Agar Gel Double Diffusion Tests Research Articles

Development of an immunochromatographic strip for detection of antibodies against duck Tembusu virus

Duck Tembusu virus (DTMUV), a novel flavivirus, causes severe disease in ducks. There is an urgent need for a rapid and effective diagnostic method to control the spread of DTMUV. We chose the envelope (E) protein from DTMUV as an antigen and combined it with colloidal gold particles as tracers to specifically detect anti-DTMUV antibodies. Based on the double-antigen sandwich format, an immunochromatographic strip (ICS) for the rapid detection of anti-DTMUV antibodies was developed. The ICS showed a high specificity and no cross-reactivity with other sera. By detecting a serially diluted duck anti-DTMUV serum, the sensitivity of the ICS was 16-fold higher than that of the agar gel double diffusion test. Moreover, the ICS was both stable and reproducible, maintaining the same performance at 4°C for at least 6 months. To evaluate the effectiveness of the ICS, 217 duck serum samples were tested with the ICS and an indirect enzyme-linked immunosorbent assay (iELISA). The consistency ratio of positive and negative results between the two methods was 97.87% and 97.06%, respectively. The agreement between the ICS and the ELISA was 97.24%. The ICS developed in this study offers a specific, sensitive, and rapid method to detect anti-DTMUV antibodies.

Microscopic and polyclonal antibody-based detection of yellow leaf disease of arecanut (Areca catechu L.)

Phytoplasma, the pathogen of yellow leaf disease (YLD) of arecanut (Areca catechu L.) was detected by transmission and scanning electron microscopy. Tissues of YLD affected palms contained phytoplasmas in the phloem sieve elements, but not in symptomless healthy palm tissues. Phytoplasma was purified from tissues of diseased palms employing percoll density gradient centrifugation and confirmed by transmission electron microscopy. Using the purified phytoplasma preparation, a polyclonal antiserum was raised in rabbits and used for standardisation of agar gel double diffusion (Ouchterlony) test and DAC-ELISA. Clear precipitin line was observed in Ouchterlony test between the antigen from diseased palms and the pathogen-specific antibodies after 48 h incubation and only undiluted antiserum showed best result in the test. However, in ELISA, 1:10 antigen dilution and 1:400 pathogen-specific antibodies dilution produced sensitive detection of the pathogen with a difference of >3.5 times absorption values between healthy and diseased samples. The results thus confirmed the ability of antiserum to distinguish healthy and infected plants and utility of ELISA for effective diagnosis of YLD.

Comparisons of complete RNA-2 sequences, pathological and serological features among three Japanese isolates of Arabis mosaic virus

Arabis mosaic virus lily and narcissus isolates (ArMV-Li and ArMV-Na) induced severe necrotic spots on Chenopodium quinoa, whereas ArMV butterbur isolate (ArMV-Bu) caused symptomless infection in the plant. The accumulation level of ArMV-Bu in upper non-inoculated leaves of C. quinoa was comparable to that of ArMV-Li or -Na. The agar gel double-diffusion test using an antiserum against ArMV-Li showed ArMV-Li was closely related to ArMV-Na, but not to ArMV-Bu. The RNAs-2 of ArMV-Li, -Na, and -Bu consist of 3707, 3709, and 3789 nucleotides, and they contain one open reading frame encoding a putative polyprotein of 1083, 1084, and 1122 amino acids, respectively. The overall identity of RNA-2 of ArMV-Li displayed more than 90% with ArMV-Na, but less than 70% with ArMV-Bu. A phylogenetic analysis of 2A sequences from ArMV isolates revealed ArMV-Bu was not categorized in any cluster. ArMV-Bu is a unique isolate from the point of view of pathological and serological features, and nucleotide sequence.

동부(Vigna sinensis)의 국부병반을 이용한 Cucumber mosaic virus와 Broad bean wilt virus 2의 구별

Cucumber mosaic virus(CMV) and Broad bean wilt virus 2(BBWV2) were isolated from Gentiana scabra plants showing typical mosaic and yellowing symptoms. When the inoculum of mosaic symptom propagated in Nicotiana benthamiana was inoculated to primary leaves of Vigna sinensis, the local lesions of different types was developed. Type one produced a small necrotic spot(SNS) of pinpoint type, while the other one showed a large necrotic spot(LNS) of halo type. LNS on primary leaves of V. sinensis was also induced by inoculum from yellowing symptom on G scabra. Single lesion from SNS induced a typical mosaic symptom on N. Benthamiana. On the other hand, LNS produced a chlorotic ring symptom on inoculated leaves and mosaic plus necrotic ringspot on upper leaves of N. benthamiana. An isolate of CMV from SNS and BBWV2 from LNS were detected by using dsRNA analysis, RT-PCR and agar gel double-diffusion test. Thus, our results should provide a tool of a simple method for discrimination from mixed infected plants by CMV and BBWV2.

