Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus
© Shang et al; licensee BioMed Central Ltd. 2011
Received: 16 February 2011
Accepted: 15 May 2011
Published: 15 May 2011
Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, can be transmitted by seeds and infects many cucurbit species, causing serious yield losses in cucumber and watermelon plants. In this paper, five serological methods including antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), Dot-immunobinding assay (DBIA), direct tissue blot immunoassay (DTBIA) and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) were described for detection and diagnosis of CGMMV.
Using the purified CGMMV particles as immunogens, six murine monoclonal antibodies (MAbs) were produced. Five serological methods were established using the MAb 4H1 and detection sensitivity was compared using purified preparations and infected-plant tissue extracts. The detection sensitivity of ACP-ELISA was 0.16 ng of purified CGMMV, whereas TAS-ELISA was more sensitive than ACP-ELISA with a minimum detection of 0.04 ng of purified CGMMV. The sensitivities of TAS-ELISA and DBIA were similar for detecting CGMMV in infected-plant tissue extracts, and were four times higher than ACP-ELISA. The IC-RT-PCR was the most sensitive method, which could detect as little as 0.1 pg of purified virus. The detection sensitivity of IC-RT-PCR for CGMMV-infected plant tissues was about 400 times higher than that of TAS-ELISA and DBIA.
The established ACP-ELISA, TAS-ELISA, DBIA and DTBIA are suitable for routine CGMMV detection of large-scale samples in the field survey, while IC-RT-PCR is more sensitive and suitable for acquiring information about the viral genome.
Cucumber green mottle mosaic virus (CGMMV) is a species of the genus Tobamovirus and is an economically significant seed transmitted pathogen, which causes yield losses of about 15% in cucurbitaceous vegetable crops [1, 2]. The virion of CGMMV is rod-shaped, approximately 300 nm in length and 18 nm in diameter . CGMMV contains a single 6.4 kb plus-strand genomic RNA . The most characteristic symptoms of the disease in cucurbit plants are systemic mosaic and mottling on leaves, and blistering and deterioration of fruit pulp . CGMMV was first reported in the United Kingdom in 1935 . Subsequently, it had been reported in Germany, Finland, Israel, Saudi Arabia, India, Pakistan, Korea and Japan [7–10]. To date, several isolates of CGMMV from Korea, Israel, Japan, Greece and Spain have been characterized based on serology and genomic sequences [1, 4, 11–15]. In 2003, a new disease with green mottle and mosaic symptoms occurred at watermelon and cucumber fields in northeast China . In 2005, this disease developed an epidemic in watermelons in Liaoning province of China and caused considerable economic damage. The serological and reverse transcription-polymerase chain reaction (RT-PCR) detection results confirmed that the disease was caused by CGMMV . CGMMV is an alien invasive pathogen  and it remains a potential serious threat to the production of cucurbitaceous crops in China.
A variety of techniques have been established for the detection and diagnosis of CGMMV: RT-PCR [4, 15, 19, 20], real time RT-PCR , transmission electron microcopy (TEM) [1, 22], immune capture (IC)-RT-PCR , ELISA using polyclonal antibodies (PAbs) [1, 11, 23] and monoclonal antibodies (MAbs) [2, 5]. Among those detection methods, enzyme-linked immunosorbent assay (ELISA), Dot-immunobinding assay (DBIA) and direct tissue blot immunoassay (DTBIA) are more suitable for routine detection of large-scale samples in the field survey, while IC-RT-PCR is more sensitive and suitable for acquiring information about the viral genome . In this study, six MAbs were produced and MAb-based ACP-ELISA, TAS-ELISA, DBIA, DTBIA and IC-RT-PCR methods for CGMMV detection were established.
Materials and methods
Virus sources and Virus purification
A CGMMV Liaoning isolate was kindly provided by Qing Chen (Xiamen Entry-Exit Inspection and Quarantine Bureau, Fujiang province, China) and used as antigens for raising PAbs and MAbs. The CGMMV isolate was maintained on Cucumis sativus cv. Aohagauri by mechanical inoculation in an insect-proof greenhouse. Tobacco mosaic virus (TMV), Odontoglossum ringspot virus (ORSV) and Tomato mosaic virus (ToMV) were characterized and maintained by author's laboratory.
