Development Of Magnetic Microbead-based Enzyme Immunoassay For Detection Of Schistosoma Japonicum Antibody In Human Serum

Schistosomiasis japonica, a disease caused by small parasitic blood flukes, has afflicted humankind for thousands of years. It is endemic mainly in Southeast Asia. Diagnosis is a key step in the control of schistosomiasis. Present diagnosis methods can be mainly divided into direct parasitological techniques and indirect immunodiagnostic techniques. The microscopic examination of excreta remains the gold standard for the diagnosis of schistosomiasis, but it is time-consuming and less cost-effective.And immunodiagnostic assays have relatively high false negatives and poor sensitivity, especially in patients with light infections.Magnetic microbead-based immunoassay (MEIA) was first developed in the l980s. The main advantages of bead-based immunoassay include increasing the surface area for immobilization of antigen or antibody, reducing the incubation time and increasing sensitivity. At present, magnetic bead-based immunoassay has been widely used in disease diagnosis field. However, to our knowledge, few studies have been focused on the application of magnetic bead-based immunoassay in Sj-Ab detection area.The purpose of this research is to develop magnetic bead-based immunoassay for detection of Sj-Ab in human serum with high sensitivity and high accuracy to prevent false negatives.In this reaseach, the performance of two different kinds of domestic magnetic beads was compared; blocking condtions of magnetic beads were optimized, and coupling technology was studied. FITC was used to label soluble egg antigen (SEA), anti-FITC monoclonal antibody was coated to magnetic beads by using 1-Ethyl- 3-(3- dimethyl lam- inopropyl) carbodiimide hydrochloride(EDC), and goat anti-human IgG was labeled with Alkaline Phosphatase (AP) using Succinimidhyl 4-(N-maleimidomethl) cyclohexane-1-carboxylate(SMCC) in the optimized conditions. FITC-labeled soluble egg antigen (SEA) and polymer-coated magnetic beads, to which anti-FITC monoclonal antibodies were immobilized, were used as separation support in MEIA. Immunoassay parameters were optimized based on a direct immunoreaction of SEA on the magnetic microbead and Sj antibody in serum samples. The laboratory experimental results showed that MEIA method was more sensitive and more precise than traditional ELISA. In the field test, human sera collected from 513 infected humans and 2260 uninfected humans were tested with indirect haemagglutination assay (IHA), dipstick dye immunoassay (DDIA) and MEIA. IHA and DDIA were then compared with MEIA and the lower false negative rate (0.97%) was obtained. With the high sensitivity and low FNR, MEIA could offer a rapid and reliable diagnosis. We conclude that MEIA is a new tool for the diagnosis of schistosomiasis, especially for large scale screening in the field with human populations.