The Use of Monoclonal Antibodies to Detect Wheat Soil-borne Mosaic Virus

Transcription

1 J. gen. Virol. (1988), 69, Printed in Great Britain 1317 Key words: monoclonal antibodies~wheat soil-borne mosaic virus/elisa The Use of Monoclonal Antibodies to Detect Wheat Soil-borne Mosaic Virus By Z. BAHRANI, J. L. SHERWOOD,* M. R. SANBORN 1 AND G. C. KEYSER Department of Plant Pathology and ~ Department of Botany and Microbiology, Oklahoma State University, Stillwater, Oklahoma 74078, U,S.A. (Accepted 3 March 1988) SUMMARY Stable hybridoma cell lines secreting monoclonal antibodies (MAbs) to wheat soilborne mosaic virus (WSBMV) were produced by fusing spleen cells of immunized BALB/c mice and mouse myeloma cell line P3-X63-Ag Hybridoma clones produced antibodies of the IgG2a, IgG2b and IgG3 subclasses. Epitope analysis suggested that all the antibodies tested reacted with the same or closely adjacent antigenic sites. The MAbs reacted with particles of four isolates of WSBMV, but not with particles of 13 other viruses. The serological reactivities of the MAbs were compared with those of rabbit polyclonal antibodies for the detection of WSBMV in leaf tissue by ELISA. ELISA using both polyclonal antibodies and MAbs was superior to assays using either source of antibody alone. The MAbs of the IgG2b subclass worked well in a Protein A sandwich ELISA, and a dot immunobinding assay using only MAbs was also satisfactory for the detection of WSBMV. INTRODUCTION Infection with wheat soil-borne mosaic virus (WSBMV) causes mosaic, stunting and rosetting symptoms that are most prominent on spring growth of winter wheat. Yield losses of up to 50 have been attributed to WSBMV infection in the United States, Japan and Italy (Brakke, 1971 ; Palmer & Brakke, 1975; Campbell et al., 1975). The virus has a bipartite RNA genome which is encapsidated as two rods with lengths of 281 nm and 138 nm or less by a single coat protein species (Shirako & Brakke, 1984). The plasmodiophoraceous fungus Polymyxa graminis Led. acts as a vector for the virus which persists in the fungus in infested fields (Estes & Brakke, 1966; Rao & Brakke, 1969; Brakke et al., 1965). The symptoms of WSBMV infection in wheat are similar to those of other viruses and also to symptoms produced as a result of agronomic problems such as nutrient imbalance. Hence, screening germplasm entries for resistance to WSBMV by visual assessment is not always reliable (Hunger & Sherwood, 1985a, b, 1986). Serology provides a useful technique for the routine detection of plant viruses. Monoclonal antibody (MAb) technology provides a means to produce a supply of highly specific uniform antibody which can be useful in the detection of plant viruses and which is required for a disease resistance screening programme (Halk & DeBoer, 1985). The purpose of this study was to produce hybridomas secreting WSBMVspecific MAbs, to test the reaction of selected MAbs to isolates of WSBMV, and to develop an ELISA and dot immunobinding assay for the detection of WSBMV in leaf tissue. Preliminary reports of this work have been published (Bahrani & Sherwood, 1985; Bahrani et al., 1986). METHODS Propagation and purification of viruses. The WSBMV isolate (WSBMV-OK) used for MAb production was maintained in wheat (Triticum aestivum cv. Vona) and purified as previously described (Hunger & Sherwood, 1985b). Other isolates of WSBMV were obtained from M. K. Brakke (University of Nebraska, Lincoln, Ne., U.S.A.), C. J. D'Arcy (University of Illinois, Urbana, I11., U.S.A.) and S. A. Lommel (Kansas State University, SGM

