Assay for recombinant hepatitis B surface antigen using reversed-phase high-performance liquid chromatography.
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.
O'Keefe
D O
DO
Department of Analytical Research, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
Paiva
A M
AM
D001643:Bridged Bicyclo Compounds; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes; D006514:Hepatitis B Surface Antigens / Q000032:analysis*; D016014:Linear Models; D011994:Recombinant Proteins / Q000032:analysis; D012441:Saccharomyces cerevisiae; D013056:Spectrophotometry, Ultraviolet; D013439:Sulfhydryl Reagents
D001643:Bridged Bicyclo Compounds; D005456:Fluorescent Dyes; D006514:Hepatitis B Surface Antigens; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; C028381:monobromobimane