Department of Biochemistry, Showa University School of Medicine, Hatanodai 8-5-1, Shinagawa-ku, Tokyo 142-8555, Japan.
Queen Mary University of London, School of Biological & Chemical Sciences, Mile End Road, London E1 4NS, UK.
Gen Comp Endocrinol. 2021 Sep 1;310:113831. doi: 10.1016/j.ygcen.2021.113831. Epub 2021 Jun 4.
A relaxin-like gonad-stimulating peptide (RGP) acts as a gonadotropic hormone in starfish. In this study, antibodies to Asterias rubens RGP (AruRGP) were used for the development of a specific and sensitive enzyme-linked immunosorbent assay (ELISA) to measure AruRGP. Biotin-conjugated RGP (biotin-AruRGP) that binds to peroxidase-conjugated streptavidin was synthesized chemically so that it could be specifically detected using 3, 3', 5, 5'-tetramethylbenzidine (TMB)/hydrogen peroxide as a substrate. Similar to AruRGP, biotin-AruRGP bound to AruRGP antibodies. In binding experiments with biotin-AruRGP using wells coated with AruRGP antibodies, a displacement curve was obtained using serial dilutions of AruRGP. Using this ELISA system, AruRGP could be measured in the range 0.01-5.0 pmol per 50 µl test solution. Furthermore, 0.22 ± 0.03 and 0.20 ± 0.04 pmol AruRGP/mg wet weight tissue were detected in the radial nerve cords and circumoral nerve-rings of A. rubens, respectively. Smaller amounts of AruRGP were detected in tube feet, pyloric stomach and cardiac stomach but AruRGP was not detected in pyloric caeca, ovaries and testes. Analysis of the specificity of the AruRGP antibodies revealed that the A- and B-chains of AruRGP, Patiria pectinifera RGP, Aphelasterias japonica RGP, and human relaxin exhibit little or no cross-reactivity in the ELISA. We conclude, therefore, that we have successfully generated an ELISA system that is highly sensitive and specific for detection of AruRGP.
一种类松弛素性腺刺激肽（RGP）在海星中充当性腺激素。在这项研究中，使用针对 Asterias rubens RGP（AruRGP）的抗体开发了一种特异性和灵敏的酶联免疫吸附测定（ELISA）来测量 AruRGP。化学合成了与过氧化物酶缀合的链霉亲和素结合的生物素化 RGP（biotin-AruRGP），使其可以使用 3,3',5,5'-四甲基联苯胺（TMB）/过氧化氢作为底物特异性检测。类似于 AruRGP，biotin-AruRGP 与 AruRGP 抗体结合。在使用 AruRGP 抗体包被的孔进行的与 biotin-AruRGP 的结合实验中，使用 AruRGP 的系列稀释液获得了一个置换曲线。使用这种 ELISA 系统，可以在 50μl 测试溶液中 0.01-5.0 pmol 的范围内测量 AruRGP。此外，在 Asterias rubens 的桡神经索和围口神经环中分别检测到 0.22±0.03 和 0.20±0.04 pmol AruRGP/mg 湿重组织。在管足、幽门胃和心脏胃中检测到较少量的 AruRGP，但在幽门盲囊、卵巢和睾丸中未检测到 AruRGP。对 AruRGP 抗体特异性的分析表明，AruRGP、Patiria pectinifera RGP、Aphelasterias japonica RGP 和人松弛素的 A 和 B 链在 ELISA 中几乎没有或没有交叉反应。因此，我们得出结论，我们已经成功地生成了一种高度敏感和特异性的 ELISA 系统，用于检测 AruRGP。
