comparison of elisa and immunoblotting techniques for the detection of cherry mottle leaf virus

Ann. appl. B id . (1996), 129:013-023 Printed in Great Britain

Comparison of ELISA and immunoblotting techniques for the detection of cherry mottle leaf virus

13

By D JAMES and S MUKERJI Centre for Plant Health, Agriculture and Agri-Food Canada, 8801 East Saanich

Road, Sidney, British Columbia, Canada V8L I H3

(Accepted 20 May 1996)

Summary Formaldehyde treated cherry mottle leaf virus (ChMLV) and the isolated coat

protein were used successfully for the production of polyclonal and monoclonal antibodies. The monoclonal antibodies had a titre of 1 5 1 200 and consisted of IgG1 and IgG2. The antibodies reacted with all 11 isolates of ChMLV, from five locations in Canada and the USA, included in this study.

Several serological procedures were assessed to compare their sensitivity for detecting ChMLV. Plate-trapped antigen ELISA (PTA-ELISA) and dot-blot immunobinding assay (DBIA), using virus specific MAbs, were the most sensitive tests in this study. Triple antibody sandwich ELISA (TAS-ELISA) and Western blot were found to be less sensitive. Dilution of the samples appeared to increase the sensitivity of both PTA-ELISA and Western blot detection. Young leaves and flowers of Prunus avium were the best tissue for detecting the virus which could also be detected in the fruit and leaves of P. tomentosa. April and May were optimal for detection of the virus in the field, whereas both April to May and August to September were optimal for screenhouse-grown plants.

Key words: Cherry mottle leaf virus, polyclonal antibody, monoclonal antibody, PTA-ELISA, TAS-ELISA, Western blot, DBIA, virus detection

Introduction Cherry mottle leaf (CML) has been identified as one of the most severe diseases of cherry

in some regions of North America (Nemeth, 1986). The disease was first observed in Oregon in 1920 (Zeller, 1934) and recently a filamentous virus was identified as the causal agent of the disease (James & Mukerji, 1993). CML is a disease of quarantine significance and any rapid accurate diagnostic test will be an asset in indexing for this diseases. Serological assays are commonly used for the detection and identification of plant viruses (Van Regenmortel, 1986). This involves the use of polyclonal andlor monoclonal antibodies. The techniques for hybridoma generation and monoclonal antibody production were developed by Kohler & Milstein (1 975). Polyclonal antibodies consist of heterologus populations of antibodies with variable specificities (Ball, Hampton, De Boer & Schaad, 1990). In contrast, monoclonal antibodies are highly specific for a single antigenic site, and also a constant supply of well characterised antibodies can be obtained (Van Regenmortel, 1986; Jordan, 1990). The specificity of monoclonal antibodies is a desirable feature as it may allow the unambiguous identification of some plant viruses. 0 1996 Association of Applied Biologists

14 D JAMES AND S MUKERJI

The objective of this study was to develop polyclonal and monoclonal antibodies which could be used as tools for virus identification and for rapid and accurate detection of the virus associated with mottle leaf disease in cherry.

Materials and Methods

Virus sources The Saanichton isolate of cherry mottle leaf virus (ChMLV, Sall62-21) was maintained in

Chenopodrum quinoa and Bing cherry (James & Mukerji, 1993). The isolate SA1162-22 from Saanichton, was also included in the study. Isolates C27-4B, SP4-14, and SP6-15 in cherry were obtained from T Li, Research Centre, Agriculture and Agri-Food Canada, Summerland, BC, Canada. Isolates CMLAE-88-15, BCAE-89-7, and BCAE-89-14 were obtained from J Uyemoto, and G Oldfield, University of California, USA (Davis and Riverside, respectively). Isolates 103-5 and 105-16 were obtained from Bill Howell, USDA, Prosser, Washington, USA; and isolate FD#2 in P. tornerzfosa was obtained from V Damsteegt, USDA, Fort Detrick, Maryland, USA. Samples N 12T4-N 12T 14, N 18T1, N 18T2 were sent by Uyemoto and Oldfield as “blind” samples of healthy and infected Bing cherry, i.e. these samples were identified by numbers with no information on their disease status. Samples PT13-24 were sent as “blind” samples of healthy and infected Prurtus fomentosa.

