Characterization of monoclonal antibodies to ovine tumor necrosis factor-α and development of a sensitive immunoassay

Abstract Monoclonal antibodies (mAbs) and a polyclonal rabbit antiserum were raised against recombinant ovine tumor necrosis factor-α (rovTNFα). Ten mAbs specific for rovTNFα were isolated and designated TNF1–10. All mAbs were of the IgG1 isotype and reacted with rovTNFα in Western blot analysis. Eight of the ten mAbs, TNF1, TNF3–7 and TNF9 and 10, completely blocked the activity of rovTNFα and macrophage derived native ovTNFα, as measured by their ability to inhibit TNFα-mediated lysis of WEHI-164 or L929 cells. In addition, TNF3, -7, -9 and -10 blocked the cytolytic activity of recombinant human TNFα (rhuTNFα). However, when tested for the ability to inhibit TNFα 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 rovTNFα. The mAbs were used to develop a sensitive sandwich immunoassay for the detection of ovTNFα. 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 rovTNFα 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 rovTNFα. This immunoassay also detected glycosylated ovTNFα in the supernatant of COS-7 cells which had been transfected with an ovTNFα cDNA.