Monoclonal antibodies for affinity purification of IL-6/IFN- beta 2 and for neutralization of HGF activity.
Two types of recombinant human IL-6 (rIL-6) were used for the development of specific monoclonal antibodies. The first was produced in E. coli and used for immunization, the second was produced in Chinese Hamster Ovary Cells (CHO) and used for screening. The complete translated sequence of the cDNA coding for human IL-6 was fused, in phase, to protein-A and the hybrid gene was fused to the strong lambda PR promoter. This protein was purified from bacterial extracts by chromatography on rabbit IgG-Sepharose columns. After six injections of the purified protein into mice, sera were tested for their binding titer in a solid phase radioimmunassay (sRIA) and for the specificity of binding by Western blots. In the sRIA, crude supernatants of CHO cells (harboring a plasmid containing the human IL-6 gene and expressing high levels of IL-6 but no protein-A or any bacterial antigen) were bound to a solid support, reacted with supernatants of the hybridomas and finally detected with [125I]-goat anti-mouse antibodies. Spleen cells derived from a mouse showing the highest binding titer were fused to mouse myeloma cells. The hybridomas were screened by the sRIA and several positive clones were isolated and characterized. One of the clones was found to neutralize the hybridoma growth factor activity of the rIL-6 from both sources. The same clone was also used for Western blots and for affinity purification of both natural and recombinant IL-6 (E. coli and CHO).
Novick
D
D
Department of Virology, Weizmann Institute of Science, Rehovot, Israel.
Eshhar
Z
Z
Revel
M
M
Mory
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002460:Cell Line; D002846:Chromatography, Affinity; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005347:Fibroblasts; D006801:Humans; D007163:Immunosorbent Techniques; D015850:Interleukin-6 / Q000302:isolation & purification*; D009500:Neutralization Tests; D010053:Ovary; D011971:Receptors, Immunologic / Q000652:urine; D019947:Receptors, Interleukin-6; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D015850:Interleukin-6; D011971:Receptors, Immunologic; D019947:Receptors, Interleukin-6; D011993:Recombinant Fusion Proteins