| Foot-and-mouth disease (FMD) is a highly contagious disease in cloven-hoofed animals and the most economically important disease of livestock all over the world. Outbreaks of FMD always evoke severe economic and social effects. Vaccination is a key strategy for the prevention and control of FMD in many countries, especially in developing countries. To validate the efficacy of the vaccine, a rapid and simple test is needed in routine field practice to monitor antibody titers against FMDV induced by vaccines. Serological tests used most commonly for FMD antibody determination are virus neutralization test, liquid-phase blocking enzyme-linked immunosorbent assay (LPBE) and solid-phase competition ELISA (SPCE). Although these assays provide accurate and sensitive detection of anti-FMDV antibodies, they require specialized equipments and technical expertise. As intact virus is involved in these tests, there is the risk of incomplete inactivation and escape of virus from laboratories. So, it is very important to develop an assay with the merits of safety, simplicity and rapidity. In this study, 3 monoclonal antibodies binding neutralization site of FMDV type O were produced with the expressed protein VP1 and synthetic peptides as immunogen and reactionogen respectively. And an immunochromatographic strip was developed for the serological detection of type FMD O in swine. In the strip, synthetic peptide conjugated to bovine serum albumin (BSA-Pep2) was labelled with colloidal gold as a detector, the staphylococcal protein A (SPA) and IgG extracted from monoclonal antibody were blotted on the nitrocellulose membrane for the test and control line, respectively. This technique has several advantages over traditional immunoassays, such as simple procedure, rapid operation, immediate results, low cost and no requirements for skilled technicians or expensive equipments. Because of these characteristics, the immunochromatographic test strip is suitable for on-site detection of FMDV antibodies. 1. Prokaryotic Expression and analysis of O-type Foot-and-mouth Disease Virus VP1 GeneThe gene fragment for VP1 (639 bp) was amplified by PCR from the recombinant plasmid pMD18T-O-VP1 and ligated into the pET-32a vector. The ligated plasmid was transformed to E. coli BL21 competent cells. After inducing by 0.5 mM Isopropyl-β-D-thiogalactopyranoside (IPTG) for 5h, the E.coli BL21 (DE3) carrying pETVP1 plasmid was found to express a recombinant protein with a molecular weight around 45 kDa analysized by SDS-PAGE, after sonication and centrifugation, indicating that the expressed protein existed mainly as insoluble aggregates. After purifing by Ni-chelation chromatography, the inclusion bodies were denatured and refolded by 8 M guanidine buffer and refolding buffer, respectively. Western-blot and indirect ELISA revealed that the renatureed protein can bind swine O-type FMDV positive serum with high specificity. It will lay the foundation for the production of FMDV monoclonal antibody and development of antibody detection methods.2. Synthesis and analysis of pig FMDV serotype O specific epitope peptidesFive FMDV serotype O specific epitope peptides were synthesized by 12-Symphony Quartet peptide synthesizer according to Fmoc solid phase synthesize principle. Molecular Characteristics analysized by Quattro Micro LC-MS indicated that all of the 5 peptides were synthesized successfully. Peptide-BSA conjugates were analysized by Dot-blot and indirect ELISA, and the results showed that Pep1, Pep2 and Pep3 can bind to both FMDV type O infected sera and immunized sera specifically. It provides materials for the development of immunochromatographic strip for the detection of FMDV antibody and the preparation of FMDV monoclonal antibody.3. Preparation and characterization of monoclonal antibody against neutralization site of FMDV serotype OThree hybridoma lines of 2C-A9, 3D-A11, 5G-E11 were screened with expressed protein VP1 and synthetic peptide (BSA-Pep2) as immunogen and reactionogen respectively. The indirect ELISA titers of them were 1:2.56×10~2, 1:6.4×10~2, 1:3.2×10~2 in cell supernatant; 1:1×10~5, 1:1×10~6, 1:1×10~4 in ascites, 1:1.28×10~5, 1:1.02×10~6, 1:512×10~5 in purified mAb IgG. WestGold results showed these mAbs can bind expressed protein VP1 and synthesized peptides specifically with no cross-reaction with BSA and peptide control. All of the 3 mAbs can react with FMDV serotype O determined by liquid phase blocking ELISA and sandwich ELISA. mAbs obtained were to establish immunoassay for the rapid detection of FMDV antibody.4. Development and evaluation of an immunochromatographic strip for the detection of antibodies against foot-and-mouth disease virus serotype OAn immunochromatographic strip was developed for the serological detection of type O foot-and-mouth disease (FMD) in swine based on the principle of gold immuno-chromatography assay (GICA). In this strip, the BSA-pep2 conjugates labelled with colloidal gold was used as the detector, 1.0 mg/mL staphylococcal protein A (SPA) and 0.54 mg/mL purified mAb IgG against neutralization site of FMDV serotype O were blotted on the nitrocellulose membrane for the test and control line, respectively. The sensitivity of the strip to vaccinated pig serum after immunizing 8 days with FMDV peptide vaccine for 8 days reached to 1:12800. Specificity tests showed that there was no cross-reactivity with other pathogenic antibodies, such as CSF, PRRS, SVD, PR and PPI. Cross-reaction of the immunochromatographic strip with antibodies against different serotypes of FMD was determined by measuring the test responses to type O, A, Asia1 positive samples. The results showed all the strips tested with type O anti-sera gave a clear positive. However, other antisera gave no visible line in the test zone. Immunization and field experiments indicated that the new assay gave a good correspondence result with commercial ELISA kit. This suggests that the immunochromatographic strip is an acceptable alternative for using in clinical laboratories lacking specialized equipment and for field diagnosis.
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