title: A simple method of extracting Cap antigen of PCV2b from emulsified vaccines for testing its stability and antigenicity.
abstract: Oil-based emulsions are commonly used adjuvants for veterinary vaccines. After formulation, it is required to extract protein antigens from emulsified vaccines for testing their stability and antigenicity. To establish a simple method to extract the protein antigens, two emulsified vaccines, designated as Cap-206 and Cap-35, were prepared by formulating a 28-kDa capsid protein (Cap) of PCV2b with ISA 206, a water-in-oil-in-water emulsion, or ISA 35, an oil-in-water emulsion. We found that the freeze-thaw-centrifugation method with steps of freezing at -20 °C for 12 h, thawing at room temperature and centrifuging at 9000×g for 10 min could separate the aqueous phase from Cap-206, and a centrifugation method by centrifuging at 9000×g for 10 min could isolate the aqueous portion from Cap-35. The Cap proteins were recovered from the aqueous phase and could be evaluated for their stability and antigenicity by SDS-PAGE, Western blot and ELISA. The freeze-thaw-centrifugation or the centrifugation method could also be used to recover recombinant mycobacterial heat-shock protein 65, a larger protein with molecular weight of 57-kDa, from ISA 206 or ISA 35 emulsions. The methods could be used to recover protein antigens from oil-based emulsion formulated vaccines for monitoring their stability and antigenicity during vaccine manufacture and storage.
authors.affiliation: Department of Molecular Biology, College of Basic Medical Sciences, Norman Bethune Health Science Center, Jilin University, Changchun, 130021 Jilin, China; Department of Epidemiology and Biostatistics, School of Public Health, Jilin University, Changchun, 130021 Jilin, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Yu
authors.affiliation: Department of Molecular Biology, College of Basic Medical Sciences, Norman Bethune Health Science Center, Jilin University, Changchun, 130021 Jilin, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Molecular Biology, College of Basic Medical Sciences, Norman Bethune Health Science Center, Jilin University, Changchun, 130021 Jilin, China.
authors.forename: Wenzhen
authors.initials: W
authors.lastname: Diao
authors.affiliation: Department of Immunology, College of Basic Medical Sciences, Norman Bethune Health Science Center, Jilin University, Changchun, 130021 Jilin, China.
authors.forename: Jiwei
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Molecular Biology, College of Basic Medical Sciences, Norman Bethune Health Science Center, Jilin University, Changchun, 130021 Jilin, China.
authors.forename: Yunpeng
authors.initials: Y
authors.lastname: Yao
authors.affiliation: Department of Immunology, College of Basic Medical Sciences, Norman Bethune Health Science Center, Jilin University, Changchun, 130021 Jilin, China.
authors.forename: Li
authors.initials: L
authors.lastname: Hua
authors.affiliation: Department of Epidemiology and Biostatistics, School of Public Health, Jilin University, Changchun, 130021 Jilin, China.
authors.forename: Yaqin
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Department of Immunology, College of Basic Medical Sciences, Norman Bethune Health Science Center, Jilin University, Changchun, 130021 Jilin, China. Electronic address: ylyu@jlu.edu.cn.
authors.forename: Yongli
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Department of Molecular Biology, College of Basic Medical Sciences, Norman Bethune Health Science Center, Jilin University, Changchun, 130021 Jilin, China. Electronic address: wlying@jlu.edu.cn.
authors.forename: Liying
authors.initials: L
authors.lastname: Wang
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D002498:Centrifugation / Q000379:methods; D017925:Circovirus / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004355:Drug Stability; D004655:Emulsions; D004797:Enzyme-Linked Immunosorbent Assay; D007106:Immune Sera / Q000276:immunology; D008807:Mice, Inbred BALB C; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D014765:Viral Vaccines / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D004655:Emulsions; D007106:Immune Sera; D011994:Recombinant Proteins; D014765:Viral Vaccines
keywords: Emulsion; Extraction; Protein antigen; Vaccine