Dot Immunobinding Assay Research Articles

Dot immunobinding assay method with chlorophyll removal for the detection of southern rice black-streaked dwarf virus.

Southern rice black-streaked dwarf virus (SRBSDV), a new virus from Fiji, has seriously damaged rice crops in southern China and northern Vietnam in recent years. This virus is difficult to diagnose in the early stages of infection, and is very destructive at the late stage. In the present study, a dot immunobinding assay (DIBA) that has a high sensitivity for diagnosing SRBSDV was developed. Two kinds of treatment for the DIBA were evaluated to determine the most effective one for removing chlorophyll interferences via rice extraction. The first included several reagents to remove chlorophyll, namely, the alkaline reagents like magnesium oxide and alumina oxide, the adsorbent reagents like activated carbon and bentonite, as well as the extraction agent acetone. The second and third treatments, which were used to remove chlorophyll in blot membrane-nitrocellulose and polyvinylidene fluoride (PVDF), included several organic solvents containing methanol, ethanol, acetone, ethyl acetate, and diethyl ether. The results showed that activated carbon and methanol yielded the best contrasting purple color for the infected samples by decreasing the chlorophyll content.

Current Status of Fig Mosaic Disease in Iran

AbstractDuring 2009–2010, a survey was conducted in gardens and commercial fig orchards throughout Iran to determine the prevalence of Fig leaf mottle‐associated virus 1 (FLMaV‐1), Fig leaf mottle‐associated virus 2 (FLMaV‐2), Fig mild mottle‐associated virus (FMMaV), Fig latent virus 1 (FLV‐1) and Fig mosaic virus (FMV). Reverse transcription‐polymerase chain reaction and dot immunobinding assay (DIBA) were conducted on 104 fig samples collected from seven provinces. FLV‐1, FLMaV‐1 and FMV were found in 14.5, 11.5 and 8.6% of the samples, respectively, but FLMaV‐2 and FMMaV were absent. The overall average of infection reached 18.3%, with a peak of 42.9% in Semnan Province, followed by Golestan (40%), Tehran (32.3%), Lorestan (28.6%) and Mazandaran (25%) provinces. No infection was found in Fars and Gilan provinces. Fig samples from Varamin and Khorramabad districts showed high levels of mixed infections, 35.7 and 28.6%, respectively. The presence of FMV and FLV‐1 in the sap of symptomatic fig leaves was also ascertained by DIBA. Sequence analysis of amplified DNA from the partial RNA‐dependent RNA polymerase gene of two FMV isolates from Iran showed a low level of nucleotide variability (5%). The Iranian isolates shared a common phylogeny with other Mediterranean FMV isolates and in particular with those originating from Turkey already reported in GenBank. This is the first report on the presence of FLMaV‐1 and FLV‐1 in Iran and offers a preliminary insight into the unsatisfactory health status of fig in this country.

Distribution of Viruses Infecting Cucurbit Crops and Isolation of Potential New Virus-Like Sequences from Weeds in Oklahoma.

Field surveys were conducted from 2008 to 2010 to detect and determine the incidence of viruses in the major cucurbit-growing areas of Oklahoma. In total, 1,049 symptomatic leaf samples (890 from cucurbits, 109 from weed species, and 50 from crop plants [agricultural crops]) were collected from 90 fields in four counties (Atoka, Blaine, Jefferson, and Tulsa) of Oklahoma. Samples were tested against seven viruses, including Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV), Melon necrotic spot virus (MNSV), Papaya ringspot virus-watermelon strain (PRSV-W, formerly known as Watermelon mosaic virus-1), Squash mosaic virus (SqMV), Watermelon mosaic virus-2 (WMV-2), and Zucchini yellow mosaic virus (ZYMV), using dot-immunobinding assay (DIBA). Results showed the highest incidence for PRSV (51%), followed by WMV-2 (14%) and ZYMV (10%) among the collected samples. SqMV, MNSV, and CMV were detected in 3.8, 3.3, and 1.1% of the samples, respectively. None of the samples collected during surveys reacted positively against the antiserum of CGMMV. Mixed virus infections were common involving two (5.18%) or three (4.61%) viruses in various combinations. New weed host species were found to be infected with PRSV when confirmed by both DIBA and reverse-transcription polymerase chain reaction (RT-PCR). Some weed species contained possible new viruses when analyzed by random RT-PCR, followed by cloning, sequencing, and BLAST analysis with sequences in GenBank.