tr?zmunisation and hybridoma production Four %week old BALB/c mice were immunised by intraperitoneal injections, using

formaldehyde-treated preparations of purified ChMLV (Sall62-21, 50-100 pg). The virus was purified using the procedure described by James & Mukerji (1 993). The purified virus was then treated with formaldehyde by adjusting the virus suspension to 1% formaldehyde, incubated at room temperature for 10 min, and dialysed in phosphate buffered saline (PBS; 0.15 M sodium chloride, 0.02 M sodium phosphate, pH 7.2). The antigen was adjusted to lOOp1 with PBS, and emulsified with 1 0 0 ~ 1 of Freund’s complete adjuvant. A second injection of 50 pg of the treated antigen was given two weeks later. A test bleed was carried out 4 wk after the initial injection to determine antibody titre. Four weeks after the second injection. mice showing the highest antibody titre were selected and given a series of three consecutive intraperitoneal injections, using the coat protein of the virus. The virus coat protein was isolated by a SDS-agarose gel procedure described by Sakakibara, Tominaga, Sakai & Ishiguro (1987). Spleens were harvested the day following the final injection (Stahli et al., 1980).

Spleen cells (lo*) were fused with P3-X63-Ag8.653 mouse myeloma cells (lo’) using a procedure similar to that described by Galfre & Milstein (1981). Hybridoma cells secreting antibodies, which reacted positively to ChMLV infected tissue but not to healthy tissue, were cloned and recloned by plating the cells in semi-solid medium containing methylcellulose and other components of the HAT selection system. Positive hybrid clones were selected for in L’irro propagation and liquid nitrogen storage. This procedure provides a single step technique For both selecting and cloning hybridomas without the need for limiting dilutions (Davis, Pennington, Kubler & Conscience, 1982).

Monoclonal antibody purificatiori and isotyping Pristane primed BALB/c mice were given an intraperitoneal injection with 1 x lo7

hybridoma cells in PBS. After about 10-14 days, ascitic fluid was collected and the antibody

Detection of cherry mottle leaf virus 15

titre determined by indirect ELISA. The immunoglobulins were purified by ammonium sulfate precipitation and dialysed against PBS. Immunoglobulin class and sub-class of the monoclonal antibodies were determined by ELISA using the Mouse Typer Isotyping Kit (Bio-Rad).

Polyclonal antibody production Formaldehyde-treated purified ChMLV-Sall62 (approximately 100 pg in 300 pl PBS) was

mixed with Freund’s complete adjuvant and used for the initial injection of a New Zealand white rabbit. The virus was purified using the procedure of James & Mukerji (1993). Subsequent injections were made using the virus coat protein isolated as described above. Immunisation was carried out by mixing the melted gel containing the disassociated virus protein coat (0.2 mg purified virus used), with an equal volume of Freund’s incomplete adjuvant. Intramuscular injections were given and these were repeated at 2-week intervals. Serum was collected 5 days after the second booster injection.

Enzyme-linked immunosorbent assay (ELISA) Two types of indirect ELISA were used in the process of selecting for ChMLV-specific

antibody producing hybridomas and for general screening (Clark & Adams, 1977; A1 Moudallal, Altschuh, Briand & Van Regenmortel, 1984; Torrance, 1992). In the plate trapped antigen (PTA) ELISA, 96-well microtitre plates (Linbronitertek, ICN Biomedicals Inc.) were coated with virus preparations diluted in 0.05 M carbonate buffer, pH 9.6, with 2% polyvinylpyrrolidone (PVP), and incubated overnight at 4°C.