Identification of Tomato mosaic virus Infection in Lisianthus in Taiwan

In recent years, Lisianthus (Eustoma russellianum (Don.) Griseb) has become popular as potted plants and cut flowers in Taiwan. They are grown in the central and southern regions of the island. Since 1998, diseased plants with mosaic symptoms, followed by necrosis of leaf tissues, were observed in commercial greenhouses and field-grown lisianthus. Newly emergent leaves were curled and smaller compared with those on healthy plants. These symptoms greatly decreased the commercial value of the crop. Rigid rods similar to tobamoviruses that measured 300 × 18 nm were found consistently associated with symptomatic plants. In July 2002, a virus culture was isolated from diseased lisianthus from Chiayi County, Taiwan and established and maintained in systemic hosts Nicotiana tabacum L. and N. benthamiana Domin. Chlorotic and necrotic spots developed on lisianthus leaves 1 to 2 weeks after inoculation with the virus; symptoms eventually became systemic. Virions were purified from inoculated N. tabacum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the virus contained one 18-kDa (Mr) polypeptide. The virus reacted positively in agar gel double diffusion tests and enzyme-linked immunosorbent assays with antisera prepared to Tobacco mosaic virus (TMV) or Tomato mosaic virus (ToMV) (gifts of S. D. Yeh, National Chung Hsing University, Taichung, Taiwan). A viral coat protein (CP) gene approximately 0.5 kb was amplified by reverse transcription-polymerase chain reaction from total RNA prepared from infected N. benthamiana using 5'-GCGAGCCATGGATTCTTACTCAATTACT as a forward primer and 5'-ACTCTCGGATCCTTAAGATGCAGGTGCAGA as a reverse primer. Comparison of the 480-nt CP gene region with that of ToMV-OM (GenBank Accession No. X02144) (3) revealed 99.2% nucleotide identity and 99.4% amino acid identity. It shares, however, 74.4% nucleotide identity and 83.9% amino acid identity with CP genes of TMV-U1 (GenBank Accession No. AX040174) and TMV-vulgare (GenBank Accession No. J02415) (1). The virus induced local lesion responses similar to ToMV on inoculated N. tabacum cv. White Burley, N. sylvestris Speg. & Comes, and Datura stramonium L. Inoculation of TMV, however, resulted in a systemic infection in these plants. Results from sequence analysis and diagnosis based on host reaction to the virus inoculation indicated that the tobamovirus infecting lisianthus in Taiwan is an isolate of ToMV. The virus is economically important to lisianthus and tomato in Taiwan. To our knowledge, this is the first report of ToMV causing disease on lisianthus in Taiwan. The disease was previously observed on lisianthus in Italy (2).

Evaluation of agar gel double diffusion test for detection of nuclear polyhedrosis virus of silkworm, Bombyx mori L.

The agar gel double diffusion test was evaluated for detection of nuclear polyhedrosis virus infecting the silkworm, Bombyx mori L. (BmNPV). The test was successful using alkali-dissolved BmNPV as antigen. The antiserum raised against dissolved BmNPV gave positive results with a wider range of antigenic concentrations compared to antiserum raised against undissolved BmNPV. Gel plates prepared using phosphate buffered-saline as dissolving medium for agarose gave sharper and clearer precipitation bands compared to those made with sodium carbonate-saline as diluent. Sharp bands were obtained when the experiment was carried out at 25°C, whereas at higher temperature (35°C), the bands were diffused. The test was able to detect antigen concentration as low as 2 × 106 OBs/ml (18.9 μg/ml of antigen protein).

