Inouye, Narinobu

Maeda, Takanori

Okayama University Scientific Achievement Repository

Ber. Ohara Inst. landw. Boil.， Okayarna Univ. 20: 19-26 (1991) RAPID DETECTION OF CUCUMBER MOSAIC VIRUS BY A SIMPLIFIED F(ab')z ELISA USING PEG Takanori MAEDA and Narinobu INOUYE INTRODUCTION 19 After the introduction of the enzyme-linked immunosorbent assay (ELISA) in plant virology， the method has been widely used for the detection of many plant viruses as being a method suitable for large-scale disease-indexing programs. However， itis laborious and time-con-suming to analyze large numbers of samples. To overcome this， several modified forms of ELISA have been developed. Flegg and Clark (1979) described a modified ELISA based on simultaneous incubation of sample and enzyme conjugate for the detection of apple chlorotic leafspot virus. This procedure has been successfully applied to diagno-sis of some other plant viruses，tooz，7，8，12). In this report we describe the comparison of direct and indirect procedures of ELISA with simultaneous incubation of sample and con-jugate in relation to their suitability for rapid detection of cucumber mosaic virus (CMV). MATERIALS AND METHODS 1. Virus and Antiserum The yellow strain of CMVll) was propagated in Nicotiana tabacum cv. White Burley and was purified by the method described previouslylO). Polyclonal antiserum10) to the virus was prepared in a rabbit by four intramuscular injections of purified virus emulsified with an equal volume of Freund's complete adjuvant. The titer of the anti-serum was 1 :256 in an agar-gel double diffusion test. Before use the antiserum was absorbed with insolubilized proteins of healthy tobacco leaves5) . Research Institute for Bioresources， Okayarna University， Chuo 2-20-1， Kurashiki， Okayarna 710 ]apan Received January 7， 1991 ・Supportedin part by a Grant-in-Aid for Developrnental Scientific Research (No. 6186∞05) frorn the Ministry of Education， Science and Culture of Japan 20 T. Maeda and N. Inouye 2. Pr仰 rationof Immunoglobulins and Conjugates The )' -globulin fraction (lgG) from rabbit antiserum was purified by ammonium sulfate precipitation followed by DEAE-cellulose column-chromatography. F(ab')2 fragments of IgG were obtained by digestion of IgG with pepsin (1 :60.000， Sigma)l・別.Anti-CMV IgG was conjugated with horseradish peroxidase (HRP) (Type トC，3，000 u/mg， RZ:3.29， Toyobo， Osaka) by a periodate-oxiclation method1• 9). Protein A (N akarai Pure Chem. Co.， Kyoto) was conjugated simultaneously with HRP and bovine serum albumin (BSA) at a ratio of 0.5:1:0.75 (w/w) by the same method. Initial concentra-tions of IgG-HRP and Protein A -HRP conjugates were adjusted to 1 mg IgG/ml and 1.25 mg Protein A/ml， respectively. HRP-conjugates were stored at 4t after addition of 0.5% BSA and 0.02% sodium merthiolate (w/v). 3. Simplified ELISA Simplified direct ELISA (D-ELISA) and indirect ELISA (I-ELISA) were essentially based on DAS ELISA4) and F(ab')2 ELISA 1)， respectively. Wells of polystyrene microtiter plates (Nunc-Immuno Plate UII， Inter Med) were incubated for 3 hr at 30t with IgG (2μg/ml， D.-ELISA) or F(ab')2 (2μg/ml， I-ELISA) in coating buffer (0.05 M carbonate buffer， pH 9.6). After washing the wells with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBST)， 75μ1 each of sap and IgG・conjugate(final dilution: 1/800) (D-ELISA) or the mixture of native IgG (final concentration: 2μg/ml) and protein A-conjugate (final dilution: 1/2，000) (1-ELISA) were added to the wells at the same time， and then incubated at 30t or 37t. The sap was prepared from infected and healthy tobacco plants by homogeniz-ing leaf tissue in PBST containing 0.2% BSA and 2% polyvinyl pyrro-lidone. After washing of the plates the enzyme substrate (0. 