Preparation Of Monoclonal Antibody Against Human Interleukin-15 And Preliminary Application

Objective: Interleukin-15 (IL-15) is a kind of new interleukin, which has similar biological functional activities but without sequence homology to Interleukin-2 (IL-2). Its distribution is far wider than that of IL-2 in vivo. IL-15 plays great important role in the immune regulation and immune response, especially in the immune response to tumor. The anti-human-IL-15 monoclonal antibody (McAb) is very essential for the identification and investigation of IL-15. The present study was intended to prepare a kind of specific McAb against human IL-15 by the cell engineering method, to identify its biological, immunological, physicochemical characteristics, and to try its application primarily. Methods: The GST-IL-15 was extracted from the genetic engineering E.coli and identified by SDS-PAGE. The gel strip corresponding to the band of protein whose density was 50μg/ml on the SDS-PAGE was cut off as immunogen. The BALB/c mouse was immunized with GST-IL-15 for five times. The immune interval was four weeks for the first and second time and was two weeks forthe others. And the mouse was attacked by the rhIL-15 inclusion body protein (rhIL-15IBP) three days before the hybridization. The spleen cells of the immunized mouse were hybridized with Sp2/0 cells by 50 % PEG. The hybridoma cells were selected with HAT culture medium. The antibody-secreting hybridoma cells were primary detected by the indirect ELISA using rhIL-15IBP as the antigen, and coloned by limited dilution to obtain the hybridoma cell lines which can stably secret the McAb against rhIL-15IBP. The McAb was identified by the functional recombinant human Interleukin-15 (rhIL-15) protein purchased from the USBiological. The chromosomes of hybridoma,Sp2/0 and the mice bone marrow cells were counted. The stability of the McAb-positive cell lines were detected by thirty subcultures in vitro,being frozen and thawed repeatedly for 5 times and thawed after frozen for 3 months. The ascites McAb, the culture supernatant McAb and the serum-free culture supernatant McAb were prepared. The Ig class of McAb was identified by the indirect ELISA using the serum-free culture supernatant McAb. The antibody titers of ascites and culture supernatant McAb were measured. The stability tests of ascites McAb for the heat-resisting,freezing-thrawing and under different pH were performed. The heat-resisting test was done at 56℃for 30 minutes, 37℃ for 1-7 days and 4℃ for 7 days, respectively. Thefreezing-thrawing test was that the ascites McAb was frozen at -20℃, then thawed at room temperature naturally, so repeatedly for 6 times. The stability test of the McAb under different pH was as following: the ascites mAb was diluted with the buffers of pH2.2,pH7.4 and pH9.6 ( the normal mouse serum as the negative control) , then kept at 4℃ for 24 hours and 48 hours, respectively. The rhIL-15-binding specificity and cross-reaction with rhIL-2 and IL-6 of the McAb were detected by the competitive ELISA. The New Zealand rabbits were immunized with the rhIL-15IBP to prepare the serum polyclonal antibody. The ascites McAb was purified by PEG precipitation. The IgG of serum polyclonal antibody was purified by the saturated ammonium sulfate. The indirect Sandwich ELISA was established with the purified monoclonal and polyclonal antibody, and used to detect the functional rhIL-15 protein to determine the sensitivity. Results: The GST-IL-15 was abstracted from the genetic engineering E.coli and identified by SDS-PAGE. The mouse was immunized successfully using the gel strip. Three wells of antibody-secreting hybridoma cells against rhIL-15IBP were primary obtained by cell hybridation, HAT selection and antibody detection. One hybridoma cell line can secret the McAb against rhIL-15IBP continuously after coloning. The McAb can bind to the functional rhIL-15 protein purchased from the USBiological .Thehybridoma cell line was named 5B11, and its stability was well. The McAb can be secreted by 5B11 after thirty subcultures in vitro,being frozen and th...