CHO-S expression of a novel human recombinant IgG1 of anti-ALD antibody isolated by phage mutation display.

Abstract

Aldosterone (ALD) is elevated in the serum and plasma of patients with various metabolism-related diseases, and anti-ALD antibodies are important materials in early diagnostic kits. However, current anti-ALD antibodies for clinical diagnosis are not specific, resulting in high false positives. We have developed an anti-ALD-601# rabbit monoclonal antibody that is commercially available and licensed for diagnosis. To obtain monoclonal antibodies with better stability and affinity, a phage mutation library was constructed by site-specific mutagenesis of the CDR3 region in the V_L and V_H domain of the anti-ALD-601# antibody. Anti-ALD3302#, a single-chain variable fragment (scFv), with enhanced affinity, was screened by enzyme-linked immunoassay (ELISA). To improve the less stable and shorter half-lives of scFvs, anti-ALD-scFv-3302# was inserted into a eukaryotic expression vector (pCHO1.0-Fc) and then transfected into CHO-S cells to express anti-ALD-scFv-Fc. Then, cells were cultured by reducing culture temperature (33.5 °C) and application of CuSO₄ (50-200 μM) treatment. The expression-purified antibody was detected with competitive-ELISA, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and capillary electrophoresis. Non-reducing SDS-PAGE electrophoresis showed a single band and the percentage of the highest peaks in capillary electrophoresis was more than 90 %. The coefficient of variation in the competitive-ELISA assay was reduced to 2 % from 5 % before optimization. The correlation between the assay using this monoclonal antibody and the HPLC-FLD determination was also greater than 0.99. In conclusion, the designed anti-ALD-specific antibody (3302#) has been successfully developed for use in metabolism-related diseases to detect the concentration of ALD in early diagnostic.