5g/1 0-phenylenediamine in 0.025 M sodium acetate buffer， pH 5.5， contain-ing 0.06% hydrogen peroxide) was added and incubated for 20 min at 30t in the dark. The reaction was stopped by the addition of 50μ1 of 3 M sulphuric acid to each well and the absorbance of the diluted reac・tion mixture was measured at 490 nm using a spectrophotometer. RESULTS 1. Comμ付sonof Simplified D-ELISA and I-ELISA In preliminary experiments the optimum concentrations of anti-bodies and conjugates for the detection of CMV in sap from infected plants by both simplified procedures of ELISA were determined (see 21 materials and methods). Both procedures were compared for their ability to detect CMV in serially diluted extracts from infected tobacco leaves and for the pro-duction of nonspecific reactions with healthy controls. As shown in Fig. 1， similar dose-response curves with a depression of the absorb-ance at the lowest dilution (at the highest virus concentration) of in-fected samples were obtained for both procedures. Although CMV could be reliably detected by both procedures， 1 -ELISA gave higher positive ELISA values and lower backgrounds than D-ELISA. The re-sults of these experiments indicated that 1-ELISA is superior to D-ELISA for detecting CMV， and therefore in subsequent experiments only 1 -ELISA was tested. Detection of Cucumber Mosaic Virus by Simplified F(ab')2 ELISA 0.5 ? ? 。 。??? ? ????〈10-1 10-1 10-' Dilution of sap FIG.1. Comparison of simplified D-ELISA and I-ELISA with simultaneous incubation of samples組 dconjugates for the detection of cucumber mosaic virus目 D-ELISAand I-ELISA were based on standard DAS ELISA and F(ab'h ELISA， respectively. * : D-ELISA， ・:I-ELISA， 一ー一:infected， …… : healthy. ?? ????? ? ????????? ????Effect of PEG in Simjが'ifiedF(ab?2 ELISA It is well known that immune reactions can be accelerated in the presence of polyethylene glycol (PEG). Various concentrations (1 to 6%) of PEG 6，000 were included in the reaction mixtures containing sample (plant extract diluted 1: 100)， IgG and protein A-conjugate， and the mixtures were incubated for 2 hr at 30"C. The ELISA values of in-fected extracts considerably increased by adding PEG to the reaction mixtures. The positive ELISA values increased with increasing PEG concentrations up til 3% PEG and decreased somewhat at higher con-centrations. On the other hand nonspecific reactions markedly in-creased with increasing PEG concentrations (TABLE 1). Including 2 to 3% PEG in the reaction mixtures seemed to be optimal for the detec-tion of CMV by simplified 1-ELISA. 22 2. Effect of addition of PEG in reaction mixtures in simplified F(ab'h ELISA for the detection of cucumber mosaic virus') TABLE 1 Experiment 2 Experiment 1 Concentration Infected 0.76 1.22 1.35 1.25 1.21 1.18 1.10 Healthy 0.02 0.04 0.04 0.04 0.07 0.12 0.43 Infected 0.60 0.97 1.34 1.36 1.34 1.33 1.26 Healthy O.Olb) 0.02 0.02 0.03 0.04 0.08 0.43 of PEG (%) ?????????F(ab')2-coated wells of microplates were incubated simultaneously with sap， IgG and protein A-conjugate for 2 hr at 30t:. Absorbance values at 490 nm a) b) The use of PEG in D-ELISA caused severe nonspecific adsorp-tion of conjugate on the solid phase (data are not shown). The influence of incubation time on ELISA values for infected and healthy extracts was tested by incubating reaction mixtures con-taining 2% PEG at 30"C for various times. The ELISA values of in-fected samples with PEG increased with incubation time and reached a maximum at 2 hr after initiating the reaction. On the other hand the positive ELISA values of reaction mixtures without PEG gradually in-creased up to 3 hr and were lower than those obtained with PEG at the various incubation times (FIG. 2). The results indicated that an incuba-tion time of 2 hr in simplified 1-ELISA with PEG is sufficient for a re-liable detection of CMV. 23 Detection of Cucumber Mosaic Virus by Simplified F(ab')2 ELISA l.0 0.5 ??。。??????』』