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 P۳۲ protein (Tr.P۳۲) of goat pox virus. The P۳۲ protein was expressed in Rosetta strain of E. coli using pET۲۴a+ vector and evaluated by SDS-PAGE and Western blotting. Then, Tr.P۳۲ 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 P۳۲ recombinant protein was negligible, while Tr.P۳۲, a ~ ۳۱ kDa recombinant protein, was expressed up to ۰.۲۷۰-۰.۳۰۰ mg/۲۰۰ mL of culture media. The results of checkerboard titration revealed that ۶۷۵ ng/well of Tr.P۳۲ antigen and ۱:۱۰ 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.P۳۲ 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, ۰.۳۹۷, ۹۴% and ۹۶.۶%, respectively. These findings indicate that the
ELISA system based on Tr.P۳۲ protein could potentially be used in sero-surveillance of all capripoxviruses; however, further investigations are required.