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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.

SUBMITTER: Miyoshi S

PROVIDER: S-EPMC7159214 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Miyoshi Shogo S Tokunaga Soh S Ozawa Tatsuhiko T Takeda Hiroyuki H Aono Mitsuo M Miyoshi Takanori T Kishi Hiroyuki H Muraguchi Atsushi A Shimizu Shin-Ichi SI Nozawa Akira A Sawasaki Tatsuya T

PloS one 20200415 4

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 ...[more]

PMID: 32294099

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Project description:The rapidly evolving nature of antibody drug development has resulted in technologies that generate vast numbers (hundreds to thousands) of lead antibody candidates during early discovery. These candidates must be rapidly pared down to identify the most drug-like candidates for in-depth analysis of their safety and efficacy, which can only be performed on a limited number of antibodies due to time and resource requirements. One key biophysical property of successful antibody therapeutics is high specificity, defined as low levels of nonspecific binding or polyspecificity. Although there has been some progress in developing assays for detecting antibody polyspecificity, most of these assays are limited by poor sensitivity or assay formats that require proprietary antibody surface display methods, and some of these assays use complex and poorly defined polyspecificity reagents. Here we report the PolySpecificity Particle (PSP) assay, a sensitive flow cytometry assay for evaluating antibody nonspecific interactions that overcomes previous limitations and can be used for evaluating diverse types of IgGs, multispecific antibodies and Fc-fusion proteins. Our approach uses micron-sized magnetic beads coated with Protein A to capture antibodies at extremely dilute concentrations (<0.02 mg/mL). Flow cytometry analysis of polyspecificity reagent binding to these conjugates results in sensitive detection of differences in nonspecific interactions for clinical-stage antibodies. Our PSP assay strongly discriminates between antibodies with different levels of polyspecificity using previously reported polyspecificity reagents that are either well-defined proteins or highly complex protein mixtures. Moreover, we also find that a unique reagent, namely ovalbumin, results in the best assay sensitivity and specificity. Importantly, our assay is much more sensitive than standard assays such as ELISAs. We expect that our simple, sensitive, and high-throughput PSP assay will accelerate the development of safe and effective antibody therapeutics.

Project description:BackgroundCassava mosaic disease (CMD) is one of the most devastating viral diseases for cassava production in Africa and Asia. Accurate yet affordable diagnostics are one of the fundamental tools supporting successful CMD management, especially in developing countries. This study aimed to develop an antibody-based immunoassay for the detection of Sri Lankan cassava mosaic virus (SLCMV), the only cassava mosaic begomovirus currently causing CMD outbreaks in Southeast Asia (SEA).MethodsMonoclonal antibodies (MAbs) against the recombinant coat protein of SLCMV were generated using hybridoma technology. MAbs were characterized and used to develop a triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) for SLCMV detection in cassava leaves and stems. Assay specificity, sensitivity and efficiency for SLCMV detection was investigated and compared to those of a commercial ELISA test kit and PCR, the gold standard.ResultsA TAS-ELISA for SLCMV detection was successfully developed using the newly established MAb 29B3 and an in-house polyclonal antibody (PAb) against begomoviruses, PAb PK. The assay was able to detect SLCMV in leaves, green bark from cassava stem tips, and young leaf sprouts from stem cuttings of SLCMV-infected cassava plants without cross-reactivity to those derived from healthy cassava controls. Sensitivity comparison using serial dilutions of SLCMV-infected cassava sap extracts revealed that the assay was 256-fold more sensitive than a commercial TAS-ELISA kit and 64-fold less sensitive than PCR using previously published SLCMV-specific primers. In terms of DNA content, our assay demonstrated a limit of detection of 2.21 to 4.08 × 106 virus copies as determined by quantitative real-time PCR (qPCR). When applied to field samples (n = 490), the TAS-ELISA showed high accuracy (99.6%), specificity (100%), and sensitivity (98.2%) relative to the results obtained by the reference PCR. SLCMV infecting chaya (Cnidoscolus aconitifolius) and coral plant (Jatropha multifida) was also reported for the first time in SEA.ConclusionsOur findings suggest that the TAS-ELISA for SLCMV detection developed in this study can serve as an attractive tool for efficient, inexpensive and high-throughput detection of SLCMV and can be applied to CMD screening of cassava stem cuttings, large-scale surveillance, and screening for resistance.

Dataset Information

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

Publications

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

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