Development&Preliminary Application Of Direct Competitive ELISA For Monitoring Rabies Antibody In Canine

Rabies virus（RABV）is the typical member of the zoonotic viruses, belonging to genusLyssavirus of family Rhabdoviridae which is responsible for fatal encephalitis in animals andhumans. Once the symptoms start appearing, the mortality rate is almost100%. Over60,000human deaths occur globally, most of which in Africa and Asia. Almost all warm-blooded animalscan be infected by rabies virus. However, stray dogs represent the major reservoir of RABV inChina. More than95%cases of human rabies occurred due to the bite or licking on wounded skinby the rabid dogs.According to the current World Health Organization (WHO) and World Organization forAnimal Health (OIE), it is generally accepted that a protective response is related to the titer ofvirus-neutralizing antibody higher than0.5IU/mL. We can effectively control the spread of rabiesby immunizing70%of animals. Thus, enhancing the vaccination of animals and monitoringantibody levels can effectively prevent the immune failure and leak-free phenomenon. All of thesefactors have an important significance to control the spread of rabies. The rapid fluorescent focusinhibition assay (RFFIT) and the fluorescent antibody virus neutralization (FAVN) arerecommended by WHO and OIE which can quantify the neutralizing antibodies. However,performance of these tests needs viable rabies virus and cell, and requires suitable bio-containmentfacilities. These procedures take multiple days to complete, making the process highly inefficientand difficult to conduct in a large serum. Enzyme-linked immunosorbent assays (ELISA) can notonly avoid the above mentioned disadvantages, but also achieve rapid detection of large samples.Horseradish peroxidase (HRP) own its popularity as labels for ELISA conjugates to its lowmolecular weights and maintaining stability by chemical modification. In this article, the IgG wasisolated from canine serum by ammonium sulfate precipitation (ASP) followed by Protein Aaffinity chromatography, then identified the purity by PAGE electrophoresis. HRP was labeled toIgG by oxidation using sodium iodide, which acts as a competitive enzyme-labeled antibody indirect competitive ELISA (c-ELISA) assay to quantify the antibody titers of sample serum, so asto avoid non-specific binding in the indirect ELISA when detecting the antibodies.Purification of anti-rabies virus IgG in canine serum: The anti-rabies virus IgG in canineserum was firstly purified by a modified ASP, and then further purified by Protein A affinitychromatography. SDS-PAGE and ELISA analysis indicated that the recovery of IgG after ASP was 96%, titer was38×102, and Protein A affinity chromatography was39×102.Conjugation of HRP to IgG and selection of the optimal working concentration: The purifiedIgG and HRP was conjugated by sodium iodide oxidation method, and then obtained afterprecipitation by45%ASP. The precipitation was detected by UV, whereas the combined rate was39%. The optimal reaction conditions in ELISA determined by phalanx were as follows: optimalantigen coating concentration was50μ g/mL, optimum dilution of HRP labeled IgG was1:1000.There was no cross-reactivity between the labeled antibody and the susceptible pathogens incanine.Foundation of the standard curve which can be use in antibody detection by c-ELISA: Thestandard curve of serum antibody titers and the absorbance was established by c-ELISA aftermaking two-fold serial dilutions of the standard positive serum. The linear regression equationwas Y=－6.387X＋5.617, R2=0.940. The detected limit was0.25IU/mL and the detected rangewas0.25-4IU/mL. Monitoring the antibody titer in serum samples by c-ELISA and FAVN findingthat there was no significant difference (P=0.092, P>0.05). Precision detection revealed that theintra-assay and inter-assay standard deviation was4.76%and5.89%, respectively.