RESUMO
In this study, we describe an optimized method of obtaining virus-like particles (VLPs) of the recombinant hepatitis C virus (HCV) core protein (HCcAg) expressed in yeast cells (Pichia pastoris), which can be used for the construction of diagnostic test systems and vaccine engineering. The described simplified procedure was developed to enable in vitro self-assembly of HCcAg molecules into VLPs during protein purification. In brief, the HCcAg protein was precipitated from yeast cell lysates with ammonium sulfate and renatured by gel filtration on Sephadex G-25 under reducing conditions. VLPs were self-assembled after the removal of the reducing agent by gel filtration on Sephadex G-25. Protein purity and specificity were evaluated by SDS-PAGE and immunoblotting analysis. The molecular mass of VLPs and their relative quantity were measured by HPLC, followed by confirmation of VLPs production and estimation of their shape and size by transmission electron microscopy. As a result, we obtained recombinant HCcAg preparation (with ~90% purity) in the form of VLPs and monomers, which has been used to produce hybridomas secreting monoclonal antibodies (mAbs) against HCcAg.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Hepacivirus , Anticorpos Anti-Hepatite C/imunologia , Saccharomycetales , Vacinas de Partículas Semelhantes a Vírus , Proteínas do Core Viral , Vacinas contra Hepatite Viral , Animais , Feminino , Hepacivirus/genética , Hepacivirus/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Saccharomycetales/genética , Saccharomycetales/metabolismo , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Proteínas do Core Viral/isolamento & purificação , Vacinas contra Hepatite Viral/biossíntese , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologia , Vacinas contra Hepatite Viral/isolamento & purificaçãoRESUMO
This article reexamines some opinions concerning pH requirements for optimal immobilization of monoclonal antibodies (mAbs) by passive adsorption in antigen capture ELISA. It was discovered that substitution of "classical" sodium phosphate (pH 7.5) and carbonate (pH 9.5) coating solutions by acid (pH 2.8) buffers maximized antigen capture 4 out of 10 different tested anti-HBsAg mAbs, resulting in a 1.5-2.5 increase of binding curve coefficients. By measuring both mAbs amounts and functionality, the enhancement effect was attributed to the better preservation of solid phase antibodies activity.