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

Christy Ambarsari; Herman Suryadi; Arry Yanuar

doi:10.24198/ijpst.v10i3.40641

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

Christy Ambarsari, Herman Suryadi, Arry Yanuar

Abstrak

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.

Kata Kunci

Indirect ELISA, glycerol and m-cresol, method validation, rabbit IgG polyclonal antibody, recombinant human insulin.

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Referensi

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DOI: https://doi.org/10.24198/ijpst.v10i3.40641