In the triple antibody sandwich (TAS) ELISA, plates were coated with rabbit polyclonal antiserum diluted 1:2000 in 0.05 M carbonate buffer, pH 9.6, and incubated for 3 h at 37°C. This was followed by incubation with the virus-containing samples overnight at 4°C. Both procedures were identical from this point onwards. The plates were incubated with a blocking solution of PBST-BSA (PBS containing 0.05% Tween 20, and 1% bovine serum albumen), for 1 h, at room temperature. The ChMLV-associated monoclonal antibody (MAb 1 162-A2) was diluted 1:2000 in PBST-BSA, and the plates loaded, then incubated for 3 h at 37°C. This was followed by a 3 h incubation with goat anti-mouse IgG alkaline phosphatase conjugate (Bio-Rad) diluted 1:2000 in PBST. The substrate consisted of 1 mg ml-’ p-nitrophenyl phosphate in 10% diethanolamine buffer, pH 9.8. Between each incubatedion step, the plates were rinsed three times with PBST. The absorbance at 405 nm was measured using a Titertek Multiskan plate reader.

Dot-blot immunobinding assay (DBIA) The DBIA method as described by Lazarovits (1990) was used, but with a few

modifications. Crude sap extracts, healthy and infected, were prepared by grinding 1 g of leaf tissue in 10 ml of ice cold phosphate buffered saline (PBS, pH 7.4) containing 2% polyvinylpyrrolidone (PVP). Samples were centrifuged at 10000 x g for 10 min and the supernatants collected and diluted as desired. Purified preparations of the antigen were also used. Samples (1-2 pl) of the purified antigen or crude extract were spotted on pre-wetted nitrocellulose, 0.45 pg pore size (Schleicher & Schuell Inc.). The membranes were air-dried then placed in a blocking solution consisting of 1% gelatin in Tris buffered saline with 0.05% Tween 20 (TBST, pH 7.4) and stored at 4°C. The blots were incubated for 2 h in the ChMLV- associated MAb (1 162-A2) diluted 1:2000 with TBST containing 2% gelatin. The membranes were then washed three times in TBST, followed by a 90 min incubation in goat anti-mouse alkaline phosphatase conjugate (diluted 1 :2000 in TBST). The membranes were washed as described above and then rinsed in the substrate buffer consisting of 0.1 M Tris, 0.1 M NaCl


Table 1. PTA-ELISA to determine the spec@city of a monoclonal antibody to cherry mottle leaf virus

Sample E L S A values”

C. quinoa, healthy Purified ChMLV’ C. quinoa with ChMLV C. quinoa with ASGV c. qlkiMJa with ACLSV

0.13 f 0.01 0.49 i 0.05 1.67 2 0.05 0.12 2 0.01 0.12 f 0.01

‘Mean absorbance values (hoS) i standard deviation, n = 12. MAb 1162-A2 was used at a dilution of 1:5000. h ~ . 6 ~ pg of virus particles per well. ChMLV = cheny mottle leaf virus, ASGV = apple stem grooving virus, and ACLSV = apple chlorotic leaf spot virus.

and 5 mhi MgC122, pH 9.5. The membranes were stained by immersion in 10 ml of substrate buffer containing 100 pl of a nitroblue tetrazolium stock solution (30 mg m1-l) and 100 p1 of a 5-bromo-4-chloro-3-indolyl phosphate stock solution (15 mg ml-I). The development of a purple colour on blots was indicative of a positive result, usually observed within 15 min. The membranes were washed in distilled water and air-dried for storage.

Western blotting Proteins of the purified antigen and healthy and infected crude sap were separated by SDS-

PAGE as described by Laemmli ( I 970). The proteins were then transferred, by electroblotting, to nitrocellulose membrane (Schleicher & Schuell, 0.45 pm pore size) using the procedure described by Towbin, Staehelin & Gordon (1979). Prestained markers (Bio- Rad) were used to assess transfer of the proteins from the gel to the membrane. Diluted ChMLV-associated MAb was used as the primary antibody and goat anti-mouse IgG alkaline phosphatase (Bio-Rad) was used as the secondary antibody. NBTBCIP substrates were used as recommended by the supplier (Bio-Rad).