Serological and molecular detection of Macrophomina phaseolina, causing root rot of Citrus reticulata

Polyclonal antibodies (PAbs) were raised against mycelial antigens of Macrophomina phaseolina a causal organism of root rot disease of mandarin plants. IgG was purified and further packaged into immunological formats such as immuno diffusion, Plate trapped antigen (PTA)-ELISA, dot immunobinding assay, Western blot analysis and indirect immunofluorescence for quick and accurate detection of pathogen from soil. Indirect staining of mycelia and sclerotia of M. phaseolina with homologous PAb and labeling with goat antirabbit IgG conjugated with FITC developed strong fluorescence in young hyphal tips and sclerotia of M. phaseolina. Genomic DNA prepared from mycelia of M. phaseolina was purified and PCR amplification of 18S rDNA was done using ITS region specific primer pair. The amplified DNA was sequenced and aligned against ex-type strain sequences from NCBI GenBank using BLAST and phylogenetic analysis was obtained using MEGA4 software. Amplification of ITSI region of the rDNA can be considered as a rapid technique for identifying pathogens successfully in all cases.

Five Proteins of Laodelphax striatellus Are Potentially Involved in the Interactions between Rice Stripe Virus and Vector

Rice stripe virus (RSV) is the type member of the genus Tenuivirus, which relies on the small brown planthopper (Laodelphax striatellus Fallén) for its transmission in a persistent, circulative-propagative manner. To be transmitted, virus must cross the midgut and salivary glands epithelial barriers in a transcytosis mechanism where vector receptors interact with virions, and as propagative virus, RSV need utilize host components to complete viral propagation in vector cells. At present, these mechanisms remain unknown. In this paper, we screened L. striatellus proteins, separated by two-dimensional electrophoresis (2-DE), as potential RSV binding molecules using a virus overlay assay of protein blots. The results, five L. striatellus proteins that bound to purified RSV particles in vitro were resolved and identified using mass spectrometry. The virus-binding capacities of five proteins were further elucidated in yeast two-hybrid screen (YTHS) and virus-binding experiments of expressed proteins. Among five proteins, the receptor for activated protein kinase C (RACK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH3) did not interact with RSV nucleocapsid protein (NCP) in YTHS and in far-Western blot, and three ribosomal proteins (RPL5, RPL7a and RPL8) had specific interactions with RSV. In dot immunobinding assay (DIBA), all five proteins were able to bind to RSV particles. The five proteins' potential contributions to the interactions between RSV and L. striatellus were discussed. We proposed that RACK and GAPDH3 might be involved in the epithelial transcytosis of virus particles, and three ribosomal proteins probably played potential crucial roles in the infection and propagation of RSV in vector cells.

Arabidopsis is Susceptible to Rice stripe virus Infections

AbstractRice stripe virus (RSV), a member of the genus Tenuivirus, causes rice stripe disease in East Asia and is one of the most economically important rice pathogens. The pathogenesis of RSV and the molecular basis of plant responses to the pathogen are poorly understood. We investigated the process of RSV infection in Arabidopsis thaliana which is highly susceptible to the virus. A simple inoculation method using viruliferous small brown planthoppers was developed to infect A. thaliana plants with RSV. The symptoms were developed within 2 weeks of inoculation. One month after inoculation, all infected plants showed stunted growth and vein chlorosis in newly emerged leaves. Forty‐five days after inoculation, RSV‐infected plants showed severely stunted growth and distorted flower stalks. RSV replication in A. thaliana was confirmed using a dot immunobinding assay, reverse transcription‐polymerase chain reaction, and a protein gel blot assay. RSV infection strongly induced PR1, PR2 and GST1 but not PDF1.2 expression, indicating that the pathogen activated salicylic acid‐dependent basal defence mechanism, which is consistent with the characteristics of plant RNA virus. This Arabidopsis–RSV pathosystem provides an approach for analysing interactions between RSV and plants.

Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus

BackgroundCucumber 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.ResultsUsing 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.ConclusionsThe 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.

Spiroplasma citri: A Wide Host Range Phytopathogen

Spiroplasmas are helical motile filamentous, wall-less and culturable mollicutes. Thirty six spiroplasma species have been identified. Only S. citri, S. kunkelii and S. phoeniceum have been identified as plant pathogens. Spiroplasma citri, the causal agent of citrus stubborn disease, have a wide host range. S. citri infects most citrus species and cultivars and a wide range of non-rutaceous plant species. Citrus srubborn disease widely distributed in the southwestern united states of America, northern Africa and Mediterranean countries. It is naturally transmitted by phloem-feeding leafhopper vectors. S. citri can be detected by grafting to citrus indicators, culturing on artificial media, serological, DNA probes, dot-immunobinding assay, Immunocapture Polymerase Chain Reaction (I C- PCR), Polymerase Chain Reaction (PCR) and real-time PCR. There is genetic variability among isolates of S. citri. © 2011 Asian Network for Scientific Information.

