Development Of Colloidal Gold Immunochromatography Assay Strip For Animal’s Schistosomiasis Japonica Diagnosis

Schistosomiasis is a serious zoonotic parasitic disease. Schistosoma japonicum’s infection can be detected by parasitological methods and immunological methods. However, the sensitivity of the traditional tests is compromised for subjects with low-intensity infections and in area with a low prevalence.To meet the needs of the grassroots screening, it is necessary to develop a rapid detection kit for a variety of animals. The colloidal gold immunechromatography assay strip is suitable for mass screening because it is simple, rapid, sensitive, specific, and no special equipment. This study aims to develop quick and easy GICA strip methods for schistosomiasis diagnosis.Streptococcal protein G can bind to IgG of Fc domain about many species. This function was widely used in immunology experiments. To reconstruction of a streptococcal protein G(SPG) and identification its biological function, it made SPG more useful. The SPG domain was subcloned into PET-28a(+) and pGEX-2T and expressed in supernatant of E. coil. BL21(DE3) cell and E. coil. BL21 cell, respectively. Then, the His–tagged fusion protein and GST-taggged fusion protein were obtained. Western blotting showed that the recombinant SPG(His-rSPG and GST-rSPG) had the ability to bind with IgG. The affinity constant assayed by ELISA, and the affinity constanted about rSPG in four species IgG had obtained. It showed the affinity of GST-rSPG is higer than that of the standard streptococcal protein G(SPG). The affinity of His-rSPG is basically the same that of the standard SPG.The optimization pH determination and the concentration determination of His-rSPG and GST-rSPG were labeled with colloidal gold. Finally, the His-rSPG was prepared and labeled labeled with colloidal gold. The soluble egg antigen(SEA) and His-rSPG were blotted on the nitrocellulose membrane for the test line and control line, respectively. Besides, the optimal concentration of the lines and the optimal dilution of serum were determined. As a result, the optimal pH was 5.0, the best labeled amount of His-rSPG was 10 μg/mL, the test and control lines’ optimal concentration were 0.5 mg/mL and serum best dilution of the serum was 1:20.Specificity and sensitivity of the strip method were detected with the serum of BALB/c mice, New Zealand white rabbits, buffalo and goat. The cross-reaction of the strip method were detected with the serum of paramphistomiasis buffalo, haemonchosis goat and orientobilharziasis goat and compared with the ELISA.To further evaluation of the test strip, GICA and ELISA were detected the buffalo serum in different intensity of infection. Meanwhile, the blood papers of buffalo were detected by GICA and ELISA.The colloidal gold immunochromatography assay strips has been assembled and they can detect successfully the S.j infected serum of BALB/c mice, New Zealand white rabbits, buffalo and goat. Besides, the sensitivity of GICA strip was 100.00% in serum of mice and rabbits with infection of S.j. The specificity was 100.00% in serum from mice and rabbits with free of infection. The sensitivity of GICA was 100.00% in serum from goat with miracidia of S.j hatching from the stool with the same results as ELISA and the specificity was 88.64% in serum from uninfected goat that higher than the result of 75.00% with ELISA. The sensitivity was 100.00% in serum from buffalo with miracidia of S.j hatching from the stool with the same results as ELISA and the specificity was 94.23% in serum from uninfected buffalo that higher than the result of 84.62% with ELISA. The cross-reaction rate was 14.29% with buffalo in paramphistomiasis, 16.67% with goat in haemonchosis and 33.33% with goat in orientobilharziasis the results were slightly lower or as same as the ELISA method. When the infection was less than 20 worms per buffalo, the sensitivity of GICA was 75.00% lower than the 100.00% of ELISA. When the infection was more than 20 worms per buffalo, the sensitivity of GICA and ELISA were both 100.00%. Both the sensitivity and specificity of GICA were 100.00% in blood papers from buffalo with the same results as ELISA. Besides, there was no statistical difference between the two methods in buffalo blood papers from epidemic area.On the whole, this study successfully expressed recombinant streptococcal protein G. Meanwhile, the GICA strip can successfully detect variety of Schistosoma japonicum infected domestic animal and may be a useful tool for screening on large scale in the endemic areas.