Field evaluation A number of ChMLV isolates from sources in Canada and the USA were obtained for

screening (See Virus sources above). Some of these were received as “blind” or unidentified samples. Different types of tissue, including bark, leaves, flowers and fruit, were tested for the presence of the antigen. These samples were tested by PTA-ELISA andor DBIA. Also, ChMLV-infected cherry plants in the field and screenhouse were tested from April to September to determine the optimum time for detection of the virus.

Results

Hybridoma production Two mice, M1 and M2, were selected for fusion after assessment of the antibody titre in

their serum by PTA-ELISA. Absorbance readings at 405 nm were 13 x (M1 serum) and 10 x (M2 serum) that of the pre-immune sera at 1:40 dilution. The MI and M2 sera gave positive reactions at dilutions of 1:5120 (absorbance readings greater than twice the value of the pre- immune sera were considered positive). Of 80 wells plated after methylcellulose cloning, over


Fig. 1 . Dot blot assay to determine the specificity and reactivity of 1162-A2 ascitic fluid. Each row contains four replicates of each of the following samples: A, purified ChMLV; B, C. quinoa infected with ChMLV, diluted 1:20; C, C. pinon infected with ASGV; D, C. quinou infected with ACLSV; E, healthy control; and F, C. quinoa infected with ChMLV, undiluted.

50% of these wells produced hybridomas. Thirty eight of these hybridoma lines produced antibodies that reacted positively with the viral antigen or infected crude sap, and negatively with the healthy controls when screened by DBIA. Two of the stable positive cell lines, with sera giving high reaction levels (1162-A2 and 1162-C2), were cloned for expansion and further testing.

In ELISA tests using dilution series analyses, the ascitic fluids of 1162-A2 and 11 62-C2 were found to have titres of 1 5 1 200 when compared with pre-immune sera. When tested for specificity by TAS-ELISA, the absorbance values for Chenopodium quinoa plants infected with the homologus antigen were approximately 13 times higher than the values recorded for healthy controls. C. quinoa infected with apple stem grooving virus (ASGV) or apple chlorotic leaf spot virus (ACLSV) were negative (Table 1). The specificity of the monoclonal antibodies was also checked by DBIA assay. The ascitic fluid was used at a dilution of 1:10 000 in PBST with 1% BSA. Positive reactions were observed with the purified antigen and infected leaf sap extracts, no reaction was observed with healthy sap or crude sap from plants infected with ASGV, or ACLSV (Fig. 1).

Antibody isotyping Two subclasses of immunoglobulins, IgG1 and IgG2 specific for ChMLV, were identified

in tissue culture supernatants and in ascites fluid of MAbs 1162-A2 and 1162-C2. Colour development occurred almost immediately and the positive clones were 8 to 10 times as reactive as the negative controls.

Comparison of PTA-ELISA and TAS-ELISA All comparisons were carried out using ChMLV, Sa1162-21 isolate. The sensitivity of

PTA-ELISA and TAS-ELISA for the detection of the anitgen were compared using known amounts of purified ChMLV diluted in healthy Bing cherry leaf sap, dilutions of crude leaf


Table 2. Comparison of plate trapped antigen (PTA)-ELISA, triple antibody sandwich (TAS)- ELISA, dot blot immunobinding assay (DBIA), and Western blot for detecting purified

samples of cherry mottle leaf virus Virus Absorbance Values * S D ~ Concentration r \

