Development Of ELISA Methods To Detect Antibodies Against Parvovirus B19 In Human Serum

The parvovirus B19 is an erythrovirus included in the family of Parvoviridae. It can be transmitted through respiratory route or by blood-derived products; and it can also be transmitted vertically from mother to the fetus. For healthy immunocompetent people, B19 may cause erythema infectiosum in children, arthritis or joint disease among the grown-ups. For immunocompromised hosts, B19 infection will manifest as pure red cell aplasia or chronic anemia. Pregnant woman who was infected with B19 would get an increased risk of hydrops fetalis or even fetal death in utero.It is necessary to develop a serological detection method of B19 infection since such methods are still absent in China.In this study, we attempted to express and purify B19 VP2 protein firstly, and then use it as the detection antigen to establish the enzyme-linked immunosorbent assay (ELISA) methods to determine IgG or IgM against B19 in human serum.1 Preparation of B19 VP2 proteinObjective: To express B19 capsomer protein VP2 using prokaryotic or eukaryotic expression system. Methods: Codon-optimized VP2 gene was synthesized with overlap-extension PCR method, and then subcloned into pET30a(+) or pET44a(+) prokaryotic expression vector. Isopropyl-β-D-thiogalactoside (IPTG) in various concentrations was added to the culture system to induce the expression of VP2. Ion exchange chromatography (IEX) or immobilized metal ion affinity chromatography (IMAC) were employed to purify the VP2 protein. The methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were used to evaluate the result of purification; New Zealand white rabbit was immunized with purified VP2 protein to produce anti-VP2 IgG. Adenoviral expression sytem was chose to express VP2 in eukaryotic cells. VP2 gene was subcloned into the pShuttle-CMV plasmid. The resulted plasmid was co-eletroporated with pAdEasy-1 into E.coli BJ5183 strain to form the adenoviral plasmid. Recombinant adenovirus was rescued and amplified in 293 cells. SDS-PAGE and Western blot were employed to evaluate VP2 expression in recombinant adenovirus-infected 293 cells. Results: VP2 coding sequence was synthesized and confirmed by seqencing. Four kinds of prokaryotic expression vectors were successfully constructed. The results of SDS-PAGE indicated that VP2 protein was expressed in the form of inclusion bodies. After purifying the VP2 protein with ion exchange chromatography (IEX) under denaturing condition, soluble VP2 protein was obtained by refolding. Rabbit anti-B19-VP2 IgG was purified from serum and labelled by horseradish peroxidase. On the other hand, recombinant adenovirus carrying B19 VP2 gene was successfully constructed. However, the quantity of VP2 expressed in 293 cells is too low to form VLPs. Conclusion: Purified soluble VP2 protein was obtained through prokaryotic expression. Rabbit anti-B19 VP2 IgG was prepared and labelled with HRP, which makes it possible to establish ELISA methods to detect the antibodies against B19 virus in human serum.2 Establish ELISA methods to detect anti-B19 virus antibody in serumObjective: To establish ELISA methods to detect IgG or IgM antibody against B19 virus in human serum. Method: For IgG detecting ELISA, commercial human IgG was used as an antigen to coat microtiter plate to determine the appropriate dilution of HRP-conjugated anti-human IgG. Thereafter, the concentration of VP2 antigen and dilution of human serum was optimized with checkerboard titration method. Indirect ELISA was then developed by coating VP2 antigen on microplate to detect anti-B19 IgG. For IgM detection, we attempted to Establish capture ELISA. Commercial anti-human IgM antibody was coated on microplate, and then diluted human serum was added. After washing, soluble VP2 protein was added to the microplate. After further washing, HRP-labeled anti-VP2 antibody was used to detect the absorbed VP2. Finally, 355 human serum samples were subjected into anti-B19 IgG or IgM detection. Results: For IgG-detection ELISA, the optimized procedure was as following: use VP2 with a concentration of 5μg/ml to coat microtiter plate; block it with PBS plus 5% NCS for 2 hours; incubate with 1:100 diluted human serum at room temperature for 1 h; after washing, add 1:10000 diluted HRP-labeled goat anti-human-IgG; after further washing, add TMB for color development. Anti-B19 IgG was 54.1% positive among 257 samples. For IgM detection ELISA, the optimized procedure is to use 5μg/ml rabbit anti-human-IgM to coat the microtiter plate, 5μg/ml VP2 protein and 1:6000 diluted rabbit anti-human-B19 VP2-HRP. 4.1% were anti-B19 IgM positive among 172 samples. Conclusion: ELISA methods were established for parvovirus B19 antibody-detection (IgG or IgM) in human serum.In conclusion, we synthesized the codon-optimized VP2 gene and cloned it to the prokaryotic expression vector. Soluble VP2 protein was prepared and purified, and ELISA methods to detect anti-B19 IgG or IgM was finally established. Evaluation of the reliability of these methods is under way.