????〈::::::ニヰ::::::::_~t:::二二2二三-- --主---_.........主3 2.5 2 l.5 0.5 lncubation time in hr Relationship between ELISA values and incubation time in simplified I-ELISA with or without PEG in the reaction mixtures. F(ab')2-coated wells were filled with the mixture of sap (diluted 1: 100)， IgG and protein A-conjugate， and incubated at 30't. ・:with PEG (3%)， * : without PEG， 一ー一:CMV-infected， 一一:healthy. FIG.2. Detection of CMV 均SimplifiedF(ab ')2 ELISA Nonspecific reactions in simplified 1-ELISA with PEG were con-siderably decreased by addition of 1% Triton X -100 to the reaction mixtures without reducing specific ELISA values (data are not shown). The simplified 1-ELISA described above was tested for the detection of CMV in infected tobacco plants_ Serially diluted extracts from in-fected and healthy tobacco leaves were incubated with IgG and conju‘ gate simultaneously in the presence of 2% PEG and 1% Triton X -100 for 2 hr at 30t. The virus could be detected in extracts diluted 1: 1，000，000 (FIG. 3). 3. T. Maeda and N. Inouye 1.0 ? ? 。 。 ? ? ? ?? ?』?〈24 Dilution of sap FIG. 3. Detection of cucumber mosaic virus by simplified I-ELISA. PEG (2%) and Triton X-lOO (1%) were in-cluded in reaction mixtures and incubated at 30'C for 2 hr. 一一一:infected， :ーhealthy. DlSCUSSION Many modified ELISA procedures have been described that re-duce th'e assay time required for plant virus diagnosis. Simplified direct DAS ELISA with simultaneous incubation of virus samples and conjugates was first reported by Flegg and Clark (1979) to detect apple chlorotic leafspot virus which could not be detected by the standard procedure. This procedure has been successfully applied for the di・agnosis of some plant viruses without reducing the sensitivity of the assay2. 7. 8. 12) In this study we compared direct and indirect procedures of simpli-fied ELISA to evaluate their suitability for rapid detection of CMV. Reduced ELISA values in undiluted plant extracts as compared to di・Detection of Cucumber Mosaic Virus by Simplified F(ab')2 ELISA 25 luted ones were obtained in both procedures. This phenomenon may be due to the reaction of antibodi"es with free virus particles which are present at a high concentration in those reaction mixtures， which in the end results in a decrease of conjugate (in D-ELISA) or IgG (in 1 -ELISA) molecules which bind to captured virus and immune com-plex on the solid phase3J • 1 -ELISA showed better detection of CMV wi th less nonspecific reactions than the direct procedure. The higher background in D-ELISA seemed to be caused by the different type of conjugate used in that procedure. Barbara and Clark (1982) evaluated three types of Fc-specific conjugates for use in F(ab')2 ELISA and demonstrated that the two immunoglobulin-based conjugates gave higher values with healthy sap than the protein A-conjugate. In our experiments a serially diluted IgG-conjugate (direct procedure) or a mixture of IgG and pro-tein A-conjugate (indirect procedure) were tested. The ELISA values for infected sap increased with the conjugate concentrations in both procedures. However， the increase of nonspecific reactions with various conjugate concentrations was much less for the protein A-conjugate than for the IgG-conjugate. Furthermore， the protein A-conjugate could be used at higher dilutions than the IgG-conjugate (unpublished data). The addition of PEG to the reaction mixtures in F(ab')2 ELISA gave higher positive ELISA values and it reduced the time required to reach the highest ELISA values. It is not clear whether PEG acceler-ates the reaction between IgG and virus or/and between IgG