Screening And Application Of Antigen For Diagnosis Of Schistosomiasis Japonica Base On IgG4-oriented Proteomics

Objective Worldwide schistosomiasis continues to be a serious public health problem.Over a few decades, China has made remarkable progress in reducing Schistosomajaponicum infections in humans to a relatively low level. At present, schistosomiasis isabout to be brought under control in china. The direct parasite examination gives rise toa relatively insensitive outcome because of low worm burdens, which leads to lessefficiency in low transmission settings. Now, it is urgent that a search for a diagnosticapproach with a simple, affordable, sensitive, and specific assay for field diagnosis ofSchistosoma japonicum, is essential and should be given high priority towards fullcontrol and elimination of schistosomiasis.Methods Adult worms were collected from infected rabbits with Schisitosomajaponicum. Adult worm antigen (AWA) extract was prepared and subjected to two-dimension electrophoresis (2-DE). The reactive proteins were screening, excised,digested by trypsin and further identified by LC-MS/MS following IgG4-orientedWestern blotting. The candidate molecules for diagnosis of schistosomiasis was clonedand expressed from the transformed E. coli after bioinformatics analysis. Furthermore,the recombinant protein was applied with enzyme linked immunosorbent assay (ELISA)and evaluated for its performance in ELISA for diagnosis for schistosomiasis.Results This IgG4-oriented serological proteome assay yielded more than40immunodominant spots. Sixteen of these spots were precisely matched with ahomologous2-DE gel and successfully identified by LC-MS/MS as corresponding to ten different proteins. These proteins were S. japonicum aconitase (SjAA), heat shockprotein70(HSP70), pyruvate kinase M2isoform (SjPKM2), phosphoglycerate kinase(SjPGK1), α-L-fucosidase (SjALF), inorganic pyrophosphatase (SjIPP), glutathionetransferase (SjGST),21.7kDa antigen (Sj21.7), tegument membrane-associated antigen(Sj22.6) and sarcoplasmic calcium-binding protein of S. mansoni (SmSCP).Bioinformatics analysis displayed that SjIPP possesed variety of biological functionsand abundant antigen epitopes for B cell, which implied potential insight aboutdiagnosis for schistosomiasis. Furthermore, SjIPP was successfully expressed, and therecombinant protein was further applied in the diagnosis of human schistosomiasisusing IgG4-ELISA. The ELISA results revealed sensitivities of100%and87.1%foracute schistosomiasis and chronic schistosomiasis, whereas the assays showed aspecificity of95.0%with rSjIPP recombinant protein. Remarkably, rSjIPP showed lowcross reaction with these sera infected with Paragonimus westernami(lung fluke), C.sinensis (liver fuluke)or nematodes(roundworms). The cross reaction rates wererespectively3.7%,4.5%and4.2%.Conslusions SjIPP was successfully screened as a diagnostic candidate from AWA ofSchistosoma japonicum by IgG4-oriented proteomics followed by expression. Ourpresent study confirmed that rSjIPP was a useful diagnostic biomarker for Schistosomajaponicum infection.