PMID: 8585629Sep 1, 1995Paper

Assay for recombinant hepatitis B surface antigen using reversed-phase high-performance liquid chromatography

Analytical Biochemistry
D O O'Keefe, A M Paiva

Abstract

Several HPLC assays are reported for monitoring the mass of recombinant hepatitis B surface antigen (rHBsAg) in yeast cell lysates. The assays utilized either a polymeric resin column containing a phenyl ligate or a silica-based octadecyl micropellicular column. Prior to chromatography on the polymeric column, the samples were derivatized with the thiol-specific fluorescent probe monobromobimane to discriminate the rHBsAg from nonfluorescent cellular components. Using a dual gradient of acetic acid and acetonitrile the derivatized rHBsAg eluted with a retention time equal to 17 min. Chromatography on the micropellicular column did not require prederivatization and utilized an isopropanol gradient with increasing amounts of acetonitrile. Operating this column at elevated temperature with a high flow rate resolved the rHBsAg from yeast components within 5 min and allowed a new sample injection every 10 min. All the assays displayed useful linear ranges for analyzing rHBsAg in cell lysates and had detection limits for rHBsAg between 10 and 50 ng per injection.

Citations

Sep 24, 1998·Journal of Chromatography. B, Biomedical Sciences and Applications·D Tleugabulova
Jan 19, 1999·Journal of Chromatography. B, Biomedical Sciences and Applications·D TleugabulovaE Pentón
Feb 17, 2000·Journal of Chromatography. B, Biomedical Sciences and Applications·D TleugabulovaY Fleitas
Oct 31, 2018·Molecular Pharmaceutics·José Vicente González-AramundizNoemi Stefánia Csaba
Oct 12, 1999·Analytical Chemistry·M A WinklerH Aboleneen

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