The Establishment And Application Of Sandwich ELISA For Detection Of Prdx6 From Sheep(Ovis Aries)

Peroxiredoxin 6(Prdx6) has been confirmed that widely exists in cytoplasm of prokaryotic and eukaryotic organisms. The members of that family have a variety of biological functions, for example, related with cell proliferation and differentiation, the protection of free radicals from sensitive proteins, enhanced the activity of NK cell, mediated signal conduct of mammalian cells,etc. Using thioredoxin to reduce peroxidase and superoxide and eliminate peroxide produced by metabolism is the main function.Studies have shown that when pathogens enter the living organism, phagocytes could swallow them and lead to the generation of ROS aiming to kill pathogens. Most of pathogens can be cleared by this proces, but there will still be some intracellular parasites escaped by regulating the expression of peroxiredoxin. It has been reported that the escaping mechanism is due to the expression of Prdx6 makes ROS inactive, thus reducing oxidative damage caused by ROS. This mechanism could be an important reason that intracellular parasites could survive in the body’s immune response. In addition, in recent years, studies also find that except antioxidant effect,Prdx6 is also associated with diabetes, cancer, brain and lung disease, and a variety of cytokine secretion and immune injury, so Prdx6 could be a biomarker molecules for some disease diagnosis.Therefore, the establishment of an accurate and rapid detection method for detecting Prdx6 could provide an effective tool for assessing the health of the body and the level of oxidative stress capacity.Brucellosis is a zoonotic diseases which could endanger public safety seriously, this disease is widely distributed throughout the world, the incidence is increasing in our country, additionally,it poses a great threat to human health and livestock breeding. Therefore, establishing a rapid and accurate diagnostic tests for brucellosis has great significance for the prevention and treatment of brucellosis effectively. Based on previous studies, the gene expression profiles in sheep leukocyteis different between the virulent strain group and the vaccination group, Real-time PCR analysis showed that: compared with healthy group, Prdx6 is upregulated significantly in virulent strain group,gene expression level of Prdx6 in vaccination group is dissimilar with virulent strain group.It can be inferred, Prdx6 may have some contact with Brucella intracellular parasitism and proliferation.In this study, we prepared r Oa Prdx6 with high concentration and purity, anti-Prdx6 with high titer and specificity, which can be used in ELISA. The result shows that the highly specificity of the sandwich ELISA which had high sensitivity,detection range is 8 ng/m L ~ 500 ng/m L, the standard curve equation: y = 1.3349x-0.7978, the correlation coefficient: R2 = 0.9946, the recovery rate is 94.84% ~ 102.9%, The lowest detection limit was 5.95 ng/m L; the double-antibody sandwich ELISA method has good repeatability. According to optimized ELISA detection method,standard protein, antibody detection, sample dilution, concentrated washing liquid and other ingredients have been prepared to assembled the sandwich ELISA kit. This kit has highly sensitivity, detection range is 8 ng/m L ~ 500 ng/m L and excellent repeatability, there is no difference between the same batch and different batches. The 37 ℃damage experiments show that this kit could be stored at 4 ℃ for more than one year. The clinical samples have been detected by the established ELISA and the results were statistically analyzed.We found that the diagnostic of Prdx6 is 31.9 ng/m L between brucellosis vaccine and unvaccinated sheep. At this concentration, the sensitivity is 0.711, specificity is 0.739, the maximum of Youden index is 0.45.Because of the limited of clinical practice sample source, virulent infected, vaccinated and unvaccinated sheep clinical samples needs further accurately collection. therefore, whether this ELISA, which we have established, could be used to identify the difference between brucellosis vaccination and natural infection needs further research. But, this sandwich ELISA is able to detect the content of Oa Prdx6 from sheep leukocytes with highly sensitivity and specificity. The ELISA kit has highly sensitivity and reproducibility,it could be stored for more than one year in4 ℃ and used for scientific research and applications.