Abstract
Foot-and-mouth disease (FMD) is an acute and contagious vesicular disease affecting wild
and domesticated cloven-hoofed animals. The disease is caused by foot-and-mouth disease
virus (FMDV) of the genus Aphthovirus under Picornaviridae family. FMD is a significant trans
boundary disease with extensive economic impact. FMDV exists as seven distinct serotypes;
O, A, C, Asia 1, SAT (Southern Africa Territories) 1, SAT 2 and SAT 3, each with multiple
subtypes which are not equally distributed around the world. FMD is endemic mostly in
cattle of Bangladesh and the prevailing FMDV serotypes are O, A and Asia 1. Seromonitoring is necessary to determine the vaccination strategies for the control of FMD. ELISA
is most commonly used for seroprofiling. However, ELISA kits are not produced in
Bangladesh and imported kits are highly expensive. Now a days, recombinant DNA
technology is a widely used tool for in vitro expression of recombinant proteins for the
development of diagnostics and vaccines. The aim of the present study was to express in vitro
the structural protein VP1 of FMDV serotype type 'O' and to develop an indirect ELISA for
the detection of antibodies to FMDV serotype 'O' in cattle serum using this recombinant
protein as antigen. VP1 gene of a local isolate of FMDV serotype 'O' was directionally cloned
in an expression plasmid vector (pET161/GW/D-TOPO) and used for transformation of
TOP10 E. coli. For expression of recombinant VP1 (rVP1) BL21star(DE) E. coli was ring
transformed with the recombinant plasmids isolated from TOP10 E. coli. The expression of
rVP1 was induced with IPTG. The rVP1 was successfully expressed as a fusion protein of
~29.0kDa size with 6X Histidine tag and tetra Cysteine Lumio tag. The Histidine tag and
Lumio tag were used for nickel-agarose column purification and fluorescent detection of
expressed protein, respectively. Purified rVP1 was used as an antigen for the development of
an indirect ELISA (rVP1 iELISA) for the detection of antibodies to FMDV serotype 'O' in
cattle serum and the test was validated against a commercial competitive ELISA kit (IDVET
cELISA). For the assay rVP1 antigen dilution of 1:100 (40 µg/ml) and the test serum dilution
of 1:500 were found to be optimum. The result was best expressed as the percent positivity
against a reference positive serum and the positive/negative cutoff threshold for percent
positivity was found to be 47.36%. The inter-assay coefficient of variation was between 2.02%
to 3.12%. The developed rVP1 iELISA was found to be 93.10% sensitive and 94.44% specific
when compared with the commercial IDVet cELISA using 74 field sera. This newly developed
rVP1 iELISA is the first in-house developed ELISA system in Bangladesh for the detection of
antibody to FMDV serotype O and can be used in FMDV seromonitoring programme.