Comparison of the nucleotide sequence of the coat protein open reading frame of nine isolates of wheat streak mosaic rymovirus

Wheat streak mosaic rymovirus (WSMV) is an important pathogen of wheat (Triticum aestivum L.). The coat protein region of nine isolates of WSMV were cloned by RT-PCR using primers that were inclusive of nucleotides 398–1825 (1) and sequence analysis indicated four regions of variability among the isolates.     

The production and characterisation of monoclonal antibodies to wheat gliadin peptides

BALB/c mice maintained on a gluten-free diet were immunised with Frazer's Fraction III (FFIII, a peptic tryptic digest of wheat gluten that exacerbates coeliac disease) in order to overcome oral tolerance. A control group was maintained on normal diet. Serum antibody titres to FFIII were higher in the mice on a gluten-free diet (P less than 0.05). Three monoclonal antibodies to FFIII were produced from splenocytes obtained from mice maintained on a gluten-free diet. The antibodies were characterised by ELISA, immunodot assay and immunoblotting with prolamins from cereals toxic to coeliac patients (wheat, rye, barley, oats) and the non-toxic prolamins from maize and rice. The binding characteristics of the three antibodies to the cereal prolamins were different, implying that the antibodies recognise different cereal protein epitopes. Immunoblotting revealed FFIII to be comprised of antigenically dissimilar peptides.     

Monoclonal antibody based two‐site enzyme immunoassays for wheat gluten proteins. 1. Kinetic characteristics and comparison with other Elisa formats

Solid‐phase two‐site enzyme immunoassays were developed using a number of monoclonal antibodies with differing specificities for wheat grain gluten proteins. While polystyrene microwells performed well in the assay, very high levels of non‐specific binding occured with PVC (Polyvinylchloride) microwells in the absence of ‘capture’ (solid‐phase bound) antibody. This was due to binding of complexes of antigen and labelled antibody, and could only be reduced by the use of rather stringent blocking conditions. Assays involving either the simultaneous or sequential addition of antigen and labelled antibody were developed, the latter providing superior results. A number of successful two‐site assays were constructed using single monoclonal antibodies, suggesting that repeating epitopes, known to occur in gluten proteins, were commonly recognized. Pairs of monoclonal antibodies with dissimilar antigenic specificites did not function in the two‐site assay. A comparison of monoclonal antibody based two‐site and antigencompetition immunoassays for gliadin was made. Several antibodies performed considerably better in one format than the other; thus, both assay formats will be of use in quantification of specific gluten protein subclasses.     

Three distinct mechanisms facilitate genetic isolation of sympatric wheat streak mosaic virus lineages

Cross-protection and vector transmission bottlenecks have been proposed as mechanisms facilitating genetic isolation of sympatric viral lineages. Molecular markers were used to monitor establishment and resolution of mixed infections with genetically defined strains of wheat streak mosaic virus (WSMV). Two closely related WSMV strains from the U.S. (Type and Sidney 81) exhibited reciprocal cross-protection in wheat, confirming this classic phenomenon as a mechanism of genetic isolation. In contrast, cross-protection between either U.S. strain and the divergent El Batán 3 strain from Mexico was unilateral, erratic, and only partially effective. Distribution of WSMV strains within individual leaves of plants supporting a mixed infection of Type and Sidney 81 was spatially nonuniform. Strain distribution among individual tillers of coinfected plants also was heterogeneous, with some containing either Type or Sidney 81 alone and some containing both. Transmission by wheat curl mites, acquiring virus from source plants simultaneously infected with both Type and Sidney 81, often resulted in test plants bearing only a single WSMV strain. Spatial subdivision of virus strains within coinfected plants likely contributed to vector transmission bottlenecks during acquisition. Collectively, these three distinct mechanisms enhance genetic isolation of individual viral lineages, and together with stochastic processes, may explain generation and maintenance of genetic diversity in field populations.     

