Production of a rabbit monoclonal antibody for highly sensitive detection of citrus mosaic virus and related viruses

Abstract

Citrus mosaic virus (CiMV) is one of the causal viruses of citrus mosaic disease in satsuma mandarins (Citrus unshiu). Prompt detection of trees infected with citrus mosaic disease is important for preventing the spread of this disease. Although rabbit monoclonal antibodies (mAbs) exhibit high specificity and affinity, their applicability is limited by technical difficulties associated with the hybridoma-based technology used for raising these mAbs. Here, we demonstrate a feasible CiMV detection system using a specific rabbit mAb against CiMV coat protein. A conserved peptide fragment of the small subunit of CiMV coat protein was designed and used to immunize rabbits. Antigen-specific antibody-producing cells were identified by the immunospot array assay on a chip method. After cloning of variable regions in heavy or light chain by RT-PCR from these cells, a gene set of 33 mAbs was constructed and these mAbs were produced using Expi293F cells. Screening with the AlphaScreen system revealed eight mAbs exhibiting strong interaction with the antigen peptide. From subsequent sequence analysis, they were grouped into three mAbs denoted as No. 4, 9, and 20. Surface plasmon resonance analysis demonstrated that the affinity of these mAbs for the antigen peptide ranged from 8.7 × 10 −10 to 5.5 × 10 −11 M. In addition to CiMV, mAb No. 9 and 20 could detect CiMV-related viruses in leaf extracts by ELISA. Further, mAb No. 20 showed a high sensitivity to CiMV and CiMV-related viruses, simply by dot blot analysis. The anti-CiMV rabbit mAbs obtained in this study are envisioned to be extremely useful for practical applications of CiMV detection, such as in a virus detection kit.

Major Citrus Viruses in Japan

“…The type isolate Ci-968 (Iwanami et al 1993a) was collected from a satsuma mandarin tree in Wakayama Prefecture. CiMV is often found in other parts of Japan (Iwanami et al 1991a(Iwanami et al , 1993aMiyoshi S. et al 2020) as well as in Korea and China (Hyun et al 2017, Yan et al 2020. To date, CiMV has not been detected in Turkey.…”

“…A comparison of a partial RNA2 nucleotide sequence revealed a 95.5%-96.2% sequence identity between Korean CiMV isolates and the Japanese isolate Ci-968 (Hyun et al 2017). In Japan, some novel CiMV isolates that are serologically distinct from Ci-968 have been reported (Iwanami et al 1993a, Miyoshi et al 2020. For example, Az-1 is serologically distinct from Ci-968, but its nucleotide sequence is highly homologous to that of Ci-968 (Iwanami et al 1993a, Iwanami et al 2001.…”

“…For example, Az-1 is serologically distinct from Ci-968, but its nucleotide sequence is highly homologous to that of Ci-968 (Iwanami et al 1993a, Iwanami et al 2001. Isolates (e.g., B291) that are serologically similar to Az-1 are widespread and seriously affect satsuma mandarin cultivation in Ehime Prefecture, resulting in considerable economic losses (Miyoshi et al 2020). At the amino acid level, a Chinese isolate (CiMV-SE1) was revealed to be the most similar to Az-1 (Yan et al 2020).…”

“…Many lines of polyclonal and monoclonal antibodies targeting SDV and CiMV have been raised in Japan and elsewhere; they have been used for serological diagnoses, typically in DAS-ELISA platforms (Hirashima et al 1990(Hirashima et al , 1991(Hirashima et al , 1994Nozu et al 1986;Usugi & Tsuchizaki 1982;Wu et al 2012;Zhou et al 1996). The anti-CiMV rabbit monoclonal antibodies developed recently may be very useful for detecting CiMV in infected plant material (Miyoshi et al 2020).…”

Development of a Dot-Elisa Assay for Diagnosis of Southern Rice Black-Streaked Dwarf Disease in the Field

“…Producing specific polyclonal antibodies using antigenic peptides has been previously studied to detect plant viruses through immunoassay (Gallo et al, 2013;Ahmed et al, 2019). The peptides chosen to use as antigens were designed based on virus coat protein sequences with different lengths and could be situated on the N-terminus or C-terminus depending on the structural characteristics of the corresponding proteins, and specific research purposes (Wang et al, 2012;Gallo et al, 2013;Ahmed et al, 2019;Miyoshi et al, 2020). For rice, Wang et al (2012) produced specific polyclonal antibodies for SRBSDV from three peptides of 20 amino acids each, located at the positions 20-39, 140-159, and 319-339 in the P10 protein sequence.…”