Echinococcus multilocularis: molecular and immunochemical characterization of diagnostic antigen II/3-10

Abstract

SUMMARYA recombinant Echinococcus multilocularis antigen (II/3-10), which had previously been shown to exhibit immunodiagnostic characteristics highly specific for human alveolar echinococcosis, and the corresponding native parasite antigen, were further characterized with immunochemical and molecular biological methods. Immunoblot analysis using a polyclonal antiserum raised in rabbits against the recombinant protein, and subsequent Northern hybridization analysis, revealed that the native antigen was expressed by E. multilocularis at the adult as well as at the metacestode stage. In metacestodes, the antigen was shown by using indirect immunofluorescence and the same antiserum to be localized within the germinal layer and membrane structures of developing protoscolices. Electrophoretic analyses revealed remarkable differences in the apparent molecular weight of the antigen under reducing and non-reducing conditions. In further immunoblot analyses, anti-II/3-10 antibodies identified the corresponding epitopes on bands with identical M T in all E. multilocularis isolates investigated (European, Asian and North American). By Southern hybridization analyses of the respective gene, phylogenetically related sequences were shown to be present in other helminth species such as E. granulosus and several Taenia spp. In the same respect, immunoblotting revealed that anti-II/3-10 antibodies reacted with antigens of different M r from various E. granulosus isolates and some other cestode species, indicating the presence of shared and thus potentially cross-reacting epitopes. The relevance of these findings for the immunodiagnostic performance of the recombinant antigen is discussed.

Comparative analysis of full-length antigen II/3 fromEchinococcus multilocularisandE. granulosus

“…These analyses revealed the presumed identity of antigen II/3-10 with an antigen EmlO recently described by Frosch et al (1991), which seemed to be related to the antigen EM4 described by Hemmings & McManus (1991). Southern and Northern hybridization analyses and immunoblotting using a specific anti-II/3-10 hyperimmune serum raised in rabbits demonstrated that sequences encoding antigen II/3-10 seemed to be present not only in E. multilocularis, but also in E. granulosus, and that related antigens were expressed in both species, respectively (Felleisen & Gottstein, 1993).…”

“…Recently we characterized this recombinant antigen II/3-10 and the corresponding native metacestode antigen using molecular biological and immunochemical methods (Felleisen & Gottstein, 1993). These analyses revealed the presumed identity of antigen II/3-10 with an antigen EmlO recently described by Frosch et al (1991), which seemed to be related to the antigen EM4 described by Hemmings & McManus (1991).…”

“…The immunodiagnostic E. multilocularis recombinant antigen 11/3-10 allowed detection of anti-£\ multilocularis antibodies in human patients with high diagnostic sensitivity and specificity . Although homologous antigens are also synthesized by E. granulosus (Felleisen & Gottstein, 1993), very few sera from patients (6%) with cystic echinococcosis had serum antibodies against the recombinant II/3-10 antigen .…”

“…For detection of GST fusion proteins, polyclonal hyperimmune sera directed against antigen II/3-10 (Felleisen & Gottstein, 1993) and against Fasciola hepatica protease Fcpl fused to GST (Heussler & Dobbelaere, 1994) were used. The antibodies were affinity purified against recombinant II/3-10 antigen and the GST carrier protein, respectively, using the method described by Olmsted (1981).…”

“…Expression of GST-fusion proteins was monitored by SDS-PAGE and immunoblotting using an anti-II/3-10 specific rabbit hyperimmune serum (Felleisen & Gottstein, 1993). Bacteria transformed with the recombinant plasmids produced fusion proteins of approximately 89 kDa as calculated from the deduced amino acid sequences (Fig.…”