沈降抗体検出用アスペルギルス粗抗原に関する若干の検討 補遺

We reported earlier on culture filtrate antigens from A. fumigatus that had been incubated at 25°C for 1-10 weeks (Jpn J Med Mycol 34: 439-444, 1993). Additionally, we prepared five culture filtrate antigens from A. fumigatus ATCC26430 grown by shaking at 25°C for 11-15 weeks in Sabouraud liquid medium. We also prepared a polysaccharide antigen from the same strain by extracting mycelia and conidia. The growth of the fungi became weaker in cultures after 12 weeks, and by stopped at 14 weeks. Sixteen antigens were then employed in agar gel double diffusion (DD) tests against the sera of 18 aspergilloma patients. The results of DD showed three types (A, B, C) of precipitin pattern, with 9 patients belonging to type A, 6 to type B and 3 to type C. In all cases, precipitin lines were formed with the antigens obtained after 6-15 weeks of culture. To avoid contamination, inferior cultures and accumulation of waste, the incubation time should be as short as possible. Thus, as noted in our previous letter, we found that the antigen obtained from 6-week culture filtrates at 25°C is the most useful for demonstrating precipitin reactions against the sera of aspergilloma patients.

Purification and immunoreactivity of monoclonal antibodies against myeloma-λ chain

In this study, two murine IgM-monoclonal antibodies (IgM-McAbs) against lambda chain of myeloma protein from hybridoma ascites were purified by Sephadex G200 chromatography. The eluate of the first peak on absorption at 280 nm formed one precipitation band with rabbit anti-mouse IgM or anti-mouse immunoglobulin in agar gel double diffusion test; it also formed a detectable precipitation line in the IgM reaction area in immunoelectrophoresis. These findings showed that the eluate of the first peak on absorption at 280 nm contained purified IgM-McAbs. It was found in indirect ELISA that the immunoreactivity of the eluate of the first peak was four to five fold higher than that of the original ascites with equimolar protein concentration. Double antibody sandwich ELISA analyses of the immunoreactivity of HRP-conjugated IgM-monoclonal antibodies with lambda chain denoted that the purified IgM-McAb-HRP-conjugate might be of practical value in quantitative as well as qualitative assay of lambda chains.

Simplified procedure for purification ofBordetella bronchisepticadermonecrotic toxin

Bordetella bronchiseptica dermonecrotic toxin was purified by a simplified method. The method consisted of SP Toyopearl 650M chromatography and high performance liquid chromatography on a TSK gel G3000SW column. 47.5% of the activity of the crude cell extract was recovered. The purified toxin behaved as a homogeneous protein in sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance liquid chromatography, and agar gel double diffusion tests.

Simplified procedure for purification of Bordetella bronchiseptica dermonecrotic toxin

Bordetella bronchiseptica dermonecrotic toxin was purified by a simplified method. The method consisted of SP Toyopearl 650M chromatography and high performance liquid chromatography on a TSK gel G3000SW column. 47.5% of the activity of the crude cell extract was recovered. The purified toxin behaved as a homogeneous protein in sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance liquid chromatography, and agar gel double diffusion tests.

Detection of Petunia Asteroid Mosaic, Carnation Ringspot and Tobacco Necrosis Viruses in Ditches and Drainage Canals in a Grapevine‐Growing Area in West Germany

AbstractA number of virus isolates were obtained from ditches and drainage canals in the Palatinate grapevine‐growing area (‘Rheinpfalz’) in West Germany. By means of the immunoelectron microscopical decoration and the agar gel double diffusion tests these isolates were identified as petunia asteroid mosaic virus, carnation ringspot virus and tobacco necrosis virus strain D, respectively.

A Strain of Eggplant Mosaic Virus Causing a Severe Disease of Tomato in Argentina

AbstractA virus causing a severe disease of tomatoes in Argentina was identified as a strain of eggplant mosaic virus (EMV). It resembles the type, Abelia latent and various Andean potato latent strains of EMV in its host range and its transmissibility at allow rate by Epitrix sp. It differs from these strains, however, serologically and in some of its cytopathic effects. In serological agar gel double diffusion tests it proved to be closely related to the tomato white necrosis isolate of EMV studied by Barradas (1983) in Brazil.

Antigenic characteristics of larval Paragonimus westermani.

