Preparation And Identification Of Monoclonal Antibody Against Human Insulin-like Growth Factor-1

Object IGF-1 Diagnostic kit has been only produced by minority countries,for instance America and Germany,and its price is too high to limit its clinical application. Our laboratory had once chosen many carriers to produce IGF-1 protein antigen,but the result was to be disappointed,the yield of IGF-1 protein was all low,so it brought difficulty to prepare IGF-1 monoclonal antibody and IGF-1 Diagnostic kit. To plomb the blank of Diagnostic kit in China, we plan to construct recombinant vector containing the cDNA which encode hIGF-1 and to make it express,then to purify recombinant protein which the strain expresses. Subsequently,we establish the specific hybridoma cell lines that stably secreted monoclonal antibody(mAb) against human insulin-like growth factor-1 and identificate its properties. Methods 1. By using PCR technique,we amplified the target fragment which codes Human insulin-like growth factor-1 precursor cDNA,and then inserted into the expression vector pET29a(+),pET32a(+), subsequently.2. The recombinant vector was transformed into BL21(DE3). After inducing with IPTG, the recombinant protein was purified with Ni+ affinity chromatography. 3. We identified its antigenic activity with western blot.4. Balb/c mice were immunized with purified recombinant human IGF-1 protein. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line Sp2/0 cells. The hybridoma cells that secreted human IGF-1 mAb were subcloned with limited dilution.5. The specificity of mAb was detected by Western blot. Indirect enzyme-linked immunosorbent assay(ELISA) was used to evaluate the affinity and titers of mAbs. The immunoglobulin (Ig) subtype of mAb was determined by test paper bar. Result Enzyme digestion analysis and sequencing showed that the target fragment had been successfully inserted into the vector pET29a(+),pET32a(+),and the purified recombinant protein obtained by Ni+ affinity chromatography was so purified that its purity exceeded 90%.Western blot analysis showed it had immunology specificity. Two stably secreting anti-MGF-1 mAb cell lines were obtained. Western blot analysis showed the mAb had specific combination with human IGF-1 which possessed biological activities. The titers of both mAb in ascites were 6×10~4 and their relative affinities were between 2.1×10~9/mol～7.8×10~9/mol.Two mAb cell lines can secret IgM subtype antibodies. Conclusion The recombinant vector had been constructed successfully, and it could express with hign performance. Two hybridoma cell lines that secreted anti-hIGF-1 mAb were obtained, which will be useful for clinical application and fundamental basic research in hIGF-1.