Development Of A Rapid Immunochromatographic Strip For The Detection Of Orf Virvs

Orf is a mucocutaneous, highly contagious disease of sheep and goats, which ischaracterized by maculopapular and proliferative scabby lesions in the less hair areaaround the lips, breast and groin. As the causative agent of the disease, ORFV isthought to be highly “epitheliotropic”. In addition, ORFV with strong resistance canlead to the repeated infection of flocks for many years. The conventional methodsused for the diagnosis of ORFV includes virus isolation, negative staining electronmicroscope observation, ELISA, PCR, Real-time quantitative PCR, LAMP,immunofluorescence and so on. However, these methods have some disadvantagessuch as time-consuming, laborious, requiring professional and technical personnel andspecial test equipment for clinical detection. Thus, the applications of the abovementioned methods were limited in clinical detection. At present, it is necessary todevelop a simple, fast and convenient method for the diagnosis of ORFV.In this study, ORFV particles were purified by sucrose linear density gradientcentrifugation. The immunization of Balb/c mice were performed using the purifiedparticles in accordance with conventional procedures. The splenic mononuclear cellsof the immunized mice were isolated and fused with murine myeloma cells(SP/20).The hybridoma cells were selected using indirect enzyme-linked immunosorbentassay.The three single-secreting anti-ORFV McAb were observed, which named5A5,6F2and7A3, respectively.The antibody titers of three McAbs between1:12800and1:51200. The identification of antibody subclasses showed that the three monoclonalantibodies were IgG1. The chromosome number of the three hybridoma cells werebetween92and103.The three monoclonal antibodies recognized ORFV-B2L proteinby Western blot and ELISA analysis. By indirect immunofluorescence detection, thethree monoclonal antibodies could be combined with ORFV.The three monoclonalantibodies only reacted specifieally with the ORFV and don’t reacted with othercommon virus antigen. In addition, the rabbit anti-ORFV polyclonal antibodies were successfully prepared.The colloidal gold rapid diagnostic testing for ORFV were prepared using theanti-ORFV monoclonal and polyclonal antibodies. The colloidal gold particles wereprepared using sodium citrate reduction method. The size of gold particles was about30nm by theelectron microscopy observation. The optimal pH value of the colloidalgold particles combining with5A5monoclonal antibodies was determined. And theoptimal combining concentrations was40.6μg/mL. The colloidal gold-labeled MAb5A5solution was dispensed onto glass fiber paper and the polyclonal antibody or thegoat anti-mouse antibody was dispensed at the test or the control line on the NCmembrane. The results showed that the lowest detectable limits was6.25μg/mL ofthe ORFV, and no cross-reaction with SPPV, VSV, FPV, FMDV. The strips couldstore four months in the condition of4℃or room temperature.The165samplessuspected ORFV infections were detected using the rapid immunochromatographicstrip for the dignosis of Orf virus. The positive rate was20.6%. Comparing to PCRand LAMP method, the coincidence were respectively94.4%and89.5%. The aboveresults showed that the rapid immunochromatographic strip for the dignosis of Orfvirvs had good specificity, high sensitivity, good reproducibility and stabilityadvantages. Due to simple and fast, it could be widely used clinical detection.