The characteristics of the antigens in larval flukes of Paragonimus westermani were examined. The agar gel double-diffusion test and competitive enzyme-linked immunosorbent assay, using the antiserum against the antigen extracted from larval flukes, showed cross-reactivity between the larval and adult antigens. In immunoblotting analysis, there was a decrease in reactivity of the antiserum absorbed with the adult fluke antigen against the band with a molecular weight of 26, 000. However, the bands corresponding to 34, 000 or higher molecular weights were still detected. The immunoperoxidase-staining technique revealed that the antigens reacting to the unabsorbed and absorbed antisera were both located on the surface of the gut epithelium and in the luminal contents of larval flukes. These results suggest that the extract of larval P. westermani possesses at least two antigens, originating probably from the gut, one being common to the adult and the other characteristic of the larva.

Properties of a new strain of tobacco streak virus from Clematis vitalba (Ranunculaceae)

SUMMARYA distinct strain of tobacco streak virus (TS V/Cle), isolated in Yugoslavia from wild Clematis vitalba showing chlorotic spots or yellow netting of the leaves and from many symptomless shrubs, is described.TSV/Cle was seed transmitted in C. vitalba (70%), and in the experimental hosts Chenopodium quinoa (80%), Nicotiana benthamiana and N. megalosiphon. It was also detected in the pollen of infected C. quinoa. Purified virus preparations mostly contained quasi‐spherical particles measuring 24–26 × 28, 26–28 × 28–30 and 28–31 × 32–36 mn, and sedimented in sucrose density gradient and analytical centrifugation as three components with sedimentation coefficients of 76S, 87S and 98S. The virus contained a single polypeptide species of mol. wt of c. 25 000. Unfractionated TSV/Cle preparations contained four RNA species with mol. wts, estimated by gel electrophoresis in agarose, of 1.1 × 106, 0.9 × 106, 0.7 × 106 and 0.3 × 106. In comparative experiments, TSV/Cle differed from four reference strains of TSV (TSV/B, TSV/HF, TSV/RN, and TSV/Ro) in host range and in symptoms induced in some common hosts. In agar gel double diffusion tests it was more closely related to TSV/B and TSV/M (SDI = 5) than to TSV/HF (SDI = 7), TSV/RN (SDI = 7) or TSV/Ro (SDI = 5–8). Immunoelectrophoresis experiments clearly distinguished TSV/Cle from the reference strains. TSV/Cle strain was detected in C. vitalba plants from distant and climatically different regions in Yugoslavia.

Serological Relationships among Tombusviruses

Summary Serological differentiation indices (SDIs) based on serum titrations in agar-gel double diffusion tests were determined for all ten definitive tombusviruses known at present, namely artichoke mottled crinkle, carnation Italian ringspot, cymbidium ringspot, eggplant mottled crinkle, Moroccan pepper, pelargonium leaf curl, petunia asteroid mosaic, the BS3 and type strains of tomato bushy stunt, and tombus Neckar viruses. More than 2000 titrations using 222 antisera from 36 rabbits were done. The SDIs ranged from 1 to >9, and all intermediate values were found. Simple trigonometry and computer methods were used to classify the results. There was no correlation between the serological relatedness of the particles of these viruses and the homology of their genome nucleic acids previously assessed by hybridization tests. In this respect they resemble the tymoviruses but not the tobamoviruses. The electrophoretic migration of the particles of 12 tombusviruses was studied in 1% agarose containing 20 mm-phosphate buffer pH 7.0. Some serologically indistinguishable tombusvirus strains migrated at different rates.

Serum antibodies and their role in antibody-dependent cell-mediated cytotoxicity in aspergillosis

A total of 22 sera from patients with aspergilloma and allergic bronchopulmonary aspergillosis (ABPA) were examined concomitantly for specific antibody against Aspergillus fumigatus antigen and for their activity in antibody-dependent cell-mediated cytotoxicity (ADCC) against Aspergillus antigen-coated target cells. These sera demonstrated significant precipitin bands in agar gel double diffusion test (78% of ABPA and 75% of aspergilloma sera), while in indirect immunofluorescence studies all sera showed positive reactivity with a titer distribution of 1:40 to 1:160 and 1:40 to 1:320, respectively, for ABPA and aspergilloma sera. In enzyme-linked immunosorbent assay all sera demonstrated titers varying from 1:200 to 1:6400. Several sera also displayed marked cytotoxic reactions against A. fumigatus antigen coated SB target cells in ADCC assays using normal lymphocytes as effector cells (35% of aspergilloma and 25% of ABPA sera). These findings suggest a role for ADCC activity in patients with Aspergillus infections.