Increased resistance to cucumber mosaic virus (CMV) in Lilium transformed with a defective CMV replicase gene

Lilium cv Acapulco was transformed with a defective cucumber mosaic virus (CMV) replicase gene (CMV2-GDD) construct using Agrobacterium tumefaciens. Four lines were analyzed for gene expression and resistance to CMV-O strain. Expression of the CMV2-GDD gene in the transgenic plants was confirmed by reverse transcription PCR (RT-PCR). When these four lines were mechanically inoculated with CMV-O, no signal of coat protein (CP) messages using RT-PCR was detected in newly produced leaves of two transgenic lines. Dot-immunobinding assay (DIBA) of CP was performed to examine the presence of the CMV in the newly produced leaves of challenged plants. Results, similar to those obtained with RT-PCR of the CP messages, were observed in DIBA. Therefore, our results imply that the two lines show increased levels of resistance to CMV, and CMV-GDD replicase gene is an effective construct that has protection against CMV in Lilium.

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) and dot immunobinding assay (DIBA) for the detection of Ganoderma infecting palms

The genus Ganoderma has a worldwide distribution causing root and stem rot of many plantation crops. A limiting factor in controlling the BSR disease is the lack of reliable diagnostic method(s) for early diagnosis. In this study, we developed polyclonal antiserum for Ganoderma mycelial and extracellular protein, and evaluated its efficacy with different plant samples collected from artificially inoculated coconut seedlings and Ganoderma infected field palms. We also tested the cross-reactivity with the soil-borne and saprophytic fungus collected from different parts of coconut palm. The antisera developed against the crude mycelial protein (CMP) and extracellular protein (ECP) showed a 1:1000 titre value for the detection of Ganoderma. The CMP antisera developed showed more cross-reaction when compared to ECP antisera of Ganoderma. In the DIBA test, at a 1:10 dilution of antigen, 1:1000 dilution of CMP and ECP antisera, 1:5000 dilution of secondary antibody gave clear distinctions in colour development between healthy and diseased samples. In the DIBA test, ECP antisera detected positive control (ECP of Ganoderma MTP and CRS-1 isolate), artificially inoculated roots, infected field roots, infected basal trunk and additionally lesions gave positive reactions which were not found in the CMP antisera tested. Therefore, both ELISA and DIBA tests may be useful for screening a large number of samples and help in the detection of infection at the earliest stage of disease development and this will certainly help to adopt suitable management strategies against Ganoderma disease in palm crops in advance.

1C5 antigen: cancer-associated protein expressed by cervical adenocarcinomas and beta-casein.

The monoclonal antibody 1C5 reacts with an antigenic determinant present in 90% of the cases of adenocarcinoma of the uterine cervix. The antigen defined by the 1C5 antibody exhibits immunological characteristics similar to those of skim milk (bovine buttermilk). 1C5-defined antigen obtained from tumor extract and skim milk binds specifically to wheat germ agglutinin (WGA) lectin. The 1C5-defined antigenic activity of a WGA lectin-bound fraction was eluted at 0.7-0.8 M NaCl off a Mono Q column. Use of an inhibition assay and a dot immunobinding assay revealed that the antigenic epitope defined by the 1C5 MoAb from skim milk exists within the first 28 amino acids of the beta-casein peptide.

Dot-immunobinding assay 法 (DIA) によるネギ萎縮ウイルス (OYDV), ニンニク潜在ウイルス (GLV) のネギからの検出

Dot-immunobinding assay 法 (DIA) によるネギ萎縮ウイルス (OYDV), ニンニク潜在ウイルス (GLV) のネギからの検出

In vitro Inhibition of Human MMP-2 Collagenolytic and Gelatinolytic Activity by Neutralizing Monoclonal Antibodies

Matrix metalloproteinases (MMPs) have been implicated in the degradation of the extracellular matrix in normal and pathological tissue remodelling. Among the MMPs, MMP-2 is the most commonly studied protease that has been involved in cancer, inflammation, infective diseases, degenerative diseases of the brain and vascular diseases. In this study, monoclonal antibodies (MAbs) were generated against human MMP-2, purified, characterized and tested for their ability to inhibit the enzymatic activity of MMP-2. Out of 12 positive clones generated against MMP-2, 2 clones (F2-1-11 and G8-25-5) were selected for further characterization. The selected clones react specifically with human pro and active form of MMP-2 in enzyme linked immunosorbant assay (ELISA), dot immunobinding assay (DIA) and Western blot and do not cross react with other human metalloproteinases or MMP-2 from other species. Additionally, these MAbs (F2-1-11 and G8-25-5) selectively inhibit collagenolytic and gelatinolytic activity of APMA ((p-aminophenylmercuric acetate)-activated-pro-MMP-2 and MMP-2, respectively.

