[Establishment of Magnetic Affinity Enzyme Linked Immunoassay Based on Schistosoma japonicum Recombinant Antigen Sj26 and Its Application in Detection of Serum Antibody with Low Intensity of Infection].

Abstract

Objective: To develop the magnetic affinity enzyme-linked immunoassay (MEIA) using recombinant glutathione-S-transferase of Schistosoma japonicum with a relative molecular weight of 26 000（rSj26-MEIA） for antibody detection under low intensity infection.

Methods: The recombinant plasmid pET28a-Sj26 was transformed into Escherichia coli strain BL21, and 0.6 mmol/L isopropyl β-D-1-thiogalactopyranoside （IPTG） was used to induce its expression. The expression products were purified by Ni2+ （nickel sulfate） affinity chromatography, SDS-PAGE and Western blotting were performed to examine the expression of rSj26. The purified rSj26 was coupled to magnetic beads as capture antigen and the reaction conditions were optimized to establish the rSj26-MEIA method. The method was then used to analyze 58 serum samples from patients with low-intensity S. japonicum infection, 30 serum samples from non-endemic areas as a negative control, and 6 serum samples from patients with paragonimus infection. Results were compared with those obtained with ELISA.

Results: The concentration of purified rSj26 was 2.5 mg/ml. The rSj26 had a relative molecular weight of 27 000 and was expressed mainly in the soluble form as revealed by SDS-PAGE. It could be recognized by rabbit and murine sera infected with S. japonicum as shown by Western blotting. Optimization of rSj26-MEIA revealed that use of 0.2 mg magnetic beads loaded with 10 μg rSj26 and serum sample dilution at 1 ∶ 100 yielded the highest ratio of the mean A550 of positive serum to the mean A550 of negative sample (P/N)（3.97）. For serum samples from patients with low-intensity S. japonicum infection, rSj26-MEIA and rSj26-ELISA both resulted in a positive detection rate of 24.14%（14/58）, and P/N values of 3.61 and 2.56. In addition, Pearson′s correlation analysis revealed positive correlation between A550 values detected by rSj26-MEIA and by rSj26-ELISA（r=0.658, P<0.01）. Further, no positive reaction was found in the 6 serum samples from patients with paragonimus infection and in the 30 serum samples from non-endemic areas, either by rSj26-MEIA or by rSj26-ELISA.

Conclusion: rSj26-MEIA may be used as a new technique for detection of serum antibody against S. japonicum infection with low intensity.