As notifiable
diseases, lumpy skin disease (
LSD), sheep pox (SPP), and goat pox (
GTP) are associated with a profound effect on cattle, sheep, and goat farming industries. Development of the ELISA method could effectively facilitate serodiagnosis of the infected animals. This study aimed to develop an ELISA system based on the recombinant full-length and truncated P32
protein (Tr.P32) of goat pox virus. The P32
protein was expressed in Rosetta strain of E. coli using pET24a+ vector and evaluated by SDS-PAGE and Western blotting. Then, Tr.P32 was purified by Ni-NTA affinity chromatography under denaturing conditions and used to develop a capripoxvirus-specific ELISA. Checkerboard titration and receiver-operating characteristic (ROC) analysis were used to optimize the ELISA system and determine diagnostic specificity and sensitivity, respectively. The diagnostic potential of the developed ELISA was evaluated using positive and negative control sera collected from goat, sheep, and cattle. Results showed that the expression level of full-length P32
recombinant protein was negligible, while Tr.P32, a ~ 31 kDa
recombinant protein, was expressed up to 0.270-0.300 mg/200 mL of
culture media. The results of checkerboard titration revealed that 675 ng/well of Tr.P32
antigen and 1:10 dilution of control sera (anti GTPV HIS and healthy goat sera) caused maximum difference in absorbance between positive and negative goat sera. The recombinant Tr.P32 showed good reactions with
antibodies against
GTP virus (GTPV), SPP virus (SPPV), and
LSD virus (LSDV), whereas no cross-reactions with anti-Orf virus
antibodies were detected. By comparing with the neutralization index (NI), cut off, diagnostic sensitivity and specificity of the developed indirect-ELISA were estimated, 0.397, 94% and 96.6%, respectively. These findings indicate that the ELISA system based on Tr.P32
protein could potentially be used in sero-surveillance of all capripoxviruses; however, further investigations are required.