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 60 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 micrograms/l (mean +/- standard deviation; range, 0.52-5.4 microgram/l, n = 42); no significant difference was seen in patients with chronic polyarthritis (2.3 +/- 0.79 micrograms/l; n = 15). Serum TNF-BP I was significantly elevated in patients with burns (6.5 +/- 1.7 micrograms/l; n = 10) and markedly increased in patients with renal failure (49 +/- 17 micrograms/l; n = 6). TNF-BP I was also detectable in urine from normal individuals (2.2 +/- 1.2 micrograms/l; range 0.78-4.3 micrograms/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.
Adolf
G R
GR
Ernst Boehringer-Institut für Arzneimittelforschung, Bender & Co. Ges mbH, Department of Cell Biology, Vienna, Austria.
Apfler
I
I
D000818:Animals; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins / Q000032:analysis* / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006358:Hot Temperature / Q000009:adverse effects; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D007132:Immunoglobulin Isotypes; D051379:Mice; D008807:Mice, Inbred BALB C; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D012680:Sensitivity and Specificity; D053319:Tumor Necrosis Factor Decoy Receptors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D007132:Immunoglobulin Isotypes; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D053319:Tumor Necrosis Factor Decoy Receptors; D014409:Tumor Necrosis Factor-alpha; C069521:recombinant human tumor necrosis factor-binding protein-1