2 1318 Z. BAHRANI AND OTHERS Manhattan, Kan., U.S.A.). A virus morphologically similar to WSBMV which does not infect wheat was also obtained from S. A. Lommel. Other viruses used in serological assays were maintained in appropriate hosts. Polyclonal antibody production. A rabbit was given an initial injection of 2 mg of virus with Freund's complete adjuvant (half subcutaneously, half intramuscularly). This was followed by three subcutaneous injections at weekly intervals of 1 mg of virus plus Freund's incomplete adjuvant. The antiserum titre was measured by microprecipitin tests against purified WSBMV-OK and subcutaneous boosters were given as above when the antiserum titre fell below 1/512. Hybridoma production, screening and isotyping. The mouse myeloma cell line P3-X63-Ag8.653 (653) was provided by E. L. Halk (Agrigenetics Corporation) and maintained as described previously (Sherwood et al, 1987). BALB/c mice were immunized by intramuscular injection with 250 ~tg of WSBMV-OK emulsified with Freund's complete adjuvant. Three injections were made 1 week apart, followed by an injection of 250 ~tg of virus in distilled water 3 days before fusion. The procedure for cell fusion was adapted from the method of K6hler & Milstein (1975) with the modifications of Sherwood et al (1987). Approximately 2 weeks after fusion, hybridornas were screened for antibody production by an indirect double sandwich ELISA. After coating of plates with rabbit anti-wsbmv IgG at 1 ~tg/ml for 2 h, purified WSBMV (3 I-tg/ml) was added to the wells. The plates were then stored at 4 ~C overnight, washed three times and then blocked with 1 ~ bovine serum albumin in phosphate-buffered saline (PBS) for 10 min. Undiluted culture supernatant was added and after incubation for 2-5 h, this was replaced by alkaline phosphatase-linked goat anti-mouse IgG at the manufacturer's recommended working dilution (Sigma). After an additional 2.5 h incubation the enzyme substrate was added. Hybridoma cell lines that gave positive reactions were cloned by the soft agar method (Sherwood et al., 1987). Single colonies were picked and transferred into 1 ml of SP medium in 24-well plates. The culture supernatants were retested for antibody production and positive cell cultures were recloned twice. Monoclonal antibody isotypes were determined using subclass-specific antisera (Zymed Laboratories, South San Francisco, Ca., U.S.A.). Ascites production and MAb purification. Ascitic fluid containing MAbs was produced in pristane-primed BALB/c mice by injecting approximately 106 hybridoma cells into the peritoneal cavity and then collecting ascitic fluid after 2 to 3 weeks (Halk et at., 1984). Monoclonal antibody was obtained from the ascitic fluid using an IgG affinity purification kit from HyClone Laboratories or by precipitation with ammonium sulphate. To obtain antibody from clones 5A1 and 5A24, the hybridomas were grown in HL-1 serum-free medium (Ventrex). Antibody was precipitated from 45~ ammonium sulphate, dissolved in PBS and dialysed against PBS. Monoclonal antibody was also obtained from culture fluid by precipitation with ammonium sulphate followed by fractionation on DEAE Affi-Gel Blue IgG (Bio-Rad). Epitope analysis. To determine whether the five MAbs to WSBMV recognized the same or different antigenic sites an ELISA additivity assay, modified after Friguet et al. (1983), was used. In this assay each MAb served as a first and second antibody so that each MAb was tested against all other MAbs. After coating with WSBMV at 100 I-tg/ml, the plates were incubated overnight at 4 C, washed with PBS Tween, and an amount of first antibody, known from prior tests to saturate the bound virus, was added. After a 2 h incubation at room temperature, the plates were incubated for another 2 h with the second antibody, followed by alkaline phosphatase-labelled goat anti-mouse antibody. After an overnight incubation at 4 C, the enzyme substrate was added, and the reaction was stopped by the addition of 5 M-NaOH after 30 rain. The absorbance at 405 nm was read for each well in a Bio-Tek EIA plate reader. The additivity index compares the absorbance values obtained from the first antibody alone, the second antibody alone, and the two antibodies together. The index can range from 0 to 1009o; 0% indicates that the two MAbs bind randomly to the same site and 100~ indicates the MAbs bind independently at distinct sites (Friguet et al., 1983). ELISA. Direct and indirect double sandwich ELISA procedures were modified from