Purification and serology of a Japanese isolate of barley yellow dwarf virus.

A Japanese isolate of barley yellow dwarf virus (BYDV) was purified from infected plants by an improved procedure. The procedure included extraction of the virus by grinding the frozen plant tissues in a meat grinder followed by regrinding in phosphate buffer with motar and pestle for extended periods (more than 2hr) in room temperature, clarification of the sap with chloroform, concentration of the virus by polyethylene glycol, two cycles of differential centrifugation and a sucrose density gradient centrifugation. The average yield of the virus was 44μg per 100g tissues. The Japanese isolate had particle diameter of 27.3nm (2% PTA, pH 5.0) and UV absorbance spectrum with an average A260/A280 ratio of 1.71. In ELISA using the double antibody sandwich method, the virus reacted only with homologous antiserum but not with antiserum to potato leafroll virus (PLRV), and vice versa. The virus, however, reacted slightly with heterologous antisera to other luteoviruses such as PLRV, soybean dwarf and beet western yellows viruses in agar gel double diffusion tests.

Detection of rice grassy stunt virus in planthopper vectors and rice plants by ELISA.

Filamentous particles associated with rice grassy stunt were purified from the infected rice plants. The filamentous particles were 6-12nm in width. An antiserum to the filamentous particles specifically reacted to extracts of grassy stunt-infected leaves and -viruliferous planthoppers, Nilaparvata lugens by agar gel double diffusion test. The antibody neutralized the infectivity in extracts of grassy stunt-infected rice leaves. In enzyme-linked immunosorbent assay (ELISA), the filaments were detected in extracts of grassy stunt-exposed planthopper population up to a dilution of one planthopper/8ml and also from extracts of the infected leaves at 1/100, 000 dilution. Extracts of virus-free insects and healthy rice leaves did not give a positive reaction in ELISA. The purified filamentous nucleoprotein reacted positively even when diluted to A260=1.0×10-5. The infective planthoppers stored at 25C for 1 month and the infected rice leaves stored at room temperature for 4 months gave positve reactions, but the intensity of the reactions was lower than that in fresh or frozen materials. In the exposed planthopper populations, 40% of the insects transmitted the causal agent and gave a positive reaction in ELISA, 41% failed to transmit the agent but gave a positive reaction, while both tests were negative in the remainder. The brown planthoppers that migrated across the South China Sea were collected and tested for the presence of the filaments in ELISA in 1982. About 0.1% of the total 1, 126 individuals tested gave positive reaction.

Antigenic cross-reactions between Mycoplasma arthritidis and rat tissues

An antigenic relationship between Mycoplasma arthritis and rat tissues could be demonstrated using the enzyme-immune assay and the agar gel double diffusion test. Cross-reactions were observed with joint homogenates as well as with muscle, lung, liver, kidney, spleen and brain tissue from rats. In addition, antibodies against joint homogenate were found in the sera of rats infected with M. arthritidis ISR 1 using the indirect hemagglutination test. The significance of common antigens between M. arthritidis and rat tissues for the pathogenesis of the M. arthritidis polyarthritis in rats is discussed.

Natural Infection of Tomato and Pelargonium in Germany by a Tombusvirus Originally Described from Pepper in Morocco

AbstractTwo serologically apparently identical tombusviruses were isolated from glasshousegrown tomato plants showing mottle and malformation of the upper leaves and stunting of the shoot tips and, at a low rate, from a pelargonium plant, respectively. Both plant species were received from growers in southern Germany. In agar gel double diffusion tests and immuno‐electrophoresis, the two virus isolates could not be distinguished from a tombusvirus (MPV) isolated from pepper in Morocco by Fischer and Lockhart (1977), although there were some minor differences in the reported host responses. In the tomato planting, strong symptoms were produced only temporarily. The majority of the 104 tomato plants of 15 different cultivars which had become infected by MPV following mechanical inoculation of the leaves showed only mild symptoms in contrast to a similar group of plants which developed severe stunting and foliar deformation when infected with the type strain of tomato bushy stunt virus. MPV was translocated only occasionally to the upper parts of tomato seedlings which had been grown in virus‐containing soil, although root infections were more frequent. MPV is apparently not a major threat to tomato cultivation in Germany.