Magnetic Affinity Enzyme-linked Immunoassay For Antibody Detection Of Schistosoma Japonicum

It is estimated that over 360 000 people are infected in China by the end of 2009. Currently, there is a tendency to change the epidemiology of schistosomiasis in the era of "preventive chemotherapy" in China. The lower rates of excreted eggs per gram of stool in the new endemic situation characterized by widespread, low-intensity infections influence the accuracy of prevalence assessments negatively.Parasitological diagnoses have the advantages of high specificity and of requiring relatively unsophisticated equipment and, in areas of high endemicity, personnel with only basic training. However, in areas of low-intensity infection, it has been recognized that parasitological detection methods lack sensitivity. Many lightly infected individuals are probably missed. Serodiagnosis could be a useful complement to parasitological tests and is more sensitive than conventional methods. However, these methods are labor-intensive, time-consuming, somewhat messy and less sensitive in detecting light or early infections. Recent researches showed that assays for detection of antigens may not have the expected high sensitivity and specificity when employed for diagnosis in low endemic areas or in light infections.Magnetic affinity enzyme-linked immunoassay (MHIA) was first developed in the 1980s. The main advantages of magnetic bead-based immunoassay include increasing the surface area for immobilization of antigen or antibody, reducing the incubation time and and making easy manipulation.The magnetic beads with a high binding capacity as a solid phase and the rapid reaction kineties of solutions with the simple separation of bound and unbound material on the solid phase, which provides the chance of enhancing the sensitivity of antibody detection. The microbead-based MEIA is found to provide higher sensitivity compared to microplate-based enzyme-linked immunosorbent assay (ELISA) technique.In this research, we produced soluble egg antigens (SEA) and recombinant antigens of Schistosoma japonicum (S. japonicum). We futher developed MEIA based on SEA and recombinant antigens for detection of schistosome circulating antibodies. The assay involved the use of SEA and recombinant antigens coupled to magnetic beads as capture antigens. MEIA based on SEA or recombinant antigens was used to detect antibodies in human sera infected with S. japonicum. In addition to the sera of patients with low-intensity S. japonicum infection, the detected samples include the sera of individuals with chemotherapy by praziquantel and paragonimiasis infections and those from the areas without schistosomiasis. The sensitivity and specificity of MEIA were compared in parallel with the traditional methods of ELISA.The main contents and results of this study are follows:(1) MEIA based on SEA (SEA-MEIA) for antibody detection of S. japonicum58 patients from schistosome-endemic areas were examined by using both of SEA-MEIA and SEA-ELISA. SEA-MEIA detected the positive sera, giving 96.55% positive rate, however, SEA-ELISA showed lower detection capability, giving 91.38% positive rate, there was statistical difference between the two methods (χ2=21.95, P<0.01). There were five false negative results from SEA-ELISA; of these, three patients had been detected positively by SEA-MEIA. In addition, when using Pearson's correlation in associating SEA-MEIA with SEA-ELISA, a significant correlation was showed between the two assays (r=0.845, P<0.01).15 patients with low-intensity infection were treated with praziquantel, and then the antibody IgG was detected using SEA-MEIA and SEA-ELISA for chemotherapy evaluation. The SEA-ELISA results showed that six months after treatment,33.33% of the infected individuals became negative, a value that decreased to 26.67%(χ2=10.909, P<0.01) when the SEA-MEIA results were used. Six sera from patients with paragonimiasis were tested by SEA-MEIA and SEA-ELISA. The cross-reaction rates for detection of anti-S. japonicum antibody in sera of paragonimiasis patients by these two methods were 50%(3/6).There was no difference in detection rate between the two assays.(2) Construct expression recombinant plasmidsSpecific primers were designed; Sj23, Sj23-LHD, Sj26 and Sj14-3-3 genes were amplified by PCR. The genes were cloned to pGEX and pET28a vectors respectively and sequenced, then subcloned to build prokaryotic recombinants pGEX-Sj23 mutant, pGEX-Sj23-LHD, pET28a-Sj26, and pET28a-Sj14-3-3. The E.coli BL21 (DE3) cells were transformed with the recombinant plasmids pGEX-Sj23 mutant, pGEX-Sj23-LHD, pET28a-Sj26, and pET28a-Sj14-3-3 respectively, then induced by IPTG to express the recombinant proteins.The rSj23-LHD was expressed in. Ecoli and purified through the column of affinity chromatography with GST. The rSj14-3-3 and rSj26 were expressed in. Ecoli and purified through the column of affinity chromatography with Ni2+(nickel sulfate). The molecular weight of purified rSj23-LHD, rSj26 and rSj14-3-3 were 34kDa,27kDa and 31kDa respectively. The purified rSj23-LHD, rSj14-3-3 and rSj26 were verified by SDS-PAGE and Western blotting. The rSj23-LHD, rSj26 and rSj14-3-3 can be recognized by the sera from mice and rabbits infected by S. japonicum using Western blotting, which indicated that these purified recombinant antigens might be potential for diagnosis of schistosomiasis japonicum.(3) Magnetic affinity enzyme-linked immunoassay based on recombinant antigens forantibody detection of S. japonicumThe results showed that rSj26-MEIA and rSj14-3-3-MEIA detected antibody IgG in positive sera from mice infected by S. japonicum. Moreover, the ratio of mean positive to negative value (P/N) of rSj26-MEIA and rSj14-3-3-MEIA were higher than that of ELISA at the same dilution ratio (3.92 versus 2.66,3.71 versus 2.45 respectively).We further applied rSj26-MEIA and rSj14-3-3-MEIA for diagnosis of patients with low-intensity infection S. japonicum. rSj26-MEIA and rSj26-ELISA had similarity in positive detection rates (14/58). rSj14-3-3-MEIA detected positive sera, giving 22.4% (13/58) positive rate, and rSj14-3-3-ELISA showed similar detection results. In addition, when using Pearson's correlation in associating MEIA with ELISA based on rSj26 and rSj14-3-3, significant correlations were observed respectively (r=0.658, P<0.01; r=0.618, P<0.01). The P/N of MEIA based on rSj26 and rSj14-3-3 were higher than that of ELISA at the same dilution ratio (3.61 versus 2.56,3.65 versus 2.71 respectively). There was no significant difference in positive detection rates between rSj26-MEIA and rSj14-3-3-MEIA (χ2=3.198, P>0.05).The antibody IgG was detected by using MEIA and ELISA for chemotherapy evaluation. rSj26-MEIA and rSj26-ELISA showed the similar sensitivity (2/15) and relatively high negative rate (86.67%). However, rSj14-3-3-MEIA and rSj14-3-3-ELISA showed no positive reaction. Six sera from patients with paragonimiasis were also tested. Of these patients, none of them was cross-reactive with rSj26 and rSj14-3-3 by these two methods.