Clark & Adams (1977) and Protein A sandwich (PAS)-ELISA from Edwards & Cooper (1985). Alkaline phosphatase-labelled IgGs were prepared using 5000 units of alkaline phosphatase (Sigma) and 2 ml of 1 mg/ml IgG or an equivalent ratio according to Clark & Adams (1977). For the double sandwich ELISA, plates were coated with the IgG fraction from rabbit anti-wsbmv serum or MAb for 2 h at room temperature. Samples diluted in PBS-Tween containing 2 ~ polyvinylpyrrolidone (PVP) were then added. After incubation overnight at 4 C, alkaline phosphatase-labelled anti-wsbmv IgG from polyclonal serum, alkaline phosphatase-labelled anti- WSBMV MAb, or MAb culture fluid, diluted with PBS-Tween containing 2~ PVP and 0.2~ ovalbumin, was added. In the latter case, after a 3 h incubation alkaline phosphatase-labelled goat anti-mouse antibody was added and incubated for an additional 3 h. The alkaline phosphatase-labelled anti-wsbmv IgGs were incubated for 5 h. The enzyme substrate was added, and the absorbance at 405 nm was read. The optimum concentration of reagents in double antibody sandwich ELISA were 1.0 p_g/ml of IgG from polyclonal serum or MAb for capture antibody, alkaline phosphatase-linked IgG from antiserum at a 1 : 800 dilution, and the alkaline phosphatase-linked MAb at a 1:400 dilution. In the indirect double sandwich ELISA, samples were probed with undiluted culture fluid followed by alkaline

3 MAbs to wheat soil-borne mosaic virus 1319 phosphatase-labelled goat anti-mouse antibody. Indirect non-sandwich ELISA was conducted in a similar fashion but the sample was diluted 1 : 100 in carbonate buffer and incubated at 4 C overnight prior to continuing the assay. For PAS ELISA, plates were coated with Protein A (Sigma) at 1 ~tg/ml for 2 h at room temperature. The IgG fractions from serum-free medium in which clones 5A1 or 5A24 had been grown were diluted in PBS Tween and added. After incubation at room temperature for 2 h, samples at a 1:100 (w/v) dilution were added. Plates were incubated overnight at 4 C, IgG was added as above and, after incubation at room temperature for 2 h, Protein A alkaline phosphatase (Sigma) at 1 Ixg/ml in PBS Tween was added. Plates were incubated for 2 h at room temperature, and substrate was added as above. The optimum concentration of reagents in PAS ELISA were 10 rtg/ml capture antibody and 0.1 ~tg/ml probe antibody. Dot immunobinding assay. Samples were diluted in Tris-buffered saline (TBS) and centrifuged at g for 10 rain. Samples of 4 pl were spotted on the sample supports of filter paper or nitrocellulose and allowed to dry in air. Samples on nitrocellulose were soaked in a 5% suspension of non-fat dried milk for 30 rain. Samples were then incubated in a 1:100 dilution of culture fluid for 1 h, rinsed three times with TBS, and incubated for 1 h in either Protein A-peroxidase in TBS (2 ~tg/ml) or goat anti-mouse antibody-alkaline phosphatase (1:1000 dilution in TBS). Samples were rinsed three times in TBS and then incubated in the appropriate substrate buffer for Protein A-peroxidase (Sherwood, 1987), or for alkaline phosphatase (Leary et al, 1983). RESULTS AND DISCUSSION Characterization of MAbs Fifteen of 120 stable clones producing anti-wsbmv antibody were maintained. They produced immunoglobulin of the IgG2a, IgG2b or IgG3 subclasses. Five clones (8D67, 5A1, 5A24, 8A62, 5BC4) which produced IgG2a, IgG2b, IgG2b, IgG3 and IgG3, respectively, were selected to test for reaction to three other WSBMV isolates and other viruses in ELISA. Virusinfected and uninfected plant material was used at a 1 : 100 (w/v) dilution. In an indirect double sandwich ELISA, each of the five antibodies reacted strongly with the material infected with any of the four isolates of WSBMV, but not to uninfected wheat tissue, or material infected with 13 other viruses (Table 1). This indicates that all the WSBMV isolates tested are serologically related. Similar specific reactions were observed when indirect non-sandwich ELISA was used. Thus, the specificity of the assay was not dependent