and the protein A-conjugate. The addition of PEG in each reaction step in a standard procedure will eliminate these questions. Background values which increased in simplified F(ab')2 ELISA using PEG considerably decreased by addition of Triton X -100. This may be due to Triton X -100 solubilizing green plant materials， which subsequently results in reduction of adsorption of IgG and conjugate to the solid phase. The simplified ELISA described here has the big advantage that the same conjugate can be used for al viruses， and may therefore be useful for the rapid detection of other plant viruses， too. SUMMARY Direct and indirect procedures of double antibody sandwich EL-ISA with simultaneous incubation of sample and conjugate were com-pared for their suitability to detect cucumber mosaic virus in sap from infected plants. Although CMV could be reliably detected by both pro-cedures， indirect F(ab')2 ELISA using protein A-conjugate gave lower background levels than the direct procedure. In F(ab')2 ELISA， includ-26 T. Maeda and N. Inouye ing 2% PEG in the reaction mixtures resulted in acceleration of the reaction and in higher ELISA values for infected samples. An incuba-tion time of 2 hr was sufficient for reliable detection of CMV in sap in-fected plants by the procedure described. Acknowledgement The authors are greatly indebted to Dr. H. Huttinga， a visiting professor of our institute， for his critical reading of the manuscript. REFERENCES 1. Barbara， D. ]. and Clark， M. F. 1982. A simple indirect ELISA using F(ab')2 fragments of immunoglobulin. ]. gen. Virol. 58 : 315-322. 2. Bar.]oseph， M.， Garnsey， S.M.， Gonsalves， D.， Moscovitz， M.， Purcifull， D. E.， Clark， M. F. and Loebenstein， G. 1979. The use of enzyme-linked immunosorbent assay for the detection of citrus tristeza virus. Phytopathology 69 : 190-194. 3. Clark， M. F. 1981. Immunosorbent assays in plant pathology. Ann. Rev. Phytopathol. 19 : 83-106. 4. Clark， M. F. and Adams， A. N. 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. ]. gen. Virol. 34 : 475-483. 5. Clark， M. F. and Bar.]oseph， M. 1984. Enzyme immunosorbent assay in plant virology. In Methods in Virology. 7 : 51-85 6. Flegg， C. L. and Clark， M. F. 1979. The detection of apple chlorotic leafspot virus by a modified procedure of enzyme.linked immunosorbent assay (ELISA). Ann. appl. Biol. 91 : 61骨65.7. Franken， A. A. ]. M.， Maat， D. Z. and Kamminga， G. C. 1990. Detection of squash mosaic virus in seeds of melon (Cucumis melo) by enzyme-linked immunosorbent assay (ELISA). Neth. ]. Pl. Path. 96 : 91-102. 8. Kawai， A.， Kimura， S.， Nishio， T. and Nagao， N. 1985. Detection for cucumber green mottle mosaic virus in cucumber seeds using enzyme.linked immunosorbent assay. Res. Bull. Pl. Prot. ]apan 21 : 47-53. 9. Maeda， T. and Inouye， N. 1986. Detection of cucumber mosaic virus by enzyme-linked immunosorbent assay (ELISA). -Evaluation of various conjugates in double anti-body sandwich method of ELISA-. Nogaku Kenkyu 61 : 119-127. 10. Maeda， T.， Wakimoto， S. and Inouye， N. 1983. Serological properties of cucumber mosaic virus in ]apan.Ann. Phytopath. Soc. ]apan 49: 10-17. 11. Tomaru， K. and Hidaka， Z.1960. Strains of cucumber mosaic virus isolated from tobac. co plants. II. A yellow strain. Bull. Hatano Tobacco Exp. Sta. 46 : 143-149. 12. Vuurde，]. W. L. van and Maat， D. Z. 1983. Enzyme-linked immunosorbent assay (EしISA) and disperse-dye immuno assay (DIE): comparison of some aspects of simul-taneous and separate incubation of sample and conjugate for the routine detection of lettuce mosaic and pea early-browning viruses in seed. Neth.]. Pl. Path. 91: 3-13. 

RAPID DETECTION OF CUCUMBER MOSAIC VIRUS BY A SIMPLIFIED F(ab')2 ELISA USING PEG

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