A sandwich enzyme-linked immunoabsorbent assay for measurement of gonadotropin-releasing hormone-toxin conjugates.
A sandwich enzyme-linked immunoabsorbent assay for measurement of gonadotropin-releasing hormone-toxin conjugates.
复制标题
用于测量促性腺激素释放激素-毒素缀合物的夹心酶联免疫吸附测定。
DOI：
10.1111/j.1600-0897.2005.00350.x
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Nett,TerryM
中科院分区：
文献类型：
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作者：
Yang,Wei-Hsiung;Allen,MattC;Wieczorek,Maciej;MichaelGlode,L;Nett,TerryM
目标细胞毒素的生物学有效性取决于它们的稳定性、循环半衰期、受体结合能力和细胞毒性。本研究的目的是比较用二硫化物连接物制成的促性腺激素释放激素（GnRH）毒素偶联物与使用马来酰亚胺二丁基（mb）连接物制成的偶联物的稳定性。研究方法我们开发了一种夹心酶联免疫吸收法，可以识别GnRH类似物和细胞毒素，以确保所测的偶联物是完整的。使用Anti - D - Leu6 - GnRH捕获抗美洲商陆抗病毒蛋白（Anti - PAP）或抗RNase进行定量。特异性通过与羊FSH和LH、PAP、RNase和D‐Lys6‐GnRH缺乏反应性来验证。结果二硫键制备的偶联物在体外血清中不稳定（半衰期<10 min），而mb偶联物的半衰期为10 ~ 2 hr。绵羊循环中mbGnRH - PAP的清除速度很快（t1/2<20 min）。结论该方法具有特异性、敏感性和准确性，可用于体外和体内GnRH -毒素结合物的测定。
ProblemBiological effectiveness of targeted cytotoxins is dependent on their stability, circulating half‐life, receptor binding ability, and cytotoxicity. The objective of this study was to compare stability of gonadotropin‐releasing hormone (GnRH)‐toxin conjugates made with disulfide linkers to those using a maleimidodibutyryl (mb) linkage.Method of studyWe developed a sandwich enzyme‐linked immunoabsorbent assay recognizing both GnRH analog and cytotoxin to ensure the conjugate measured was intact. Anti‐D‐Leu6‐GnRH was used for capture and anti‐pokeweed antiviral protein (anti‐PAP) or anti‐RNase for quantification. Specificity was verified by lack of reactivity with ovine FSH and LH, PAP, RNase, and D‐Lys6‐GnRH.ResultsConjugates prepared using disulfide linkages were not stable in serumin vitro(half‐lives <10 min), whereas mb conjugates had half‐lives >2 hr. Clearance of mbGnRH‐PAP from the circulation of sheep was rapid (t1/2<20 min).ConclusionThe assays were found to be specific, sensitive and accurate for measurement of GnRH‐toxin conjugatesin vitroandin vivo.