Citrus mosaic virus (CiMV) is one of the causes of citrus mosaic disease in Satsuma mandarin (Citrus unshiu). Rapid detection of trees infected with the citrus mosaic disease is important to prevent the spread of the disease.
Although rabbit monoclonal antibodies (mAbs) have high specificity and affinity, their application is limited by technical difficulties associated with the hybridoma-based technology used to enhance these mAbs.
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Here we demonstrate a possible CiMV detection system with rabbit mAb specific to the CiMV coat protein. The conserved peptide fragment of the small subunit of the CiMV envelope protein was designed and used to immunize rabbits. Antigen-specific antibody-producing cells were identified by immunospot analysis using the chip method.
After cloning the variable regions in the heavy or light chains by RT-PCR of these cells, a gene set of 33 mAbs was constructed and these mAbs were produced using Expi293F cells. Screening with the AlphaScreen System revealed that eight mAbs showed strong interactions with antigenic peptides.
Surface plasmon resonance analysis showed that the affinity of this monoclonal antibody for antigenic peptides ranged from 8.7 × 10-10 to 5.5 × 10-11M. In addition to CiMV, mAb 9 and 20 were able to detect CiMV-associated viruses in leaf extracts by ELISA.
The rabbit anti-CiMV mAbs obtained in this study are believed to be very useful for practical applications of CiMV detection, such as in virus detection kits.