on the capture of the WSBMV antigens by the polyclonal serum. The differences in the A405 values obtained from the reaction of different MAbs to different isolates of WSBMV (Table 1) could be in part due to the state of the samples used for the ELISA. The material used in the assays had been frozen prior to shipment and was frozen (-20 C) upon arrival. The freezing and thawing cycles and differences in time of shipment may have resulted in modification of the viral antigen and subsequently the results obtained in the assay. Because WSBMV is not readily transmitted mechanically, comparison of the reaction between the various MAbs to the different isolates in fresh tissue was not feasible. We have detected changes in the reaction of MAbs to material infected with WSBMV caused by repeated freezing asnd thawing. The results of ELISA additivity assay indicated that all five MAbs probably react to the same or immediately adjacent antigenic sites. In the assay the majority of pair-wise combinations resulted in additivity indexes of 0~o. Five combinations did give indices greater than 0~o. These were 5A1-8D67 (16.35~), 5A24-SD67 (48.06~), 8A62-5BC4 (9.62~), 5BC4-8A62 (18.97~) and 5A24-5A1 (15.08~). The only reciprocal combination that gave responses greater than 0~ was 8A62-5BC4. The values obtained indicate that the antigenic sites are more likely to be similar or adjacent than distinctly different. Comparable additivity index values have been interpreted to indicate that antigenic sites were similar (Friguet et al., 1983). Comparisons of ELISAs employing polyclonal antibodies and/or MAbs Five different types of ELISA using either rabbit anti-wsbmv serum alone, MAb 8D67 alone, or the two in combination were compared for detection of WSBMV in leaf samples. All assays readily distinguished between healthy and infected material (Table 2). The assay employing rabbit antibody as both the coating antibody and probe antibody gave the highest background reading. In assays in which MAb 8D67 was conjugated to alkaline phosphatase, the colour developed about three times more slowly than in the other assays regardless of whether

4 1320 Z. BAHRANI AND OTHERS Table 1. Reaction of WSMV-specific MAbs in an indirect double sandwich ELISA* MAb from hybridoma clone h Sample t 8D67 5A24 5A 1 8A62 5BC4' WSBMV-OK 1.203~ WSBMV-NE WSBMV-IL WSBMV-KS Healthy wheat Other viruses < 0.02 ~< 0.02 ~< 0-03 ~< 0.03 ~< 0.03 * Plates were coated with rabbit polyclonal anti-wsbmv IgG at 1 ~tg/ml. Samples were probed with undiluted culture fluid followed by alkaline phosphatase-labelled goat anti-mouse antibody. t Samples were extracts (1 g/100 ml) of leaves infected with WSBMV Oklahoma isolate (OK), Nebraska isolate (NE), Illinois isolate (IL), Kansas isolate (KS), healthy wheat or other viruses which were an isolate of a virus from Kansas that is morphologically similar to WSBMV but does not infect wheat, wheat streak mosaic, wheat spindle streak mosaic, peanut stripe, peanut mottle, watermelon mosaic-l, potato Y, tobacco etch, tobacco mosaic, tobacco streak, tomato spotted wilt, cucumber mosaic, and brome mosaic. :~ Values are A4o5 averages of two experiments. Duplicate values were within 5% of each other. Table 2. Reaction of rabbit antiserum (RAb) with WSBMV and anti-wsbmv MAb 8D67 in different combinations in ELISA* Sample Probes MAb-Et RAb-E:~ MAb-Et RAb-E:~ Coatings MAb MAb RAb RAb WSBMV-infected 1 ' Healthy control PBS Time for reaction (min) Anti-MAb-E MAb RAb * Plates were coated with rabbit polyclonal anti-wsbmv IgG or MAb from mouse ascitic fluid anti-wsbmv IgG at 1 ~tg/ml. Samples were added at a 1:100 dilution. t Affinity-purified MAb conjugated to alkaline phosphatase and used at a 1:400 dilution. Alkaline phosphatase-labelled goat anti-mouse antibody. Ion-exchange-purified rabbit anti-wsbmv IgG conjugated to alkaline phosphatase and used at a 1 : 800 dilution. I[ Values are averages of three experiments plates were coated with antibody from rabbit serum or MAb. This was true whether the MAb was isolated by