(per ml) PTA-ELISA TAS-ELISA Western Blot DBIA c

1 Pg 500 ng 250 ng 125 ng 50 ng 25 ng 10 ng 0 nE

1.71 2 0.13 1.42 2 0.02 0.95 i 0.03 0.71 i 0.14 0.57 2 0.04 0.36 i 0.03 0.31 +. 0.02 0.25 2 0.09 0.26 * 0.03 0.06 t 0.03 0.09 i 0.02 0.04 * 0.05 0.04 * 0.02 0.05 2 0.05 0.03 2 0.02 0.04 * 0.01

+b

+ + C -

"Mean absorbance values ( n = 16) t the standard deviation h+ = positive. ' - = negative

Table 3. Comparison of plate trapped antigen (PTA)-ELISA, triple antibody sandwich (TAS)- ELISA. Western blot, and dot blot immunobinding assay (DBIA) for the detection of cherry

mottle leaf virus in dilutions of the crude sap of infected Chenopodium quinoa Absorbance Values * S D ~

Sap dilution PTA-ELISA TAS-ELISA Western blot DBIA

1:10 (H) 0.07 2 0.02 0.07 * 0.02 - - s:10 0.65 i 0.12 1.22 i 0.34 f' + 1:lW 1.47 2 0.21 0.91 t 0.13 + 4-

1:1000 1.09 0.05 0.19 * 0.03 + + 1:soooo 0.21 i 0.02 0.08 i 0.02 -

1:20000 0.13 i 0.02 0.07 i 0.03 -

"Mean absorbance values (n = 16) t the standard deviation. '- = negative. L + = positive.

h

+ +

sap of ChMLV-infected C. quinoa, and dilutions of crude leaf sap of ChMLV-infected Bing cherry. Healthy leaf sap diluted 1 : l O was used as the negative control. For all ELISA tests, absorbance values were taken 2 h after addition of the substrate, and values that were twice the mean-plus-standard-deviation value of the healthy control, were considered positive.

PTA-ELISA was consistently more sensitive than TAS-ELISA for the detection of ChMLV. With purified ChMLV, the virus was detectable at a minimum concentration of 50 ng ml-' by PTA-ELISA and at a minimum concentration of 125 ng ml-' by TAS-ELISA (Table 2). The dilution end points for detecting ChMLV in the crude sap of C. quinoa and Bing cherry, by PTA-ELISA, were 1 : 10 000 and 1 : 1000, respectively (Tables 3 and 4). The dilution end points for detection by the TAS-ELISA procedure were 1:lOOO and 1:100, respectively .

DBIA detection DBIA was used successfully to detect the purified ChMLV at a minimum concentration of

10 ng ml-I (Table 2). The antigen could be detected in the crude sap of infected C. quinoa and Bing cherry leaves at dilutions of 1:20000 and 1:1000, respectively (Tables 3 and 4).


Table 4. Comparison of plate trapped antigen (PTA)-ELISA, triple antibody sandwich (TAS)- ELISA, Western blot, and dot blot immunobinding assay for the detection of cherry mottle leaf

virus in dilutions of the crude sap of infected Bing cherry Absorbance values f S D ~

r * Sap dilution PTA-ELISA TAS-ELISA Western blot DBIA

1 : l O (H) 0.08 f 0.01 0.15 f 0.02 - - 1 : l O 0.93 f 0.07 0.82 f 0.03 += + 1:lOO 0.69 f 0.07 0.38 f 0.03 + + 1:looO 0.27 2 0.02 0.13 f 0.02 - 1:10000 0.13 2 0.02 0.09 f 0.02 - - 1:200oo 0.09 f 0.02 0.15 f 0.03

aMean absorbance values (n = 16) f the standard deviation. b- = negative. ‘+ = positive.

b

+ - -

Western blot detection In Western blot analysis, a single prominent virus-associated band was detected (Fig. 2).

The band, which represents the coat protein subunit of the virus, has an estimated molecular mass of 20.5 kDa. This was established in a previous study (James, 1992). Purified ChMLV was detected by Western blotting at a minimum concentration of 250ng ml-’ (Fig. 2). In diluted preparations of infected C. quinoa and Bing cherry, the antigen was detected at 1:1000 and 1:100, respectively (Tables 3 and 4).

