Expression and immunological characterisation of Echinococcus granulosus recombinant antigen B for IgG4 subclass detection in human cystic echinococcosis

Abstract

A 165bp DNA fragment derived from the 12 kDa subunit of Echinococcus granulosus antigen B (AgB), a major hydatid cyst fluid antigen was cloned in the pMa1-c2 expression vector. A 52 kDa maltose binding-AgB fusion protein (rAgB.MBP) was produced and inclusion bodies containing the fusion protein were solubilised in urea and affinity purified on an amylose-Sepharose 6B column. The immunogenicity of the purified recombinant antigen for IgG4 antibody detection was tested with human serum using immunoblotting, ELISA and dot-ELISA assays and compared to native AgB. Both recombinant and native AgB preparations were highly reactive for human IgG4 antibodies in serum of cystic echinococcus (CE) patients. Recombinant AgB.MBP (rAgB.MBP) showed approximately 65% sensitivity in detection of IgG4 serum antibodies by ELISA from confirmed CE patients. Cross-reactivity (33%) occurred with alveolar echinococcosis (E. multilocularis) sera but recombinant AgB showed no seroreactivity with sera from other helminth infections tested (schistosomsis, onchocercsis, cysticercosis) or from uninfected individuals residing in CE endemic or non-endemic regions. The serologic sensitivity (63%) for IgG4 antibodies of a native AgB fraction enriched from human hydatid cyst fluid was similar to that for recombinant AgB (65%) though specificity was slightly lower (81%). A dot-ELISA for detection of total IgG, incorporating the rAgB.MBP resulted in 74% sensitivity and 88% specificity for human CE and 93% sensitivity and 65% specificity for native AgB. Recombinant AgB is a potential replacement for native antigens currently being used and could provide a better standardised E. granulosus specific test for clinical confirmation for CE especially for IgG4 antibody detection which appears to be predominantly associated with advanced disease.