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.

松弛素样性腺刺激肽 (RGP) 在海星中充当促性腺激素。在这项研究中， Asterias rubens的抗体RGP (AruRGP) 用于开发一种特异性和灵敏的酶联免疫吸附试验 (ELISA) 来测量 AruRGP。化学合成了与过氧化物酶缀合的链霉抗生物素结合的生物素缀合的 RGP（生物素-AruRGP），以便可以使用 3, 3', 5, 5'-四甲基联苯胺 (TMB)/过氧化氢作为底物对其进行特异性检测。与 AruRGP 类似，生物素-AruRGP 与 AruRGP 抗体结合。在使用涂有 AruRGP 抗体的孔进行的生物素-AruRGP 结合实验中，使用 AruRGP 的连续稀释获得了置换曲线。使用此 ELISA 系统，AruRGP 可以在每 50 µl 测试溶液 0.01–5.0 pmol 的范围内进行测量。此外，在桡神经索和口周神经环中检测到 0.22 ± 0.03 和 0.20 ± 0.04 pmol AruRGP/mg 湿重组织。A.鲁本斯，分别。在管足、幽门胃和贲门胃中检测到少量的 AruRGP，但在幽门盲肠、卵巢和睾丸中未检测到 AruRGP。AruRGP 抗体的特异性分析表明，AruRGP、Patiria pectinifera RGP、Aphelasterias japonica RGP 和人松弛素的 A 链和 B 链在 ELISA 中表现出很少或没有交叉反应性。因此，我们得出结论，我们已经成功地生成了一个对检测 AruRGP 具有高度敏感性和特异性的 ELISA 系统。