Field evaluation Samples were screened by PTA-ELISA and/or DBIA to check the reliability and efficiency

of the antisera for detecting ChMLV. All 11 ChMLV isolates obtained from five locations in Canada and the USA were detectable by PTA-ELISA and/or DBIA. The two negative control plants included did not react with the antiserum in PTA-ELISA or DBIA analysis.

A total of 24 unidentified “blind” samples were received to test the reliability of PTA- ELISA for the detection of ChMLV. Of 11 known positive samples, virus was detected by PTA-ELISA in eight, but in three (N12T7, PT17, PT19) virus was not detected (false negatives). Samples included in this study that were identified as negative by bioassay were also negative after testing by PTA-ELISA.

In studies to determine the best time for reliable detection of ChMLV, tests were made on field and screenhouse-grown infected cherry plants. PTA-ELISA was carried out every 3 wk from April to September using crude leaf sap samples. Samples with an infectedhealthy ratio (IHR) of absorbance values greater than two were considered to be positive. The virus concentration peaked in late April (IHR= 16 to 29) with gradual decline and intermittent fluctuations from May to July. The virus could not be detected in infected cherry in the field from midJuly to September. A greenhouse-grown infected cherry plant which was positive in every test made from April to September, had two peaks of increased virus concentration, one in late April (IHR = 29) and the other in mid-September (IHR = 32). A significant decrease in virus concentration was observed from May to June (IHR=2.1 to 11). The concentration increased in mid-August and stayed relatively high until September.

High concentrations of ChMLV (IHR=29 to 32) were detected in young leaves (symptomatic and non-symptomatic) and flowers of infected cherry. The virus was also detected in leaf and fruit tissue of P. tomentosa. The virus could not be detected by PTA-


2 hestern blot analysis of various concentrations of ChMLV, Sal162-21 diluted in healthy Bing lierry sap The proteins mere separated by SDS-PAGE (12% polyacrylamide), electroblotted on to

riitlocellulose, and screened with MAb 1162-A2 Lane M, pre-stained molecular mass standards (~1000 Dct. ldnes 1-8, 1 p g , 5 0 0 ng, 250 ng, 125 ng, 50 ng, 25 ng, 10 ng, 0 ng (per ml) of purified ChMLV f e\pecti\ eiy

EtlS.4. or DBIA in bark tissue taken from symptomatic trees which were positive when fiuwei5 and/or leaf tissue was assayed.

Discussion ChMLV was used successfully for the production of polyclonal and monoclonal antibodies

,when fixed in formaldehyde for the initial injections and denatured by SDS-PAGE for sjbsequent injections. The monoclonal antibodies (MAb 1162-A2 and MAb1162-C2) had a high litre ( 1 5 1 200), consisted of IgG1 and IgG2 immunoglobulins, and showed good specificity. The antibodies did not react in any of the tests with ACLSV or ASGV, two relatively common flexuous fruit tree viruses (Nemeth, 1986). This suggests that ChMLV is serologically distinct from ASGV and ACLSV. ChMLV is a very labile virus with a longevity in v i m i of 24 h (James & Mukerji, 1993). Several methods have been advocated for enhancing the production of antibodies by weak or unstable antigens; fixation in glutaldehyde (Ferguson er al., 1985), fixation in formaldehyde (Mernaugh. Memaugh & Kovacs, 1990), denaturation by SDS-treatment (Stumph, Elgin & Hood, 1974; Harlow & Lane, 1988), or by coupling the pepridt:s to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin (Hariow & Lane, 1988). In this study a combination of formaldehyde fixation and SDS- denarui.ation was used to elicit an effective immunogenic response.

(2x1 1-y mottle leaf disease is an economically important disease of cherry with quarantine nificsnce. In this investigation several detection procedures were evaluated to compare

iheir sensitivity for the detection of ChMLV. DBIA was the most sensitive test for the delection of the purified antigen (10 ng ml-I), and the antigen in serial dilutions of infected C.

