Generation and characterization of hamster monoclonal antibodies that neutralize murine tumor necrosis factors.
mAb to murine TNF (MuTNF) were produced after immunization of Armenian hamsters with purified, Escherichia coli-derived rMuTNF-alpha. Antibody produced from clone TN3-19.12, was purified and was found to inhibit 100% of the lytic activity of either recombinant or natural MuTNF-alpha at an antibody input of 25 ng/U. TN3-19.12 also inhibited all the lytic activity in culture supernatants from a variety of T cell sources, including activated T cell clones and T cell hybridomas (all of which expressed high levels of TNF-alpha and TNF-beta (lymphotoxin, LT) mRNA). Western blot analysis was used to document the physical form(s) of MuTNF recognized by TN3-19.12. Recombinant and macrophage-derived TNF displayed identical patterns of a single band with Mr 17 kDa. In contrast, T cell culture supernatants exhibited patterns consisting of two bands with Mr 17 and 24.7 kDa. The higher m.w. form was glycosylated based on its sensitivity to n-glycanase and displayed a m.w. consistent with that of TNF-beta (LT). These data suggest that TN3-19.12 recognizes both MuTNF-alpha and MuTNF-beta (LT). Monoclonal TN3-19.12 and polyvalent rabbit anti-rTNF were used to establish a MuTNF-specific ELISA capable of detecting picogram quantities of recombinant or natural TNF. This assay was used to detect TNF in the sera of mice challenged with a lethal dose of LPS. Peak TNF serum levels of 11 ng/ml were observed in these animals 90 min after i.p. LPS administration and then rapidly declined to near base line levels by 3 h. These values were confirmed by quantitating levels of TNF functional activity in the same samples. TN3-19.12 injected into mice subsequently treated with LPS prevented the detection of TNF in the circulation by either assay and protected mice from the lethal effects of endotoxin shock. Thus, TN3-19.12 effectively neutralizes endogenously produced TNF in vivo.
Sheehan
K C
KC
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
Ruddle
N H
NH
Schreiber
R D
RD
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis* / Q000493:pharmacokinetics; D000918:Antibody Specificity; D015152:Blotting, Northern; D015153:Blotting, Western; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis / Q000276:immunology; D007106:Immune Sera / Q000032:analysis; D008070:Lipopolysaccharides; D008264:Macrophages / Q000276:immunology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D011817:Rabbits; D051381:Rats; D013602:T-Lymphocytes, Cytotoxic / Q000032:analysis / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007106:Immune Sera; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha