Development of In-vitro Assays to Estimate Rabies Viral Protein in Vaccine Preparation

Abstract

Vaccine against rabies is prepared by cell culture technology and these vaccines are free from many side effects when compared to nerve tissue vaccines. The vaccine production is a continuous process involving propagation of virus, harvesting, concentration, inactivation, purification and formulation with preservatives. The quantification of viral protein in the intermediate biological product is an in-process quality control test to reduce the product loss during various process of vaccine manufacturing. The conventional in-vivo & in-vitro tests employed for the quantification of rabies viral protein are time consuming, laborious and requires laboratory animals. In this study, we attempted to develop in-house serological methods such as sandwich ELISA, Dot Blot for the detection and quantification of rabies antigen in the intermediate biological material during vaccine preparation. The hyper immune sera was prepared by immunizing two animal models i.e. Guinea Pigs and rabbits with standard Rabies antigen. The sera samples were purified by saturated ammonium sulphate precipitation and further by G50 gel column. The antirabies antibody titre in the purified preparation was estimated using Rapid Fluorescent Focus Inhibition Test (RFFIT). National Reference Rabies Vaccine received from Central Drug Laboratory, Kasauli was used to prepare the local reference standard and it was included in the in-house serological methods to validate the assay. Our in-house tests are found to be simple, rapid and cost effective and require less time when compared to in-vivo animal challenge and cell culture based in-vivo tests.

Rabies vaccine purification using HPLC, Estimation of residual vero cellular DNA by RT-PCR and potency analysis

“…The dot blot hybridization was performed to detect and quantify the rabies viral protein in the purified preparation by in-house standardized method [14].…”

“…The height of the specific HPLC peak is directly proportional to the concentration of the viral protein in the sample. Further Dot blot based quantification of rabies viral protein is more suitable method for the quantification of rabies viral protein because the reaction involves with rabies antigen with specific antibody [14]. In every step of purification the HPLC and Dot Blot methods were used for rabies viral protein detection and quantification.…”