Purified CGMMV particles were obtained from fresh infected leaf tissues as described by Zhou et al. . The purified virions were mixed with 2% (w/v, g/mL) phosphotungstic acid (PTA) and examined with an electron microscope (JEM -1200 EX, JEOL Ltd., Tokyo, Japan)).
Preparation of PAbs and MAbs against CGMMV
The purified CGMMV virions were used as an immunogen and PAbs against CGMMV were prepared in two New Zealand rabbits as described previously . The rabbits were bled one week after the fifth injection, and the PAbs were used in TAS-ELISA.
Production of hybridomas secreting MAb against CGMMV was performed as described previously . Hybridomas were injected intraperitoneally into pristane-primed syngeneic BALB/c mice to produce ascitic fluids. ACP-ELISA was used to determine the titres of ascitic fluids. MAb isotypes were determined by ELISA with the mouse MAb isotyping reagents according to the manufacturer's instruction (Sigma-Aldrich, St. Louis, MO, USA). Specificity analyses of MAbs and the purification of IgG were operated by the methods as described by Wu et al .
ACP-ELISA and TAS-ELISA
Detection of CGMMV particles in purified preparations or in sap extractions of infected leaf tissues was carried out following the standard procedures for ACP-ELISA  and TAS-ELISA . The working dilutions of the MAb and the goat anti-mouse IgG conjugated with alkaline phosphatase for ACP-ELISA were determined by phalanx tests. Briefly, the lane wells of ELISA plates coated samples were respectively added two-fold diluted MAb and incubated. The row wells of plates were respectively dispensed two-fold diluted the goat anti-mouse IgG conjugated with alkaline phosphatase and incubated. The alkaline phosphatase conjugate was detected with p-nitrophenyl phosphate. The working dilutions of the PAb and MAb for TAS-ELISA also were determined by phalanx tests. Briefly, the lane wells of ELISA plates were respectively coated two-fold diluted PAbs and incubated. After sample incubation, two-fold diluted MAbs were respectively dispensed in row wells of the ELISA plates and incubated. Goat anti-mouse IgG conjugated with alkaline phosphatase at 1:8000 dilution was subsequently applied into the wells and incubated. The alkaline phosphatase conjugate detection and the result analysis were performed as ACP-ELISA. Negative and positive controls were wells incubated with leaf extracts from healthy leaf and CGMMV-infected leaf tissues, respectively. All those samples were triturated in 0.01 mol L-1 PBS buffer (pH 7.4) and two-fold diluted in the same buffer. The sample was considered to be positive when its absorbance value was three times greater than that of the negative control.
DBIA and DTBIA
DBIA and DTBIA procedures were carried out according to the method described previously  and modified. Briefly, samples for DBIA were prepared by grinding leaf tissues in 0.01 mol L-1 phosphate buffered saline (PBS) and centrifuged at 8000 ×g for 5 min. The tissue extracts were spotted on nitrocellulose membranes (Amersham Biosciences, Bucks, UK, 2 μL/spot) and allowed to be air-dry. The nitrocellulose membrane was soaked in 5% solution of dried skimmed milk in PBS for 30 min, followed by an incubation in a suitable dilution of MAb for 1 h. Nitrocellulose was rinsed four times in PBST (0.01 mol L-1 PBS, 0.05% Tween-20, pH 7.4), then incubated in goat anti-mouse IgG conjugated with alkaline phosphatase (Sigma-Aldrich, St. Louis, MO, USA, 1:8000 in PBS) for another 1 h. After washing five times with PBST, the membrane was color-developed in a substrate solution, alkaline phosphatase buffer (0.1 mol L-1 Tris base, 0.1 mol L-1 NaCl and 0.05 mol L-1 MgCl2, pH 9.5) containing NBT/BCIP (5-Bromo-4-Chloro-3-Indolyl phosphate/Nitro-Blue Tetrazolium chloride, Promega).
Tissue prints for DTBIA were prepared by transversely cutting young stems or rolled leaves with blades and gently pressed the freshly cut surface onto nitrocellulose membranes for 3 to 5 sec. The prints were air-dried and blocked for 30 min in 5% solution of dried skimmed milk in PBS. The ensuing steps for DTBIA were same as that of DBIA.