affinity chromatography, ammonium sulphate precipitation or by DEAE Affi- Gel Blue IgG chromatography. To determine whether this problem was specific for MAb 8D67, the other four MAbs were also conjugated to alkaline phosphatase. Although every pair-wise combination of coating MAb and MAb conjugated to alkaline phosphatase was tried the results were similar to those obtained with MAb 8D67. The direct double sandwich ELISA in which plates were coated with MAb and samples were probed with antibody from rabbit serum conjugated to alkaline phosphatase gave little background reaction and required the fewest steps. The indirect double sandwich ELISA in which plates were coated with antibody from rabbit serum and samples probed with unfractionated MAb culture supernatant fluid followed by alkaline phosphatase-linked goat anti-mouse IgG gave similarly quick detection and did not require the isolation of MAb from the culture fluid. This procedure, however, requires an additional step during the assay. These results are slightly different from those of Hill et al. (1984) who used a radioimmunoassay for soybean mosaic virus that involved both rabbit polyclonal antibody and

5 MAbs to wheat soil-borne mosaic virus 1321 Dilutions ' 2. " WSBMV 0 Infected Control TBS Fig. 1. Reaction of WSBMV, WSBMV-infected wheat (infected), uninfected wheat (control) and TBS in dot immunobinding assay. Four ~tl samples were spotted and dilutions are twofold (left to right). In column 1, WSBMV was spotted at 1600 ng/ml; infected and control at 1 g/4 ml TBS. The spots were purple in WSBMV and infected rows indicating a positive reaction, and green in control row indicating a negative reaction. mouse MAb. They found that when the MAb was used as the coating antibody and rabbit polyclonal antibody used as the labelled antibody, the assay was more sensitive than when the opposite order of antibodies was used. With the WSBMV system either approach gave satisfactory results. The conjugation of alkaline phosphatase to MAb reduced its speed of the reaction in ELISA, a problem also encountered, to a greater degree, when MAb to potato leafroll (Martin & Stace-Smith, 1984) or peanut mottle viruses (Sherwood et al., 1987) were conjugated to alkaline phosphatase. Because of the affinity of IgG2b for Protein A, the immunoglobulins produced by clones 5A1 and 5A24 were tested in a PAS ELISA. WSBMV was readily detected in this assay. With both MAbs, absorbance readings (A~05) for samples positive for WSBMV were greater than 1.1 and readings for healthy wheat were less than 0.1. This assay has the advantage of using unmodified MAb, but the MAb must be obtained substantially free of medium proteins. Isolation of MAb from hybridomas growing in SP medium by ammonium sulphate precipitation resulted in coprecipitation of a contaminant that interfered with the PAS ELISA. Development of a dot immunobinding assay with MAb A dot immunobinding assay using nitrocellulose as a sample support and goat anti-mouse antibody-alkaline phosphatase as a probe to detect the MAb-antigen complex gave the most satisfactory results (Fig. 1). In assays using filter paper as the sample support followed by either Protein A-peroxidase or anti-mouse antibody-alkaline phosphatase, or when using nitrocellulose as the sample support with Protein A-peroxidase to probe the antigen MAb complex, a good distinction between healthy and WSBMV-infected samples was not evident. With a double sandwich ELISA (MAb used to coat plate and alkaline phosphatase-linked antibody from rabbit antiserum used as a probe) 1 to 2 ng/ml of WSBMV could be detected. In the dot immunobinding assay the minimum detection level was between 100 to 500 ng/ml, but this assay could be completed in a few hours. These results indicate the feasibility of using MAbs to detect WSBMV and demonstrate the relatedness of isolates of WSBMV. Monoclonal antibodies should be useful in providing rapid and sensitive detection of WSBMV in wheat. Journal Series Article no of the Oklahoma Agricultural Experiment Station.

