[Characterization of neutralizing activity of murine monoclonal antibodies for construction of anti-human TNF chimeric antibody].
OBJECTIVE
To screen high quality murine monoclonal antibodies (mAb) for construction of anti-human TNF chimeric antibody.


METHODS
3 hybridoma cells D2, E6 and F6 were selected from one group of hybridoma cells secreting anti-human TNF mAbs and then ascitic fluids were prepared according to routine protocol. After salted out with saturated ammonium sulphate, the mAb (IgG2b) secreted by D2 cells was purified through protein A affinity chromatography column. The mAbs (IgG1) secreted by E6 and F6 cells were purified through QFF anion exchange chromatography column. The titers of 3 mAbs were detected by indirect ELISA before and after purification. Inhibition of TNF induced cell death of L929 cells and ICAM-1 upregulation on ECV304 cells were assayed as indexes of neutralizing activity of the mAbs.


RESULTS
ELISA results showed that the titers of 3 mAbs D2, E6, and F6 before purification were 1x10(-6 ), 1x10(-7 ), and 1x10(-7 ), respectively, and 0.01, 0.002 and 0.002 mg/L after purification, respectively. 0.16 mg/L D2, 0.40 mg/L E6, or 0.50 mg/L F6 could protect 50% L929 cells from cell death induced by 2x10(4) U/L TNF, and the inhibition rates of ICAM-1 upregulation by 2x10(5) U/L TNF of 1.0 mg/L D2, E6 or F6 were 70.1%, 60.1%, and 60.1%, respectively. The inhibition rates still reached 60.1%,53.7% and 59.0%, respectively, when the concentrations of 3 mAbs D2, E6 and F6 decreased to 0.1 mg/L.


CONCLUSIONS
3 mAbs with high titers and neutralizing activities were obtained, which paves the way for construction of anti-human TNF chimeric antibody.
Zhu
Can-sheng
CS
Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
Ouyang
Wei-ming
WM
Liu
Xue-song
XS
Cao
Yun-xin
YX
Zhao
Ning
N
Jin
Bo-quan
BQ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D017209:Apoptosis; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1