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Updated: May 25, 2025
Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries
Published on: January 17, 2015
CHO-S expression of a novel human recombinant IgG1 of anti-ALD antibody isolated by phage mutation display
Guilin Li1, Jiazhen Liu2, Zhenzhen Guan1
1Zhengzhou Immunobiotech Co, Ltd, Zhengzhou 450001, PR China.
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 VL and VH 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.
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 VL and VH 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.
