Generation of FMDV Recombinant 3ABC Protein and Development of Monoclonal Antibodies as Reagents for 3ABC Quantitative ELISA

Order of Publishing in Issue: 
1
Volume :13
Issue :1
January, 2019 - March, 2019
Page No: 
07-19
Authors: 
Anil Kumar Jangam[1,2], Sridevi V Nimmagadda[1], Madan Mohan M, Ravi R Varma, Shukra Madhaha A[1], Praveen Alagangula[1], Premalatha Dasari[2], Lingala Rajendra[1]*
Address: 
[1]Indian Immunologicals Limited, Rakshapuram, Gachibowli, Hyderabad- 500032, Telangana, India
Address: 
[2]Department of Biotechnology, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad- 500085, Telangana, India

FMDV is one of the most contagious disease in cloven hoofed animals and it is known to cause lameness, vesicular lesions on tongue, feet, teats and snout with reduced milk production, loss of weight and high mortality in young animals. Many countries being endemic, continuous vaccination might help in attaining disease free status. The vaccination programme with a marker vaccine can establish differentiation among vaccinated and unvaccinated animals by eliciting immune response against the viral structural proteins and not against non-structural proteins (3ABC). Vaccine manufacturers should ensure the limited NSP content in their vaccine formulations, so that there will be no immune response against nonstructural proteins. This can be evaluated by determining the concentration of NSP (3ABC) in the vaccine antigen batches. In the present study, we have developed a recombinant 3ABC antigen with minor modification at the 3Cpro catalytic site and cloned the amplification products into bacterial vector for expression. The bacterial expressed purified protein was used for the generation of monoclonal antibodies. The selected monoclonal antibodies were characterized against NSP antigen. The screening of 3ABC mAb towards antigen was performed by ELISA. The specificity of the mAb was established by its non-reactivity towards other sera samples by indirect ELISA (Enzyme linked Immunosorbent assay) and competitive ELISA. To identify the mAb binding epitope site on FMDV 3ABC, Phage display dodecamer peptide library was used and selected the peptides based on the binding activity by Phage ELISA. Further analysis of the peptide sequence revealed the presence of four amino acids (QPKL) corresponding to 3ABC region of FMDV genome. We have further shown that the non-competing mAbs were used to develop immunocapture ELISA for the detection of NSP content in in-process samples during manufacture of the vaccine. This ELISA can be adapted to measure the NSP content in the final purified bulk antigen.The mAb based ELISA has the potential to use as a quality control in vaccine production.

Keywords: 
Foot and mouth disease, Monoclonal antibody, Vaccine, ELISA, Non-structural proteins.
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