A MONOCLONAL ANTIBODY-BASED ENZYME-IMMUNOASSAY FOR QUANTITATION OF HUMAN TUMOR-NECROSIS-FACTOR BINDING PROTEIN-I, A SOLUBLE FRAGMENT OF THE 60 KDA TNF RECEPTOR, IN BIOLOGICAL-FLUIDS

Three hybridoma cell lines secreting monoclonal IgG antibodies specific for human tumor necrosis factor-binding protein I (TNF-BP I), the extracellular domain of the kDa TNF receptor, were developed by fusion of spleen cells from mice immunized with TNF-BP I purified from urine. The antibodies recognize three different epitopes on TNF-BP I. Two of the antibodies were used to develop a two-site ('sandwich') enzyme immunoassay with horseradish peroxidase as the marker enzyme. The assay was able to measure TNF-BP I in serum, urine and cell culture supernatants with a sensitivity of about 200 ng/l and a precision better than 10%. TNF-BP I was detected in the serum of healthy individuals at a mean concentration of 2.1 +/- 1.0-mu-g/l (mean +/- standard deviation; range, 0.52-5.4-mu-g/l, n = 42); no significant difference was seen in patients with chronic polyarthritis (2.3 +/- 0.79-mu-g/l; n = 15). Serum TNF-BP I was significantly elevated in patients with burns (6.5 +/- 1.7-mu-g/l; n = 10) and markedly increased in patients with renal failure (49 +/- 17-mu-g/l; n = 6). TNF-BP I was also detectable in urine from normal individuals (2.2 +/- 1.2-mu-g/l; range 0.78-4.3-mu-g/l; n = 16). Culture supernatants of several human tumor cell lines also contained TNF-BP I. The assay will be a useful tool to detect activation of the TNF receptor by the physiological ligands, TNF-alpha and TNF-beta, as well as transmodulation by other mediators in various pathological conditions.