Establishment And Application Of ELISA Method For Detection Of IL2-HSA Fusion Protein

Interleukin-2 was a cytokine secreted by activated T lymphocytes which could enhance the immune ability of human bodies by promoting replication and clonal differentiation of other Helper and Cytotoxic T cell populations. It had become a dramatic biological agent in clinical therapy of tumor and autoimmune disease. In order to increase the half life of Interleukin-2, a plasmid , pPIH, which carries the coding gene of interleukin-2 and HSA and can express the fusion protein in Pichia pastoris GS115, had been constructed in our laboratory. This method could increase molecular weight of the medicamentous protein and decrease the immunogenicity of the heterogenesis protein, thereby it could diminish glomerular filtration rate and clearance rate in vivo. Furthermore, it boosted the life time of the Interleukin-2, so it would possess more extensive perspective in production and clinical application. In the coming pharmacokinetics and clinical application research, a method to quantitived detection of the fusion protein was needed to establish. It was widely used in various fields of life science for its high sensitivity and specificity . To build a double-antibody sandwich ELSIA method, which would quantitatively detect IL2-HSA fusion protein, was the main aim of this paper.At first, a strain of Pichia pastoris GS115/pPIH, which was proved to express IL2-HSA fusion protein, was cultured in BMGY medium and induced with methanol in BMMY. After fermentation, IL2-HSA fusion protein was purified and used as antigen standard. Protein quantitative analysis indicates that the concentration of standard preparation is 80μg/mL. Western Blot analysis shows that the fusion protein has immunogenicity to both IL-2 and HSA. Specific activity of the fusion protein is determined to be 1.393×107IU/mg by the CTLL-2/MTT method. Then, two antibodies respectively to IL-2 and HSA were selected and a double-antibody sandwich ELSIA method to detect IL2-HSA quantitatively was set up. The sensitivity of the method is 10 ng/mL and the measurement range is 19.53ng/mL to 1250ng/mL with correlation coefficient R2>0.988. Methodology examination indicated that the intra- and inter-assay coefficient of variation were 6.19% and 8.10%, respectively. All of these results indicate that the method has good stability. Detecting IL2-HSA fusion protein with the method can effectively determine the content of target protein and provide support to improve the technical procedures of the fermentation and purification,and it gives an optional analytic method in the coming pharmacokinetics and clinical research.