Determination of IgG subclass antibodies against wheat flour antigens by an ELISA technique

We describe the assay conditions for an enzyme-linked immunoassay for the determination of IgG and IgG subclass antibodies in serum to water-soluble wheat flour antigens. The optimal antigen coating concentration was 5 micrograms/ml for total IgG, IgG1, IgG4 and 100 micrograms/ml for IgG2. Serial dilutions of test sera were used and commercially available monoclonal mouse anti-human IgG isotype antibodies (as ascites fluid) were diluted 1:500-1:1000. Specific wheat flour antibodies belonging to the IgG1, IgG2 and IgG4 subclasses were detected. Despite the lack of standardized isotype-specific second mouse monoclonal antibodies, the subclass antibody levels between flour-exposed bakers and controls could be compared. We observed significantly higher IgG1, IgG2, and IgG4 subclass antibodies among 23 bakers than among 12 non-exposed controls, but no IgG3 antibodies were detected. The differences in biological activities of the IgG subclass antibodies may explain the clinical and pathophysiological features for fluor-induced occupational allergic diseases among bakers.     

The use of transglutaminase in the reduction of immunoreactivity of wheat flour

The immune response of wheat flour modified by the treatment with transglutaminase under different conditions of temperature, incubation periods and the ratio of enzyme/wheat flour was investigated. The particular wheat protein fractions were examined for the immune reaction by the use of an indirect non-competitive ELISA. Commercially available antibodies, namely, monoclonal antihuman IgG and monoclonal antihuman IgE conjugates with alkaline phosphatase and human sera with elevated IgG as well as rabbit sera against QQQPP peptide were tested. The highest decrease in gliadins immunoreactivity was observed for wheat flour modified under following conditions: temperature 37°C, 18 h of incubation and the ratio enzyme/wheat flour 1:10 000. For all rabbit sera examined the residual immunoreactivity of glutenins was found to be below 30% of the level measured for the untreated protein. The large decrease in allergenicity of glutenins leads to the conclusion that wheat flour modified by treatment with transglutaminase may be used as a constituent of food products destined for people with a classic food allergy, i.e. the allergy elicited by that protein fraction.     

Multiple interactions among proteins encoded by the mite-transmitted wheat streak mosaic tritimovirus

The genome organization of the mite-transmitted wheat streak mosaic virus (WSMV) appears to parallel that of members of the Potyviridae with monopartite genomes, but there are substantial amino acid dissimilarities with other potyviral polyproteins. To initiate studies on the functions of WSMV-encoded proteins, a protein interaction map was generated using a yeast two-hybrid system. Because the pathway of proteolytic maturation of the WSMV polyprotein has not been experimentally determined, random libraries of WSMV cDNA were made both in DNA-binding domain and activation domain plasmid vectors and introduced into yeast. Sequence analysis of multiple interacting pairs revealed that interactions largely occurred between domains within two groups of proteins. The first involved interactions among nuclear inclusion protein a, nuclear inclusion protein b, and coat protein (CP), and the second involved helper component-proteinase (HC-Pro) and cylindrical inclusion protein (CI). Further immunoblot and deletion mapping analyses of the interactions suggest that subdomains of CI, HC-Pro, and P1 interact with one another. The two-hybrid assay was then performed using full-length genes of CI, HC-Pro, P1, P3, and CP, but no heterologous interactions were detected. In vitro binding assay using glutathione-S-transferase fusion proteins and in vitro translation products, however, revealed mutual interactions among CI, HC-Pro, P1, and P3. The failure to detect interactions between full-length proteins by the two-hybrid assay might be due to adverse effects of expression of viral proteins in yeast cells. The capacity to participate in multiple homomeric and heteromeric molecular interactions is consistent with the pleiotropic nature of many potyviral gene mutants and suggests mechanisms for regulation of various viral processes via a network of viral protein complexes.     

Use of monoclonal antibody in a blocking ELISA to detect group specific antibodies to bluetongue virus

An enzyme-linked immunosorbent assay (ELISA) in the form of a blocking test is described for the detection of group specific antibodies to bluetongue virus (BTV). The test relies upon interruption of the reaction between BTV antigen and a group specific murine monoclonal antibody against BTV by addition of serial dilutions of bovine or ovine test sera containing specific antibodies to BTV which inhibit binding of the monoclonal antibody to the BTV antigen. This is detected as a reduction in the optical density (O.D.) reading obtained with the monoclonal antibody alone. The test is capable of specific detection of antibodies to all 22 serotypes of BTV but, unlike the agar gel precipitin (AGP) test, does not show cross-reactions with antibodies to epizootic haemorrhagic disease of deer (EHD) viruses. Furthermore, antibodies to cellular proteins which complicate interpretation of the AGP test and the indirect ELISA are not detected in the blocking ELISA. The high sensitivity and specificity of the blocking ELISA make it an ideal alternative to the AGP test. The use of a monoclonal antibody would facilitate standardisation of diagnostic testing between laboratories.     

