Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi, India; Centre for Interdisciplinary research in basic sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, India.
Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi, India.
J Immunol Methods. 2023 Nov;522:113572. doi: 10.1016/j.jim.2023.113572. Epub 2023 Sep 28.
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 ED 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 ED were improved and found to be 0.02 ng/mL, 0.086 × 10 L/mol and 2.95 ng/mL than homologous assay where Sensitivity, affinity and ED were 0.11 ng/mL, 0.02 × 10 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, ED 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 (R = 0.96) by comparing with the commercial kit and validated by measuring levels of 17α- methyltestosterone in rat serum after administering them.
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，在 17α-甲基睾酮-3-羧甲基肟和辣根过氧化物酶标记物之间引入芳族间隔物，开发了用于检测 17α-甲基睾酮的桥异源 ELISA。用于生成抗体的免疫原 17α-甲基睾酮-3-羧甲基肟-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/芳族间隔物对功能参数（即灵敏度、亲和力和 ED）的影响，比较了桥异源测定法与同源测定法。使用 17α-甲基睾酮-3-CMO-BSA 抗血清和 17α-MT-3-CMO-4,4'-二氨基二苯砜-HRP、17α-MT-3-CMO-4,4'-氧化二苯胺-HRP、17α-MT-3-CMO-联苯二胺-HRP、17α-MT-3-CMO-对苯二胺-HRP 和 17α-MT-3-CMO-对苯二磺酰胺-HRP 酶缀合物的五种桥异源测定组合进行了评估。在这五种组合中，17α-MT-3-CMO-BSA 与 17α-MT-3-CMO-联苯二胺-HRP 的组合效果最佳。灵敏度、亲和力和 ED 得到了提高，分别为 0.02ng/mL、0.086×10 L/mol 和 2.95ng/mL，而同源测定法的灵敏度、亲和力和 ED 分别为 0.11ng/mL、0.02×10 L/mol 和 5.78ng/mL。与同源测定法中看到的 8 种类固醇（6-羟基睾酮-43.75%、睾酮-38.3%、达那唑-25.14%、雄烯二酮-19.16%、诺龙-19%、美雄酮-5%、雄烯二酮-3.52%和 17α 二甲睾酮-2%）相比，这种桥异源测定组合的交叉反应性仅见于 59 种结构相关类固醇中的 4 种类固醇（6-羟基睾酮-6%、睾酮-5.14%、达那唑-0.9%和诺龙-0.85%）。因此，这项研究的结果表明，在酶缀合物中引入芳族间隔物（桥）在提高开发测定法的灵敏度、特异性、ED 和亲和力方面起着至关重要的作用。然后，通过与商业试剂盒进行比较，研究了该测定法的回收率（97.4%-108.6%）、精密度（<10%的组内和组间变异系数）和相关性系数（R=0.96）等参数，并通过测量给予大鼠后 17α-甲基睾酮在大鼠血清中的水平对其进行了验证。
