Development of a Blocking ELISA Based on a Monoclonal Antibody against a Predominant Epitope in Non-Structural Protein 3B2 of Foot-and-Mouth Disease Virus for Differentiating Infected from Vaccinated Animals

Abstract

A monoclonal antibody (McAb) against non-structural protein (NSP) 3B of foot-mouth-disease virus (FMDV) (3B4B1) was generated and shown to recognize a conserved epitope spanning amino acids 24–32 of 3B (GPYAGPMER) by peptide screening ELISA. This epitope was further shown to be a unique and predominant B cell epitope in 3B2, as sera from animals infected with different serotypes of FMDV blocked the ability of McAb 3B4B1 to bind to NSP 2C3AB. Also, a polyclonal antibody against NSP 2C was produced in a rabbit vaccinated with 2C epitope regions expressed in E. coli. Using McAb 3B4B1 and the 2C polyclonal antibody, a solid-phase blocking ELISA (SPB-ELISA) was developed for the detection of antibodies against NSP 2C3AB to distinguish FMDV-infected from vaccinated animals (DIVA test). The parameters for this SPB-ELISA were established by screening panels of sera of different origins. Serum samples with a percent inhibition (PI) greater than or equal to 46% were considered to be from infected animals, and a PI lower than 46% was considered to indicate a non-infected animal. This test showed a similar performance as the commercially available PrioCHECK NS ELISA. This is the first description of the conserved and predominant GPYAGPMER epitope of 3B and also the first report of a DIVA test for FMDV NSP 3B based on a McAb against this epitope.

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

“…This method can be used as a matching test for the marker vaccine made from the FMDV with deletion of residues 109 to 115 in the NSP 3A. A good overall concordance was observed between the 3A-Mab-bELISA and previously developed 3B-Mab-bELISA [12] and commercially available PrioCHECK ® FMDV NS ELISA. Therefore, the development of a blocking ELISA targeted to the conserved “AEKNPLE” epitope of NSP 3A will provides a suitable companion test for the marker vaccine to differentiate infected from vaccinated animals.…”

“…A polyclonal antibody against FMDV the NSP 2C epitope regions was produced by immunization of two rabbits with the purified prokaryotically expressed NSP 2C epitope region, as described previously [12]. A Mab against 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 Mabs were isotyped using Mouse Monoclonal Antibody Isotyping Kit (Sigma, USA). The reactivity of Mabs with different recombinant NSPs (3A, 3B, 3ABC, 2C3AB, and 3D) was determined by an indirect ELISA [12]. The binding sites of Mabs were analyzed using 13 synthetic peptides corresponding to the aa sequence of FMDV-3A by a peptide ELISA [12].…”

“…The procedure used for the blocking ELISA had been described previously [12]. Positive serum samples derived from cattle, sheep, and swine infected with the O/CHA/99 strain of FMDV at 30 days post-infection (DPI), and negative serum samples derived from clinically healthy, unvaccinated cattle, sheep, and swine were used in the test.…”

“…The 3A-Mab-bELISA was performed as described previously, with some modifications [12]. Briefly, 96-well ELISA plates were coated with 4.0 μg/ml purified polyclonal antibody of 2C epitope protein diluted in carbonate buffer (pH 9.6) and incubated overnight at 4°C.…”