Electrochemical Immunoassay And Chemiluminescence Biosensor For The Detection Of Schistosoma Japonicum Antibody

Schistosomiasis(bilharzia),caused by a worm of the trematode family, is one of the widespread parasitic diseases that are threatening human health.^1-3 According to the statistics of World Health Organization(WHO), the disease threatened about 5.6 billion people in 76 countries.Some diagnostic methods,such as counter immunoelectrophoresis(CIE), enzyme-linked immunosorbent assay(ELISA),indirect fluorescence immunoassay(IFIA),radiate immunoassays(RIA),etc.,have been adapted to clinical analyses.These methods are,however,of either low sensitivity,or time consuming and complicated entailing expensive instrumentation.Therefore,exploring improved alternatives for Schistosomiasis diagnosis is of considerable significance.In this paper,different methods were used to detect schistosoma japonicum antibody(SjAb),such as electrochemical immunoassay and chemiluminescent enzyme immunoassay.Biocatalytic deposition was also applied in the electrochemical immunoassay as an amplifying method.The works been done are as follows:In the first part:An ultrasensitive electrochemical immunoassay based on biocataiytic deposition has been proposed for the detection of Schistosoma japonicum antibody(SjAb) in infected rabbit serum. Schistosoma japonicum antigen(SjAg) was first immobilized on the gold electrode surface via glutaraldehyde crosslink,then incubated with the infected rabbit serum containing SjAb,and finally the goat anti-rabbit IgG labeled with alkaline phosphatase was sandwiched to form the immunocomplex on the gold electrode surface.The alkaline phosphatase convert nonelectroactive substrate into the reducing agent and the latter, in turn,reduce metal ions to form electroactive metallic product on the electrode surface.Linear sweep voltammetry(LSV) was used to quantify the amount of the deposited silver and give the analytical signal for SjAb. Scanning electron microscope(SEM) was applied to characterize the silver nanoparticles before and after silver deposition on the electrode surface.Assay conditions such as the antigen concentration and enzymatic silver deposition time were optimized.Under the optimum conditions of immunoreaction and electrochemical detection,the electrochemical immunosensor was able to realize a reliable determination of SjAb in the dilution range from 1:5000 to 1:100 with a detection limit of 1:6457 dilution ratio.The feasibility of the proposed immunosensor for possibly clinical applications was also investigated by analyzing real serum samples.In the second part:A novel chemiluminescent enzyme immunoassay based on magnetic nanoparticles(MNPs) has been developed for highly sensitive and rapid detection of schistosoma japonicum antibody(SjAb) in infected rabbit serum(IRS).Schistosoma japonicum antigen(SjAg) was immobilized onto MNPs using the N-(3-dimethylaminopropy)-N'-ethylcarbodiimidehydrochloride (EDC) and N-hydroxysuccinmide (NHS).After reacting with SjAb,the horseradish peroxidase-conjugated goat anti-rabbit IgG(HRP-IgG) was bound to the surface of nanoparticles. Followed by the separation using a magnet and washing,luminol was then added to the antgen-antibody complex and the detection of luminescent intensity was carried out.Assay conditions were optimized, including the concentration of SjAg used for labeling,the amount of SjAg-MNPs,the reaction time of SjAg with SjAb,and the concentration of HRP-IgG.A good linear relationship was obtained between the luminescent intensity and the dilution ratio in the dilution range of 1:10000 to 1:100 with a detection limit of 1:13442 of dilution ratio. In the third part:An electrochemical immunoassay based on horseradish peroxidase(HRP) has been proposed for the detection of Schistosoma Japonicum Antibody(SjAb) in rabbit serum.Schistosoma Japonicum antigen(SjAg) was first immobilized on the gold electrode surface via glutaraldehyde crosslink,then incubated with the infected rabbit serum containing SjAb,and finally the goat anti-rabbit IgG labeled with horseradish peroxidase was sandwiched to form the immunocomplex on the gold electrode surface.Assay conditions such as the antigen concentration and horseradish peroxidase labeled second antibody concentration were optimized.Under the optimum conditions of immunoreaction and electrochemical detection,the electrochemical immunosensor was able to realize a reliable determination of SjAb in the dilution range from 1:10⁶～1:10².