The forward primer (CP-F: 5'-CTTACAATCCGATCACACCTAG-3') and the reverse primer (CP-R: 5'-CTAAGCTTTCGAGGTGGTAGC-3') used for IC-RT-PCR were designed based on the most conserved part of CGMMV CP gene obtained from GenBank, which were determined based on the alignment of CGMMV CP RNA sequences using the DNASTAR package (Version 7.0, DNAStar Inc., Madison, WI, USA). The IC-RT-PCR was performed as described previously . Amplified DNA fragments were analyzed and sequenced as described previously .
Production and characterization of MAbs against CGMMV
Properties of monoclonal antibodies to CGMMV
IgG yield in ascites (mg/mL)
IgG1, κ chain
IgG2a, κ chain
IgG1, κ chain
IgG2a, κ chain
IgG2a, κ chain
IgG1, κ chain
ACP-ELISA and TAS-ELISA for CGMMV detection
The working dilutions of MAb 4H1, PAb used as coating antibody in TAS-ELISA and goat anti-mouse IgG conjugated with alkaline phosphatase (Sigma-aldrich, St. Louis, MO, USA) were determined according to the results of phalanx tests. The results of the three repeated tests indicated that the dilution of MAb 4H1 at 1:5000, goat anti-mouse IgG conjugated with alkaline phosphatase at 1:8000 were suitable for ACP-ELISA, and the dilution of PAb at 1:5000, MAb 4H1 at 1:6000 and goat anti-mouse IgG conjugated with alkaline phosphatase at 1:8000 were suitable for TAS-ELISA.
DBIA and DTBIA for CGMMV detection
IC-RT-PCR for CGMMV detection
The IC-RT-PCR amplified-fragments were cloned and sequenced. The sequences of clones were compared with the CGMMV CP sequences deposited in GenBank. The sequences of the PCR products had 97-99% homology with the CP region of the genome of CGMMV isolates in GenBank, Which confirmed that amplified products were derived from CGMMV CP gene.
Planting area of cucurbitaceous vegetable crops in China is over 3,000,000 hectare (ha), and is distributed in all provinces. The watermelon import and export trade among China, Japan and Korea is developing very quickly and might be the reason for the introduction of the virus into China. In 2005, an outbreak of a disease caused by CGMMV occurred in watermelon fields in Liaoning province, the damaged planting area was about 333 ha . Methods for detection of CGMMV, an economically important seed transmitted virus, were not well established in China. In this study, five serological methods for CGMMV detection were established and a comparative analysis of these methods was assessed for their detection sensitivities of purified CGMMV and CGMMV-infected cucumber tissues. Both ACP-ELISA and TAS-ELISA could readily and specifically detect CGMMV. TAS-ELISA was more sensitive to detect CGMMV than ACP-ELISA. Both methods could be applied to detect CGMMV in filed samples.
The limit of detection by DBIA was similar to that of TAS-ELISA for CGMMV in infected-plant tissues. Short time and low costs are the main advantages of DBIA. DTBIA is a very convenient, specific and reliable method for detecting CGMMV under field conditions, and it can provide direct information about the distribution of the virus within host plants. So in a further detection application, plant samples can be spotted on nitrocellulose membranes at fields and be delivered to detect in local laboratories. Its simple and convenient advantage of this method is very significant implications for large-scale surveys as well as long-term epidemiological or ecological studies of this virus.
As expected, IC-RT-PCR is the most sensitive assay among the five methods and it could detect 0.1 pg of purified CGMMV. The sensitivity of this method for detecting CGMMV in infected-plant extracts was about 400 times higher than that of TAS-ELISA and DBIA. Moreover, the information of viral genome can be obtained from sequencing analyses of amplified products of IC-RT-PCR.
In conclusion, DTBIA is the most convenient method, while TAS-ELISA, ACP-ELISA and DBIA are also suitable for handing large amounts of samples in routine tests. Although IC-RT-PCR is not appropriate for large scale screening, it showed the best sensitivity than the other four methods, may be valuable for acquiring information about the viral genome of samples.
This work was supported by Zhejiang Provincial Natural Science Foundation of China (Grant No. Z3090039) and National Natural Science Foundation of China (Grant No. 30871624).
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