20000). PTA-ELISA was found to be more sensitive than TAS-ELISA for the deiection of the purified antigen (50 ng ml-' vs 125 ng ml-I), the antigen in serial dilutions of


the crude extract of infected C. quinou (1: 10 000 vs 1:1000), and the antigen in serial dilutions of the crude extract of infected Bing cherry (1: 1000 vs 1: 100). This agrees with the findings of Lommel, McCain & Morris (1982) who reported that indirect ELISA was more sensitive than sandwich ELISA for the detection of some plant viruses. When serial dilutions of the crude extract of infected Bing cherry were tested, PTA-ELISA and DBIA displayed similar levels of sensitivity. This increased sensitivity of PTA-ELISA may have resulted from reduced interference at higher sample dilutions. Flegg & Clark (1979) also noted increased levels of sensitivity when ELISA detection was carried out on dilutions of the leaves, petals and fruit of apple infected with ACLSV. Western blot assay was the least sensitive test for detecting the purified antigen which was diluted in healthy Bing cherry leaf sap at a concentration of 1: 10. Dilutions of both C. quinoa and Bing cherry resulted in increased sensitivity of the Western blot assay making it comparable to TAS-ELISA but still not as sensitive as PTA-ELISA or DBIA.

Since cherry mottle leaf disease was first observed in the USA (Zeller, 1934) isolates were obtained from locations in the USA and Canada. All 11 isolates from five locations reacted positively in ELISA and DBIA assay indicating that the MAbs reliably detected conserved epitopes of various isolates in North America. Laviada, Babin, Dominguez & Sanchez- Vizcaino (1992) describes antibody sensitivity as (number of true positives - number of false negatives)/number of true positives; and specificity as (number of true negatives - number of false positives)/number of true negatives. When the unidentified samples were tested by PTA- ELISA, sensitivity of 73%, and specificity of 100% was obtained. The false negatives may have been the result of sub-detectable levels of the virus or irregular distribution of the antigen in the plants.

PTA-ELISA and DBIA were of comparable sensitivity in detecting the antigen in crude cherry leaf extracts which is the type of sample used in routine testing. We therefore recommend the use of PTA ELISA and/or DBIA for routine screening and detection of cherry mottle leaf virus. For field-grown plants, sampling should be carried out early in the season (April to May) using young leaves or flowers. Screenhouse-grown plants may be sampled at any time between April and September, but April to May and August to September appear to be optimal. The differences between field plants and screenhouse plants may be the result of differences in micro-climate, nutrient supply, or different disease status of the plants. The screenhouse plant was infected with a single virus whereas the field plants may have been infected with multiple viruses. Sample dilution appears to result in increased sensitivity of the PTA-ELISA assay. Prior to this study, detection of this disease was limited to indexing on woody indicator plants. This is a time consuming and sometimes unreliable procedure (Stouffer & Fridlund, 1989).

Both MAbs detected ChMLV in DBIA, PTA-ELISA, TAS-ELISA, and Western blot analysis. The MAbs did not react with ChMLV particles in immunosorbent electron microscopy (ISEM) studies. It is possible that these MAbs react with epitopes on the denatured particle. Further research is necessary to determine the identity of the epitopes with which the MAbs react.

Production of these antibodies has resulted in the availability of tools for rapid diagnosis of this disease. This is the first report of the production of antibodies against this virus. It is possible that this approach may be effective in the production of antibodies against other viruses.

Acknowledgements We thank Sharon Godkin for the photography, and Moira Timmons for her technical

assistance. We very gratefully acknowledge Vern Damsteegt, Bill Howell, Tom Li, George Oldfield, and Jerry Uyemoto for generously providing the various isolates and samples.


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(Received 18 December 1995)

comparison of elisa and immunoblotting techniques for the detection of cherry mottle leaf virus

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