Production of Anti-Recombinant Human Insulin Antibody and Validation by Indirect ELISA

Human insulin potential has become an interest and is important in maintaining the success of therapy in patients with the availability of chemical-based analytical methods, however, only a few have been using immunoassays. This study aimed to produce IgG polyclonal antibodies from rabbits immunized with 1 mg/mL rhINS subcutaneously and validated by indirect ELISA. Antibody was precipitated and fractioned on a HiTrap® Protein A HP column before being quantified with a UV spectrophotometer at λ 280 nm. The characterization was conducted by Dot Blot test on a BCIP-NBT substrate, as well as SDS-PAGE and Western Blot with polyacrylamide gel concentrations of 7.5% and 17.5%. Validation was performed using solutions containing glycerol and m-cresol as matrices spiked with rhINS. The linearity test in the rhINS concentration range of 80.11-200.28 μg/mL (r = 0.99) showed the linear result. The accuracy and precision obtained an average of 99.11%±5.01 and 3.91%, while the LOD and LOQ were 22.05 μg/mL and 73.51 μg/mL, respectively. Human insulin was stable at 2-8oC for 24 hours (α: 0.05, ANOVA). In conclusion, in-house produced IgG polyclonal antibodies and goat anti-IgG peroxidase conjugate can be used for routine testing of human insulin.