydroxysuccinimide mediated carbodiimide reaction method. The immunogen 17α-Methyltestosterone-3-Carboxymethyloxime-Bovine serum albumin used to generate the antibody was also prepared by the N-hydroxysuccinimide mediated carbodiimide reaction without using any spacer. We have studied the impact of bridge/aromatic spacers on functional parameters i.e. sensitivity, affinity and ED50 of the bridge heterologous assay and compared it with homologous assay. The five combinations of bridge heterologous assay using 17α-Methyl testosterone-3-CMO-BSA antiserum and 17α-MT-3-CMO-4,4\'-Diaminodiphenyl sulphide-HRP, 17α MT-3-CMO-4,4\'-Oxydianiline-HRP, 17α-MT-3-CMO-Benzidine-HRP, 17α- MT-3-CMO-p-Phenylenediamine-HRP and 17α-MT-3-CMO-Dapson-HRP enzyme conjugates were evaluated. Out of these five combinations, the combination 17α-MT-3-CMO-BSA with 17α-MT-3-CMO-Benzidine-HRP showed the best results. Sensitivity, affinity and ED50 were improved and found to be 0.02 ng/mL, 0.086 × 10-8 L/mol and 2.95 ng/mL than homologous assay where Sensitivity, affinity and ED50 were 0.11 ng/mL, 0.02 × 10-8 L/mol and 5.78 ng/mL respectively. The cross-reactivity for this bridge heterologous assay combination was seen with only 4 steroids (6-hydrotestosterone- 6%, Testosterone-5.14%, Danazol-0.9% and Nandrolone-0.85%) instead of eight steroids (6-hydrotestosterone-43.75%, Testosterone-38.3%, Danazol-25.14%, Androstenediol-19.16%, Nandrolone-19%, Metandienone-5%, Androstenedione-3.52%, and 17α dimethyltestosterone-2%) as in homologous assay out of 59 structurally related steroids. Thus, the results of this study conclude that the incorporation of aromatic spacer (bridge) in enzyme conjugate has a crucial role in improving sensitivity, specificity, ED50 and affinity of the developed assay. The assay was then studied for parameters such as recovery (97.4%-108.6%), precision (Inter and Intra-assay coefficient of variation <10%), correlation coefficient (R2 = 0.96) by comparing with the commercial kit and validated by measuring levels of 17α- methyltestosterone in rat serum after administering them.
摘要：
在这项研究中,我们通过N-羟基琥珀酰亚胺介导的碳二亚胺反应方法，通过在17α甲基睾酮-3-羧基甲基肟和辣根过氧化物酶标记之间掺入芳族间隔子，开发了桥式异源ELISA检测17α甲基睾酮。用于产生抗体的免疫原17α甲基睾酮-3-羧基甲基肟-牛血清白蛋白也通过N-羟基琥珀酰亚胺介导的碳二亚胺反应制备,而不使用任何间隔物。我们研究了桥/芳族间隔基对功能参数的影响，即灵敏度，桥异源测定的亲和力和ED50，并将其与同源测定进行比较。使用17α甲基睾酮-3-CMO-BSA抗血清和17αMT-3-CMO-4,4'-二氨基二苯基硫化物-HRP的桥异源测定的五种组合，17αMT-3-CMO-4,4'-氧二苯胺-HRP，17αMT-3-CMO-联苯胺-HRP，评估了17αMT-3-CMO-对-苯二胺-HRP和17αMT-3-CMO-Dapson-HRP酶缀合物。在这五个组合中，17αMT-3-CMO-BSA与17αMT-3-CMO-联苯胺-HRP组合显示最佳结果。灵敏度,亲和力和ED50得到改善，发现为0.02ng/mL，0.086×10-8L/mol和2.95ng/mL比同源测定灵敏度，亲和力和ED50为0.11ng/mL，0.02×10-8L/mol和5.78ng/mL。该桥接异源测定组合的交叉反应性仅见于4种类固醇(6-氢睾酮-6%,睾酮-5.14%,Danazol-0.9％和Nandrolone-0.85％）而不是八种类固醇（6-氢睾酮-43.75％，睾酮-38.3%,达那唑-25.14%,雄烯二醇-19.16%，Nandrolone-19%,美坦酮-5%,雄烯二酮-3.52%,和17α二甲基睾丸激素-2％），如在59个结构相关的类固醇中的同源测定。因此，这项研究的结果得出结论，在酶结合物中掺入芳香间隔（桥）对提高灵敏度具有关键作用，特异性，ED50和开发的测定的亲和力。然后研究分析的参数，如回收率（97.4%-108.6%），精密度(测定间和测定内变异系数<10%)，相关系数（R2=0.96），通过与市售试剂盒进行比较，并通过在给药后测量大鼠血清中17α-甲基睾酮的水平来验证。
