Establishment and application of a cell model for screening neutralizing monoclonal antibody against human TNF

    ZHU Canª²Sheng1,2, LIU Xueª²Song1, JIN Boª²Quan1

    1Department of Immunology, School of Basic Medicine, Fourth Military Medical University, Xi¬ðan 710033, China, 2Department of Laboratory, Shaanxi Provincial Institute for Endemic Disease Control, Xi¬ðan 710003, China

    ¡¾Abstract¡¿ AIM: To establish and apply a cell model to screen neutralizing monoclonal antibody against human TNF. METHODS: L929 cells were seeded into flat bottom of 96ª²well microtiter plates at a density of 1¡Á108/L in 100 ¦ÌL DMEM containing 100 mL/L FCS and cultured at 37¡æ for 16 h. Spent medium was removed and 200 ¦ÌL of experimental solutions was added to each well. The plates were similarly reincubated for 16 h, followed by removal of culture supernatants and stained with crystal violet. The absorbance at 570 nm was determined. RESULTS: In the range from 0.3 mg/L to 0.5 mg/L, antinomycin D (ActD) had stable inhibitory effects on L929 cell growth. In the concentration over 20 kU/L, TNF could induce over 95% of experimental cells apoptosis. In the range from 12.5 ¦Ìg/L to 50.0 ¦Ìg/L, monoclonal antibody could neutralize TNF in a dose dependent manner. Under the condition that the concentrations of ActD, TNF and monoclonal antibody were 0.35 mg/L, 20 kU/L, and 25.0 ¦Ìg/L, respectively, no significant difference was found between D2 and positive control antibody in aspect of neutralizing activity (P>0.05), but the neutralizing activity of D2 was significantly higher than those of E6, E11, D12 and negative control antibody (P<0.01). Compared with negative control antibody, E6 and E11 also had neutralizing activity (P<0.05), but D12 did not show any neutralizing activity (P>0.05). CONCLUSION: The cell model established and optimized in our study can be employed in screening neutralizing monoclonal antibodies against human TNF. By employing this model, we have identified that D2 is of high neutralizing activity, which can be used as a reagent for the construction of therapeutic chimeric antibody. ......