Colony establishment and maintenance of the eriophyid wheat curl mite Aceria tosichella for controlled transmission studies on a new virus-like pathogen

High plains disease (HPD) is of serious economic concern for wheat and corn production, but little is known about the virus-like causal agent. In the field, HPD is often associated with Wheat streak mosaic virus (WSMV) and both pathogens are transmitted by the same eriophyid wheat curl mite, Aceria tosichella Keifer. The objective of this study was to develop methods for establishing and maintaining HPD-transmitting wheat curl mite colonies for their use in studies on HPD. Towards this goal, mite colonies from a mixed infection source were separated into colonies either (i). not viruliferous; (ii). only transmitting WSMV; or (iii). only transmitting HPD. Maintenance of these colonies required strictly separated incubator facilities and adaptation of mite-suitable transfer techniques to permit frequent passages of mites to healthy plants. The established colonies provided reliable sources of infective material to study the progression of HPD and/or WSMV in plants using sensitive immuno-detection assays. In conclusion, we have developed reliable methods with a poorly studied arthropod vector to examine the biology and properties of a new virus-like disease.     

Characterization of the translocated chromosome using fluorescencein situ hybridization and random amplified polymorphic DNA on twoTriticum aestivum—Thinopyrum intermedium translocation lines resistant to wheat streak mosaic or barley yellow dwarf virus

Fluorescencein situ hybridization (FISH) was used to determine the breakpoint of the translocation chromosome in two bread wheat (Triticum aestivum) germplasm lines withThinopyrum intermedium chromatin carrying resistance to either wheat streak mosaic virus (WSMV) or barley yellow dwarf virus (BYDV). In addition, genome-specific random amplified polymorphic DNA (RAPD) markers were used to ascertain the genomic sources of theTh. intermedium chromosomes carrying the WSMV or BYDV resistance. CI17766, a WSMV-resistant wheat germplasm line derived from induced homoeologous pairing by using theph1b mutant, had a translocation chromosome composed of the complete 4AL and about 45% of proximal 4AS from wheat, and the entire 4ES ofTh. intermedium. The BYDV-resistant translocation line, TC14, derived from tissue culture, had a very short distal segment of 7StL fromTh. intermedium terminally attached to 56% of the proximal 7DL. These observations indicate that translocations in these wheat germplasm lines did not involve centromeric breaks and fusion but were a result of homoeologous chromosome recombination.accepted for publication by J. S. (Pat) Heslop-Harrison     

Three-site sandwich radioimmunoassay with monoclonal antibodies for a sensitive determination of human alpha-fetoprotein

The extensive use of monoclonal antibodies for the detection of human cell surface markers has necessitated the development of a simple, reproducible reagent which can be applied universally to any mouse anti-human antibody. We have developed a rosetting reagent which is composed of ox red blood cells, rabbit anti-ox RBC antibody, protein A, and rabbit anti-mouse Ig antibody. This reagent binds specifically to human lymphocytes coated with mouse monoclonal antibody, is as sensitive as immunofluorescence and gives comparable results with fresh and frozen peripheral blood lymphocytes. Sensitivity, reliability, and ease of quantitation make this an extremely practical reagent, and its versatility makes it especially useful for large scale studies of human T cell markers.     

Substitution of conserved cysteine residues in wheat streak mosaic virus HC-Pro abolishes virus transmission by the wheat curl mite

Summary Substitutions in the amino-proximal region of wheat streak mosaic virus (WSMV) HC-Pro were evaluated for effects on transmission by the wheat curl mite (Aceria tosichella Keifer). Alanine substitution at cysteine residues 16, 46 and 49 abolished vector transmission. Although alanine substitution at Cys₂₀ had no effect, substitution with arginine reduced vector transmission efficiency. Random substitutions at other positions (Lys₇ to Asn, Asn₁₉ to Ile, and Arg₄₅ to Lys) did not affect vector transmission. These results suggest that a zinc-finger-like motif (His₁₃-X2-Cys₁₆-X29-Cys₄₆-X2-Cys₄₉) in WSMV HC-Pro is essential for vector transmission.     