Development of rapid flow-through-based dot-immunoassay for serodiagnosis of leptospirosis in dogs

An IgG-ELISA used recombinant antigen and a rapid flow-through enzyme immunoassay were developed for rapid screening of leptospiral antibodies in dogs using recombinant LipL41, which is one of the conserved outer membrane proteins in pathogenic leptospires as the coating antigen. Results from this study were compared with the standard microscopic agglutination test and found that the sensitivity and specificity of the enzyme-linked immunosorbent assay were 75.46% and 93.29% and whereas that of flow-through-based dot-immunobinding assay were 87.73% and 89.63%, respectively. Relative merits of these tests were also assessed. The flow-through-based dot-immunobinding assay was thus proved to be a valid screening test for canine leptospirosis.

Sugarcane mosaic virus: The causal agent of mosaic disease on sorghum (Sorghum bicolor L.) in Tehran province of Iran

During disease diagnosing studies on sorghum fields in Tehran province, Iran through vegetation period in 2005 - 2006, 75 sorghum expressing virus-associated symptoms including mosaic, leafredding and necrosis were collected. The virus was inoculated mechanically to Sweet corn (Zea mays cv. Pars403) and grain sorghum (Sorghum bicolor cv. Kimia). The virus specifically was reacted in Double Antibody Sandwich-Enzyme Linked Immunosorbent Assay (DAS-ELISA) and Dot Immunobinding Assay (DIBA). Also relative molecular mass of virus coat protein was calculated using a densitometer via sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine electrophoretic mobility compared with protein standards. The virus reacted with anti SCMV polyclonal IgG antiserum and was detected with Goat anti-Rabbit IgG alkaline phosphatase conjugate. The total nucleic acids were subjected to reverse transcription-polymerase chain reaction (RT-PCR) using SCMV degenerate and specific primers. Sugarcane mosaic virus (SCMV) was detected in all collected samples. The capsid protein was evaluated approximately 37 kDa in size. Amplification product (approximately 900 bp) was obtained from the collected and inoculated plants but not from healthy plants. This may confirm the presence of SCMV in the symptom-expressing plants.

Híbridos de trifoliata como porta-enxertos para a laranjeira 'Valência'

O objetivo deste trabalho foi avaliar a produtividade e as características agronômicas de laranjeira 'Valência', enxertadas em porta-enxertos de híbridos de trifoliata (Poncirus trifoliata). A produção de frutos, a de sólidos solúveis totais por planta, as dimensões e a eficiência produtiva de copas de laranjeira 'Valência', enxertadas em 13 híbridos de trifoliata, cultivados sem irrigação, foram avaliados por períodos que variaram de três a oito anos. As plantas também foram avaliadas, visualmente, quanto à manifestação dos sintomas de tristeza (Citrus tristeza virus) e de declínio-dos-citros, e foi utilizado o teste diagnóstico "dot immunobinding assay" (DIBA), para detecção da ocorrência do declínio antes do aparecimento dos sintomas. As plantas tinham oito anos de idade, no início das avaliações. Verificou-se que o citrandarin 'Sunki' x 'English' induziu as maiores produções de frutos em oito colheitas, sem diferir significativamente do citrange 'Troyer'. Em três anos de análise, o citrandarin 'Sunki' x 'English', sem diferir dos citranges 'Troyer' e 'Carrizo', também induziu as maiores produções de frutos e sólidos solúveis por planta. O citrentin 'Clementina' x trifoliata, os citrandarins 'Cleópatra' x 'Swingle' (715) e (1.614), 'Cleópatra' x 'Rubidoux' (1.600) e 'Cleópatra' x 'Christian' induziram a formação de laranjeiras da cultivar Valência com alturas iguais ou inferiores a 2,5 m. Nenhuma das plantas apresentou sintomas de tristeza ou declínio-dos-citros. Foi constatada a incompatibilidade entre a cultivar Valência e o trangpur 'Cravo' x 'Carrizo'.