6 1322 Z. BAHRANI AND OTHERS REFERENCES BAHRANI, Z. & SHERWOOD, J. L. (1985). Comparison of an enzyme-linked immunosorbent assay (ELISA) and a filter paper dot-irnmunobinding assay for detection of wheat soilborne mosaic virus, Phytopathology 75, BAHRANI, Z., SHERWOOD, J. L. & SANBORN, M. R. (1986). Production of monoclonal antibodies to wheat soilborne mosaic virus (WSBMV). Phytopathology 76, BRAKKE, M. :~. (1971). Soil-borne wheat mosaic virus. CM1/AAB Descriptions of Plant Viruses, no. 77. BRAKKE, M. K., ESTES, A. P. ~ SCHUSTER, M. L. (1965). Transmission of soil-borne wheal mosaic virus. Phytopathology 55, CAMPBELL, L. G., HEYNE, E. G., GRONAU, D. M. & NIBLETT, C. (1975). Effect of soil-borne wheat mosaic virus on wheat yield. Plant Disease Reporter 59, CLARK, M. F. & ADAMS, A. N. (1977). Characteristics of the microplate method of enzyme-linked irnrnunosorbent assay for the detection of plant viruses. Journal of General Virology 34, EDWARDS, M. L. & COOPER, J. I. (1985). Plant virus detection using a new form of indirect ELISA. Journal of Virological Methods 11, ESTES, A. P. & BRAKKE, M. K. (1966). Correlation of Polymyxa graminis with transmission of soil-borne wheat mosaic virus. Virology 28, FRIGUET, B., DJAVADI-OHANIANCE, L., PAGES, J., BUSSARDE, A. & GOLDBERG, M. (1983). A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Applications to hybridomas specific for the flz-subunit of Escherichia coli tryptophan synthase. Journal of Immunological Methods 60, HALK, E. L. & DEBOER, S. H. (1985). Monoclonal antibodies in plant disease research. Annual Review of Phytopathology 23, HALK, E. L., HSU, H. T., AEBIG, J. & FRANKE, I. (1984). Production of monoclonal antibodies against three ilarviruses and alfalfa mosaic and their use in serotyping. Phytopathology 74, HILL, E. K., HILL, J. H. & DURAND, D. P. (1984). Production of monoclonal antibodies to viruses in the potyvirus group: use in radioirnmunoassay. Journal of General Virology 65, HUNGER, R. M. ~ SHERWOOD, J. L. (1985 a). Use of visual assessment and ELISA to evaluate the reaction of wheat cultivars to wheat soilborne mosaic virus. Phytopathology 75, 964, HUNGER, R. M. & SHERWOOD, J. L. (1985b). Use of symptornatology and virus concentration for evaluating resistance to wheat soilborne mosaic virus. Plant Disease 69, HUNGER, R. M. & SHERWOOD, J. L. (1986). Effect of wheat soilborne mosaic virus on yield of winter wheat cultivars. Phytopathology 76, K6HLER, G. & MILSTEIN, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, London 256, LEARY, J. J., BRIGATI, D. J. & WARD, D. C. (1983). Rapid and sensitive colormetric method visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: bio-blots. Proceedings of the National Academy of Sciences, U.S.A. 80, MARTIN, R. R. & STACE-SMITH, R. (1984), Production and characterization of monoclonal antibodies specific to potato leaf roll virus. Canadian Jot~rnal of Plant Pathology. 6, PALMER, L. T. & BRAKKE, M. K. (1975). Yield reduction in winter wheat infected with soil-borne wheat mosaic virus. Plant Disease Reporter 59, RAO, A. A. & BRAKKE, M. K. (1969). Relation of soil-borne wheat mosaic virus and its fungal vector, Polymyxa graminis. Phytopathology 59, SHERWOOD, J. L. (1987). Comparison of a filter paper immunobinding assay, Western blotting and an enzyme linked irnmunosorbent assay for detection of wheat streak mosaic virus. JournalofPhytopathology 118, SHERWOOD, J. L., SANBORN, M. R- & KEYSER, G. C. (1987). Production of monoctonal antibodies to peanut mottle virus and their use in enzyme-linked immunosorbent assay and dot-immunobinding assay. Phytopathology 77, SmRAKO, V. & ~RAKKE, M. K. (1984). Two purified RNAs of soil-borne wheat mosaic virus are needed for infection. Journal of General Virology 65, (Received 29 July 1987)