High level expression of equine herpesvirus 1 glycoproteins D and H and their role in protection against virus challenge in the C3H (H-2K) murine model

N and C-terminal truncated forms of equine herpesvirus 1 (EHV 1) glycoproteins gD and gH were expressed in baculovirus resulting in the production of secreted recombinant proteins. A carboxy-terminal histidine tag was included on each of the genes for protein isolation by nickel affinity chromatography. Recombinant gD was recognized by three gD specific monoclonal antibodies, 20C4, 5H6 and F3132. F3132 is a conformationally dependent monoclonal antibody with virus neutralizing activity. Expression of gH was confirmed by reacting the protein with the gH peptide specific antiserum R319. The truncated gD gene was also expressed as a β-galactosidase fusion protein which was purified from E. coli by nickel affinity chromatography. C3H mice were inoculated with purified recombinant gD or gH or insect cells which had been infected with recombinant baculoviruses. Mice were subsequently challenged with EHV 1. Purified recombinant baculovirus gD provided the most protection and produced high levels of virus neutralizing antibodies. The gD fusion protein was less effective at protecting mice and insect cells infected with either of the recombinant baculoviruses or purified recombinant gH were poor at conferring protection. The results emphasize the importance of using purified proteins in vaccine formulations and of including EHV 1 gD as a component of a subunit vaccine.     

抗人GCRG213单克隆抗体的制备与鉴定

目的:制备GCRG213单克隆抗体(mAb)并进行初步鉴定。方法:在大肠杆菌中表达HIS-GCRG213融合蛋白,并以所获蛋白作为免疫原制备鼠mAb。采用ELISA、Westernblot法鉴定抗体的效价及特异性。免疫组化染色观察GCRG213在胃癌和正常组织中的表达。结果:HIS-GCRG213融合蛋白在大肠杆菌中获得高效表达,经常规的细胞融合和筛选获得2株可稳定分泌抗GCRG213的杂交瘤细胞株。ELISA法检测腹水的效价可达到1∶106,Western blot证实该抗体可与重组HIS-GCRG213蛋白特异性结合。免疫组化染色显示GCRG213在胃癌组织中的表达明显强于正常胃黏膜组织。结论:成功地制备出2株抗GCRG213的mAb,为进一步研究GCRG213的生物学功能提供了有效的工具。     

Western immunoblotting of cereal proteins with monoclonal antibodies to wheat gliadin to investigate coeliac disease

Western immunoblotting was used to investigate the binding of two monoclonal antibodies raised against unfractionated wheat gliadin to different cereal protein fractions separated by SDS-PAGE. Our results confirm the presence of considerable epitope sharing between the gliadin subfractions as well as barley and rye prolamins; however, there was less binding of these antibodies to bands present in oat avenins and maize zeins. The pattern of binding of one of these two antibodies to different cereal prolamins as well as to Frazer's fraction III corresponds closely to the known toxicity of these proteins to patients with coeliac disease.     

Production of polyclonal and monoclonal antibodies against CD54 molecules by intrasplenic immunization of plasmid DNA encoding CD54 protein

DNA immunization, in theory, is of great interest as a source of specific antibodies against different antigens. In an attempt to produce polyclonal and monoclonal antibodies against cell surface molecules by using the DNA immunization strategy, intramuscular and intrasplenic routes of DNA injection were compared. Two to five, but not a single, intramuscular DNA immunizations induced anti-CD54 and anti-CD147 antibody production. In contrast, a single intrasplenic immunization of CD54-encoding DNA could induce anti-CD54 antibody production. To produce monoclonal antibody (mAb), spleen cells obtained from an intrasplenic CD54-encoding DNA immunized mouse were fused with myeloma cells using the standard hybridoma technique. A hybridoma secreting specific mAb to CD54 was established. The generated mAb reacted to CD54 protein expressed on transfected COS cells and various cell types, the same as using standard CD54 mAb MEM-111. Our results demonstrated that direct immunization of antigen-encoding DNA into spleen is an effective route for production of both polyclonal and monoclonal antibodies to cell surface molecules. This finding is very useful for the production of antibodies to cell surface molecules where the protein antigen is not available or difficult to prepare, but cDNA encoding the corresponding protein is available.     