Development of rapid, specific and sensitive detection of Cucumber mosaic virus

Cucumber mosaic virus (CMV) causes major losses to thousand agricultural and horticultural crops around the world. Unlike other plant pathogens, there are no direct methods available yet to control viruses and, consequently, the current measures rely on indirect tactics to manage the viral diseases. Hence, methods for detection and identification of viruses, both in plants and vectors, play a critical role in virus disease management. A rapid assay for diagnosis of CMV, which can be employed in both laboratory and field, is essential. Therefore, this study was undertaken to develop a procedure for detection of the CMV in infected plants using a monoclonal and polyclonal antibodies. Dotimmunobinding assays (DIBA) are useful alternatives to microtitre plate enzyme-linked immunosorbent assay (ELISA). Nine monoclonal antibodies were readily used for detected CMV by TAS-ELISA and DIBA of infected plants. DIBA has about the same sensitivity as ELISA in microtiter plates, but it has the additional advantages of simplicity, quick completion in the field or office on large numbers of samples, economy, and can be quantified using densitometry.

Molecular biology of Ganoderma pathogenicity and diagnosis in coconut seedlings

The pathogenicity of Ganoderma boninense was tested on coconut seedlings under greenhouse conditions and infection confirmed by using immunological and molecular diagnostic tools. Desiccation of older leaves and the emergence of sporophores were observed from pathogen-inoculated seedlings, whereas a control seedling does not show any pathogenic symptoms. Mature sporophores were formed within 10-13 weeks after inoculation. Polyclonal antibodies raised against mycelial proteins of Ganoderma were used for detection of Ganoderma in infected field palm and seedlings through indirect enzyme-linked immunosorbent assay technique. We adopted dot-immunobinding assay for the detection of Ganoderma from greenhouse and field samples. Under nucleic-acid-based diagnosis, G. boninense (167 bp) was detected from artificially inoculated seedlings and infected field palms by polymerase chain reaction. Apart from these, histopathological studies also support the Ganoderma pathogenicity in coconut seedlings. The pathogenicity test and combination of all the three diagnostic methods for Ganoderma could be highly reliable, rapid, sensitive and effective screening of resistance in planting material in the future.

Development of rapid, specific and sensitive detection of Cucumber mosaic virus

Cucumber mosaic virus (CMV) causes major losses to thousand agricultural and horticultural crops around the world. Unlike other plant pathogens, there are no direct methods available yet to control viruses and, consequently, the current measures rely on indirect tactics to manage the viral diseases. Hence, methods for detection and identification of viruses, both in plants and vectors, play a critical role in virus disease management. A rapid assay for diagnosis of CMV, which can be employed in both laboratory and field, is essential. Therefore, this study was undertaken to develop a procedure for detection of the CMV in infected plants using a monoclonal and polyclonal antibodies. Dotimmunobinding assays (DIBA) are useful alternatives to microtitre plate enzyme-linked immunosorbent assay (ELISA). Nine monoclonal antibodies were readily used for detected CMV by TAS-ELISA and DIBA of infected plants. DIBA has about the same sensitivity as ELISA in microtiter plates, but it has the additional advantages of simplicity, quick completion in the field or office on large numbers of samples, economy, and can be quantified using densitometry.

Dinâmica espacial de foco da clorose variegada dos citros (CVC) avaliada por meio da sintomatologia e serologia

Relationship between incidence of citrus variegated chlorosis (CVC) and causal agent Xylella fastidiosa was determined by symptoms and serology (DIBA - Dot Immunobinding Assay) for foci analysis. Experimental area consisted of an orchard of sweet orange ‘pera’, Citrus sinensis (L.) Osbeck, grafted on Rungpur lime ( Citrus limonia Osbeck) (Rutaceae). Experimental area consisted of two blocks, with 15 rows and 10 plants/row, with a total of 300 plants. Four evaluations were made and all plants in the four quadrants of each block were evaluated for symptoms. Twenty leaves of each plant were collected for serological analysis. In the first evaluation, the number of foci for symptoms was smaller than that obtained by DIBA. In the second, third and fourth evaluations the number of foci for symptoms was 3.1, 4.1 and 4.5 times higher than that obtained by DIBA. On the other hand, foci defined by DIBA were always larger than the ones defined for symptoms. Ninety per cent of foci evaluated by symptoms and seventy per cent evaluated by DIBA were smaller than 10 plants. Averages of compact foci index (CFI) were practically similar: 0.86 for the evaluation for symptoms and 0.85 for DIBA. The average of the form foci index (FFI) was 1.17 by symptoms and 0.94 by DIBA. Highest efficiency of detection of incidence for DIBA in relation to symptoms is checked by the smallest number of foci and unitary foci, as well as for the largest number of plants per foci. The importance of more sensitive methods to study the CVC epidemiology is thus enhanced