HSP60, HSP70 and HSP90 from Trichinella spiralis as targets of humoral immune response in rats

Abstract

This study identifies three heat shock proteins (HSPs) using purified preparations from Trichinella spiralis larvae. The proteins: HSP60, HSP70 and HSP90 were found to be targets of the humoral immune response in rats. Three approaches were adopted to obtain T. spiralis HSP-enriched material and/or to purify HSPs to homogeneity. The former product was prepared using affinity chromatography on gelatin sepharose and elution with ATP. Pure 90 kDa-protein was isolated from parasite extract by sequential DEAE (A50) column chromatograpy and preparative electrophoresis. Immunoblot analysis using monoclonal antibodies to HSP60, HSP70 and HSP90 detected the HSP60 and HSP70 in the affinity-purified product and HSP90 in the product obtained by sequential anionic chromatography and preparative electrophoresis. Finally, the reactivy of preimmune, T. spiralis immune and irrelevant immune rat sera on immunoblots were also examined. Only sera taken from infected rats at time-points after day 7 following the first infection exhibited activity against 60, 70 and 90 kDa proteins on blots. The fact that the serum antibodies were anti-HSP was established by immunoadsorption of HSPs to microtiter plates coated with anti-HSP60, anti-HSP70, or anti-HSP90 and using rat sera, positive on blots, to also give positive scores by continued enzyme-linked immunosorbent assay.