Immunological appraisal of wheat varieties in relation to chapatti-making characteristics

The current research work was conducted to characterize wheat proteins through immunochemical techniques and to find out their relationship with wheat quality traits. The results revealed that wheat variety AARI-11 possessed higher protein content (11.96%), wet gluten (31.39%), dry gluten (9.66%), Pelshenke value (190.52 min), and SDS-Sedimentation value (28.27 ml) than other tested varieties. The chapattis prepared from the wheat variety AARI-11 got significantly higher sensory scores owing to its higher protein contents. The wheat variety AARI-11 also exhibited significantly the highest antibody response against all the assessed protein fractions. The results of the present study suggest that anti-glutenin and anti-high molecular weight glutenin subunits (HMW-GS) antibody response was found positively correlated to the quality characteristics of flours and chapattis. The present study suggests that the use of antibodies response against glutenin and HMW-GS offers good tool for predicting quality and suitability of wheat to chapatti-making quality.     

Monoclonal antibodies to chromaffin cells can distinguish proteins specific to or specifically excluded from chromaffin granules

I have prepared a number of monoclonal antibodies to chromaffin cell membranes. One of these antibodies recognizes a number of antigenically related proteins that are present in all tissues examined. In the adrenal, these proteins are completely excluded from chromaffin granules but are present in other subcellular membrane fractions. This non-granule membrane-specific antibody has been designated NG3. A second antibody, CG7, binds to a single protein which segregates specifically into chromaffin granules. The protein recognized by CG7 is cytochrome b561, or chromomembrin B, one of the major protein components of chromaffin granule membranes. CG7 also labels a protein (the identical cytochrome b561) in bovine posterior pituitary neurosecretory vesicle membranes indicating that it functions in both peptidergic and catecholaminergic secretory granules. These two monoclonal antibodies provide useful probes of both granule and extra-granule membrane proteins for studies of membrane trafficking in chromaffin cells.     

Production and characterization of monoclonal antibodies directed against pseudorabies virus

Hybridoma cell lines producing monoclonal antibodies to pseudorabies virus (PRV) were established. The monoclonal antibodies were characterized with respect to their antigenic specifications and biological activities. Two monoclonal antibodies immunoprecipitated the 50 kDa PRV glycoprotein (gp50) and two immunoprecipitated the 82 kDa glycoprotein (gp82). The monoclonal antibodies were used to analyze the biological roles of these two glycoproteins. One monoclonal antibody directed against each glycoprotein did not require complement for in vitro viral neutralization while the other monoclonal antibody directed against the glycoprotein required complement for neutralization. The monoclonal antibodies against gp50 were shown to be directed against different epitopes within the glycoprotein. In contrast, the monoclonal antibodies against gp82 were shown to be directed against the same antigenic site on the glycoprotein. In vivo passive immunity studies in mice showed that monoclonal antibodies directed against either gp50 or gp82 could be protective.     

The development of monoclonal antibodies to respiratory syncytial virus and their use in diagnosis by indirect immunofluorescence

Twelve clones of murine hybridoma cells secreting antibody specific for respiratory syncytial (RS) virus were classified into four groups on the basis of their pattern of staining of unfixed RS virus-infected HEp-2 cells in an indirect immunofluorescence test. Three of the groups reacted with virus antigens present on the membrane of the cells, whilst the fourth group failed to stain most live cells, suggesting specificity for an antigen expressed internally. Representative monoclonals from the membrane antigen staining groups immunoprecipitated the 86K glycoprotein (G), 50K plus 19K glycoprotein (F1,2) and a 23K non-glycosylated protein (VP23). A representative monoclonal from the fourth group that appeared to stain an internally expressed protein immunoprecipitated the virion 34K phospho-protein (P). All four monoclonals stained acetone-fixed tissue culture cells infected with either the Long strain of RS virus or with strains isolated in Newcastle during the 1965, 1972, and 1983 winter epidemics. The anti-fusion protein antibody stained acetone-fixed cells from all of 26 nasopharyngeal secretions from infants with RS virus infection. The anti-G glycoprotein antibody and the anti-VP23 antibody stained cells from secretions poorly or not at all, whilst the anti-P protein antibody stained cells in half the secretions tested but reacted with only a small proportion of cells in comparison with the anti-F or polyclonal antibodies. A pool of all four monoclonals produced more intense staining than the anti-F monoclonal alone and gave a more clearly defined staining reaction than the polyclonal antiserum used for routine diagnosis in over half the secretions. These results indicate that monoclonal antibodies will be of value in the diagnosis of RS virus by indirect immunofluorescence if care is taken in the selection of a suitable pool.