猪IL-15单克隆抗体的制备及双抗体夹心ELISA方法的建立
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中图分类号：R446.61 文献标志码：A 文章编号：0366-6964（2025）07-3442-11
Abstract： A monoclonal antibody （McAb） to porcine interleukin-15 （IL-15）was prepared and a quantitative IL-15-ELISA was established. Porcine IL-15 prokaryotic expression vector pET- 28a- （20 IL15 was constructed，and the recombinant protein was expressed with Escherichia coli （ E coli ） prokaryotic expression system，and six-week-old female BALB/c mice were immunized with recombinant IL-15. On day 3 after the booster immunization，splenocytes were isolated and collected from one mouse to fuse with SP2/O cells. Positive hybridoma cells were screened and monoclonal antibodies to IL-15 secreted by the positive hybridoma cells were identified. Moreover，a sandwich ELISA for detection of porcine IL-15 based on a monoclonal antibody. Nine strains of hybridoma cells（1E8，1F4，2D3，2C10，2F3，2D10，3F4，3D4 and 3D5）secreting anti-IL-15 McAb were obtained，and all of the secreted McAb were of the IgG2bκ type. A sandwich ELISA using the prepared monoclonal antibody 2DlO as the capture antibody and 2C10 as the enzyme-labeled antibody，respectively，was able to detect a minimum of 31.25ng∙mL-1 of IL-15 protein，with no cross-reactivity with the other cytokines or protein （IL-1， IL-2， IL-4， IL-6， IL10，IL-12，Gzms-B，IFN- γ ，and TNF-α ）.The mean coefficient of variation for intra-batch and inter-batch reproducibility experiments was 2.56% and 3.39% ，respectively. This sandwich ELISA quantifies IL-15 in serum， tissue fluid and cell culture. Porcine IL-15 McAb was successfully prepared，and a double antibody sandwich ELISA method was established，which provided an important tool for IL-15 related studies.
Keywords：porcineIL-15; monoclonal antibody； double-antibody sandwich ELISA; quantitative determination
∗ Corresponding authors：SONG Qinye，E-mail： songqinye $＼textcircled { ＼omega }$ 126.com;YUAN Chen，E-mail： yuanchen060624@163.com
白细胞介素15（interleukin-15，IL-15）是1994年Grabstein在检测猿肾上皮细胞系CV-1/EBNA培养上清液时发现的一种多效细胞因子[1]，由多种细胞如基质细胞、单核细胞、巨噬细胞、树突状细胞、上皮细胞等产生。（剩余18403字）