Immuno-affinity Purification of Insect Cell Expressed Rabies Virus Glycoprotein using a Conformational Specific Monoclonal Antibody

Rajendran Ramya; Shanmugam Rajalakshmi; Bhaskaran Mohana Subramanian; Saripalli Umamaheswara Rao; Krothapalli R.S. Sambasiva Rao; Srinivasan Alwar Villupanoor

Immuno-affinity Purification of Insect Cell Expressed Rabies Virus Glycoprotein using a Conformational Specific Monoclonal Antibody

2012

.Rabies is a disease of nervous system and causes progressive encephalitis with fatal outcome. The conformation-dependent epitopes on the glycoprotein (G) of rabies virus (RV) is responsible for the induction of virus neutralizing antibodies which is ultimately required to get complete protection from viral challenge. Therefore, a suitable chromatography technique is necessary to purify the tag free recombinant rabies virus glycoprotein (rRVG) without altering its immunogenic epitopes. The present study was undertaken to purify the rRVG using a conformational specific anti-rabies virus glycoprotein (RVG) mAb, M5B4, which binds to the natively folded G. The mAb had shown a significant kinetic interaction with RVG. The mAb immobilized onto the NHS-activated Sepharose 4 fast flow™ was used for the purification of rRVG by immuno-affinity chromatography (IAC). The bound rRVG was eluted in IAC using 0.1M glycine with pH 2.5 and the identity of the purified protein was confirmed by MALDI-TOF. The IAC purified rRVG induced neutralizing antibody response and 83% of the immunized mice were protected against intra-cerebral rabies virus challenge. The results indicate that the mAb based IAC method can be an effective purification technique for tag free rRVG with significant level of purity, without compromising the protein’s immunogenic potential.

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