Egan P J, Rothel J S, Andrews A E, Seow H F, Wood P R, Nash A D
Department of Veterinary Science, University of Melbourne, Parkville, Vic., Australia.
Vet Immunol Immunopathol. 1994 Jun;41(3-4):259-74. doi: 10.1016/0165-2427(94)90101-5.
Monoclonal antibodies (mAbs) and a polyclonal rabbit antiserum were raised against recombinant ovine tumor necrosis factor-alpha (rovTNF alpha). Ten mAbs specific for rovTNF alpha were isolated and designated TNF1-10. All mAbs were of the IgG1 isotype and reacted with rovTNF alpha in Western blot analysis. Eight of the ten mAbs, TNF1, TNF3-7 and TNF9 and 10, completely blocked the activity of rovTNF alpha and macrophage derived native ovTNF alpha, as measured by their ability to inhibit TNF alpha-mediated lysis of WEHI-164 or L929 cells. In addition, TNF3, -7, -9 and -10 blocked the cytolytic activity of recombinant human TNF alpha (rhuTNF alpha). However, when tested for the ability to inhibit TNF alpha induced thymocyte proliferation, only mAbs TNF1, -3, -5, -7, -9 and -10 could completely block activity. Competitive binding analysis using unlabelled and horseradish peroxidase (HRPO) labelled mAbs indicated that the mAbs could be divided into five groups based on their reactivity with rovTNF alpha. The mAbs were used to develop a sensitive sandwich immunoassay for the detection of ovTNF alpha. All combinations of mAbs and the polyclonal antiserum were tested to determine which pair of antibodies gave the most sensitive assay. The combination of TNF5 as the capture antibody and the polyclonal antiserum gave the most sensitive result, detecting less than 0.24 ng rovTNF alpha ml-1. A similar sensitivity was obtained when TNF4 was used as the capture antibody and TNF10 HRPO labelled mAb as the second antibody. The immunoassay was more sensitive than the WEHI-164 bioassay which had a detection limit of 1 ng ml-1 for rovTNF alpha. This immunoassay also detected glycosylated ovTNF alpha in the supernatant of COS-7 cells which had been transfected with an ovTNF alpha cDNA.
制备了针对重组羊肿瘤坏死因子-α（rovTNFα）的单克隆抗体（mAb）和兔多克隆抗血清。分离出10种对rovTNFα特异的mAb，并命名为TNF1 - 10。所有mAb均为IgG1同种型，且在蛋白质印迹分析中与rovTNFα发生反应。10种mAb中的8种，即TNF1、TNF3 - 7以及TNF9和10，通过其抑制TNFα介导的WEHI - 164或L929细胞裂解的能力来衡量，可完全阻断rovTNFα和巨噬细胞来源的天然ovTNFα的活性。此外，TNF3、 - 7、 - 9和 - 10可阻断重组人TNFα（rhuTNFα）的细胞溶解活性。然而，在测试抑制TNFα诱导的胸腺细胞增殖的能力时，只有mAb TNF1、 - 3、 - 5、 - 7、 - 9和 - 10能够完全阻断活性。使用未标记和辣根过氧化物酶（HRPO）标记的mAb进行的竞争性结合分析表明，根据mAb与rovTNFα的反应性，可将其分为五组。这些mAb被用于开发一种检测ovTNFα的灵敏夹心免疫测定法。测试了mAb和多克隆抗血清的所有组合，以确定哪一对抗体给出的测定最为灵敏。以TNF5作为捕获抗体和多克隆抗血清的组合给出了最灵敏的结果，可检测到低于0.24 ng rovTNFα ml⁻¹。当使用TNF4作为捕获抗体且TNF10 HRPO标记的mAb作为第二抗体时，获得了类似的灵敏度。该免疫测定法比WEHI - 164生物测定法更灵敏，后者对rovTNFα的检测限为1 ng ml⁻¹。此免疫测定法还检测到了用ovTNFα cDNA转染的COS - 7细胞上清液中的糖基化ovTNFα。
