Development and application of triple antibody sandwich enzyme-linked immunosorbent assays for begomovirus detection using monoclonal antibodies against Tomato yellow leaf curl Thailand virus

Abstract

Background Tomato yellow leaf curl Thailand virus, TYLCTHV, is a begomovirus that causes severe losses of tomato crops in Thailand as well as several countries in Southeast and East Asia. The development of monoclonal antibodies (MAbs) and serological methods for detecting TYLCTHV is essential for epidemiological studies and screening for virus-resistant cultivars.MethodsThe recombinant coat protein (CP) of TYLCTHV was expressed in Escherichia coli and used to generate MAbs against TYLCTHV through hybridoma technology. The MAbs were characterized and optimized to develop triple antibody sandwich enzyme-linked immunosorbent assays (TAS-ELISAs) for begomovirus detection. The efficiency of TAS-ELISAs for begomovirus detection was evaluated with tomato, pepper, eggplant, okra and cucurbit plants collected from several provinces in Thailand. Molecular identification of begomoviruses in these samples was also performed through PCR and DNA sequence analysis of the CP gene.ResultsTwo MAbs (M1 and D2) were generated and used to develop TAS-ELISAs for begomovirus detection. The results of begomovirus detection in 147 field samples indicated that MAb M1 reacted with 2 begomovirus species, TYLCTHV and Tobacco leaf curl Yunnan virus (TbLCYnV), whereas MAb D2 reacted with 4 begomovirus species, TYLCTHV, TbLCYnV, Tomato leaf curl New Delhi virus (ToLCNDV) and Squash leaf curl China virus (SLCCNV). Phylogenetic analyses of CP amino acid sequences from these begomoviruses revealed that the CP sequences of begomoviruses recognized by the narrow-spectrum MAb M1 were highly conserved, sharing 93% identity with each other but only 72–81% identity with MAb M1-negative begomoviruses. The CP sequences of begomoviruses recognized by the broad-spectrum MAb D2 demonstrated a wider range of amino acid sequence identity, sharing 78–96% identity with each other and 72–91% identity with those that were not detected by MAb D2.ConclusionsTAS-ELISAs using the narrow-specificity MAb M1 proved highly efficient for the detection of TYLCTHV and TbLCYnV, whereas TAS-ELISAs using the broad-specificity MAb D2 were highly efficient for the detection of TYLCTHV, TbLCYnV, ToLCNDV and SLCCNV. Both newly developed assays allow for sensitive, inexpensive, high-throughput detection of begomoviruses in field plant samples, as well as screening for virus-resistant cultivars.

Current Developments and Challenges in Plant Viral Diagnostics: A Systematic Review

“…A study showed the detection of various plant viruses, such as the Squash mosaic virus (SQMV), Cowpea severe mosaic virus (CPSMV), Cucumber mosaic virus (CMV), Cowpea aphid borne mosaic virus (CABMV), Zucchini yellow mosaic virus (ZYMV), and Papaya lethal yellowing virus (PLYV) in squash, cowpea, and papaya plants [ 61 ]. The detection of Tomato yellow leaf curl Thailand virus, (TYLCTHV) in tomato, pepper, eggplant, okra, and cucurbit plants was analyzed using a TAS-ELISA (triple antibody sandwich enzyme-linked immunosorbent assays) [ 62 ]. The Protein A sandwich enzyme-linked immunosorbent assay (PAS-ELISA) has been used for the detection of various Badnaviruses in West African yam plants [ 63 ].…”

Development of a triple antibody sandwich enzyme-linked immunosorbent assay for cassava mosaic disease detection using a monoclonal antibody to Sri Lankan cassava mosaic virus

“…The identity and orientation of the SLCMV-CP gene in the resulting plasmid was confirmed by DNA sequence analysis. The recombinant SLCMV-CP was expressed, purified and analyzed as previously described [ 31 ]. Briefly, a bacterial clone containing the correct recombinant plasmid was grown at 37 °C in LB medium containing 100 µg/ml ampicillin to an absorbance at 600 nm (OD 600 ) of 0.6.…”

“…Recombinant SLCMV-CP was purified using a Ni–NTA agarose resin column (QIAGEN, Germany) under denaturing conditions following the manufacturer’s recommendations. Purified proteins were analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by western blotting using a commercial MAb to SLCMV (DSMZ, Germany) and MAb D2, which broadly reacts with begomoviruses [ 31 ]. The purified SLCMV-CP was dialyzed against phosphate-buffered saline (PBS), pH 7.4, before being used for mouse immunization.…”

“…The production of MAbs was performed as described previously by our group [ 31 ]. Briefly, 6-week-old BALB/c mice were immunized with purified recombinant SLCMV-CP protein by intraperitoneal injection.…”

Applications of Proteomic Tools to Study Insect Vector–Plant Virus Interactions

“…In addition to ELISA, dot blots and western blots have also been used to detect infection, which primarily include an antibody-based detection of the virus in tissue lysates that are either directly blotted or separated and transferred from a protein gel onto a nitrocellulose membrane [22]. [35] * Antiserum was used for detection in ELISA.…”