Radioimmunoassay for Bovine Tumor Necrosis Factor: Concentrations and Circulating Molecular Forms in Bovine Plasma

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Abstract
Antisera against recombinant bovine tumor necrosis factor (rbTNF) were produced in rabbits immunized with rbTNF in Freund's complete adjuvant (F314) and used in a double antibody radioimmunoassay to measure plasma TNF. Assay standards were rbTNF. Iodination of rbTNF and chromatography on G-50 Sephadex with 50 mM EDTA, 0.1 % BSA, 0.05 M phosphate buffer, pH 7.5 resulted in labelled rbTNF which was >97% TCA precipitable (specific activity of 37.5 μCi/μg). F314 (1: 80,000 dilution) bound 21% of 125I- rbTNF in a non-equilibrium assay at 4 C. Separation of bound and free 125I-rbTNF was accomplished by precipitation with goat anti-rabbit IgG prepared with 6% polyethyleneglycol (mw = 8000). Minimum detectable TNF was 4 pg/assay tube. Matrix effects of plasma were minimal. Recovery of rbTNF from plasma was linearly (recovered TNF = .932 ∗ added TNF - .12; r = .99). Displacement curves of increasing amounts of plasma from calves challenged with endotoxin to effect an increase in endogenous TNF were parallel to the rbTNF standard curve. F314 failed to crossreact with any other cytokines tested except human TNF (<1%). Neither recombinant nor native bovine TNF significantly interacted with antisera for TNF of human or murine origin. Plasma TNF was acutely elevated in calves infused with endotoxin. Changes in plasma TNF were determined in samples from calves with chronic parasitic infection. Endogenous plasma TNF existed as a monomer with a molecular weight of 17,000, and was not bound to any plasma carrier protein. These data indicate that a specific RIA for bTNF capable of detecting changes in in vivo TNF levels has been established.