Development of a Blocking ELISA Using a Monoclonal Antibody to a Dominant Epitope in Non-Structural Protein 3A of Foot-and-Mouth Disease Virus, as a Matching Test for a Negative-Marker Vaccine.

Yuanfang Fu,Xueqing Ma,Xincheng Ji,Dong Li,Pinghua Li,Yingli Chen,Qian Shi,Xingwen Bai,Zaixin Liu,Yimei Cao,Huifang Bao,Zengjun Lu,Jing Zhang,Pu Sun,Na Wang,Joanne M Devlin

doi:10.1371/journal.pone.0170560

Abstract

Foot-and-mouth disease (FMD) is a devastating animal disease. Strategies for differentiation of infected from vaccinated animals (DIVA) remain very important for controlling disease. Development of an epitope-deleted marker vaccine and accompanying diagnostic method will improve the efficiency of DIVA. Here, a monoclonal antibody (Mab) was found to recognize a conserved “AEKNPLE” epitope spanning amino acids 109–115 of non-structural protein (NSP) 3A of foot-and-mouth disease virus (FMDV; O/Tibet/CHA/99 strain), which could be deleted by a reverse-genetic procedure. In addition, a blocking ELISA was developed based on this Mab against NSP 3A, which could serve as a matching test for a negative-marker vaccine. The criterion of this blocking ELISA was determined by detecting panels of sera from different origins. The serum samples with a percentage inhibition (PI) equal or greater than 50% were considered to be from infected animals, and those with <50% PI were considered to be from non-infected animals. This test showed similar performance when compared with other 2 blocking ELISAs based on an anti-NSP 3B Mab. This is the first report of the DIVA test for an NSP antibody based on an Mab against the conserved and predominant “AEKNPLE” epitope in NSP 3A of FMDV.

Highlights

Foot-and-mouth disease (FMD) is a highly contagious viral disease that affects many clovenhoofed animals
A monoclonal antibody (Mab) against non-structural protein (NSP) 3A was produced by traditional methods [15], briefly, the prokaryotic-expressed NSP 3A used as an immunogen to immunize female Balb/c mice, the spleen cells of the mouse with the highest antibody were used to fuse with SP2/0 cells to preparation of hybridoma cell
The results of an indirect ELISA showed that 3A3 and 3A24 bound to recombinant proteins His-3A, His-3ABC, and His-2C3AB; 3A2 only bound to recombinant proteins His-3A and His-2C3AB, but not His-3ABC; none of the Mabs reacted with His-3B or His-3D; the supernatants from the Sp2/0 cell did not react with any recombinant proteins (Fig 1A)