Purificación y caracterización de las proteínas no capsidales del virus de la Fiebre Aftosa y obtención de anticuerpos policlonales: Su implicación en el control de calidad de la vacuna anti-Aftosa.

Abstract

Non-capsid proteins (PNC) of the foot-and-mouth disease virus are used as markers in the evaluation of animals that have been in contact with the virus, unlike those immunized, which should not present antibodies against these proteins, because in The vaccination process must be eliminated according to international regulations. The objective of the present grade work was to look for the best purification method for a non capsidal FMD virus protein and also induce the production of its polyclonal antibodies in vivo and monitor its immunodetection, for the realization of a standard curve to serve as a reference in an ELISA immunoenzymatic test. Isolation and purification was done from the inactive virus culture, by ion exchange chromatography. The elution of the peaks was subjected to electrophoresis for subsequent identification by mass spectrophotometry; the proteins found in the process were those belonging to the host (Rodentia), bovine albumine, the capsid protein VP3 and the non-capsidal protein 3D 3B in complex and this same 3D in its pure state in the serotype VA24C. Demonstrating the latter have a high degree of purity (> 95%) at peak three of the chromatogram; Its identification was made by the ion trap electro-spray technique. The purified 3D protein was inoculated in goats and the hyperimmune serum was precipitated and subjected to affinity chromatography to obtain immunoglobulins (IgG); the immune reaction was confirmed by immunodiffusion, Western blotting and an immunoenzymatic test that confirmed the degree of affinity between the antigen and the purified antibody. With the latter, a standard curve was made with each of the replicas showing a linear trend, with an R = 0.942 and a very low standard deviation, which allowed validating an initial model, so that in future experiments the specificity can be established and sensitivity of the method, and thus manage to measure the remaining remnants of the NCPs in each of the stages of vaccine production, which would ensure before evaluating it by traditional indirect methods, to have a high quality product without contaminants.