Development of Antibodies as a Diagnostic Tool for Plant Virus(es)

Abstract

Viral plant infections significantly reduce crop yields worldwide, especially in Pakistan. Early detection of these diseases is crucial to reduce losses and adopt focused control strategies. This present study was aimed at developing polyclonal antibodies (pAB) for the Enzyme-Linked Immunosorbent Assay (ELISA) based diagnosis of economically significant viruses such as the Sugarcane mosaic virus (SCMV), the Barley yellow dwarf virus (BYDV), and the Chili leaf curl Indian virus (ChiLCINV). The coat protein (CP) gene sequences of SCMV, BYDV and ChiLCINV were used to determine their physicochemical properties, subcellular localization, and immunogenicity through in silico analysis. The SCMV and BYDV CP genes were synthesized by Macrogen Inc. Korea while the ChiLCINV CP gene was amplified using specific primer pairs. These products were cloned into the expression vector, pET28a (+), using restriction endonuclease enzymes NdeI and HindIII, to produce the 6×His-tag recombinant fusion coat proteins rFCP-SCMV, rFCP-BYDV, and rFCP-ChiLCINV, respectively, in Escherichia coli (E. coli). The rFCP-SCMV (40 kDa) was developed in soluble form and was purified through an affinity Ni-NTA resin. Whereas rFCP-BYDV (24 kDa) and rFCP-ChiLCINV (33 kDa) were expressed as insoluble inclusion bodies and were denatured and purified with one-column refolding by affinity chromatography. The purified protein (rFCP-SCMV, rFCP-BYDV and rFCP-ChiLCINV) were utilized as an antigen in the initial response, followed by four weekly intraperitoneal boosters’ doses in mice to develop pAB antisera, which was collected by cardiac puncture. In indirect ELISA, antisera raised with rFCP-SCMV and rFCP-BYDV were able to detect 100 pg/mL of a purified protein compared to antisera raised with rFCP-ChiLCINV, which detected 50 pg/mL of the purified coat protein. Additionally, when tested against 1000-fold diluted crude protein extracts from infected leaf tissues, the antisera demonstrated high sensitivity, producing robust signals even at the maximum sera dilution in ELISA. These findings indicate the developed antisera's high specificity and sensitivity. Furthermore, serological detection and PCR identification of field samples produced identical findings, demonstrating the validity of this method. The developed antibodies serve as a valuable resource for future research and a step towards self-sufficiency in plant virus diagnosis in Pakistan, where no plant disease diagnostic mechanisms exist.