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Evaluation of enzyme-linked immunosorbent assays for the detection of cymbidium mosaic virus in orchids
JOURNAL OF FERMENTATION AND BIOENGINEERING vol. 86, No. I, 65-71. 1998
Evaluation of Enzyme-Linked Immunosorbent Assays for the Detection of Cymbidium Mosaic Virus in Orchids RENU VEJARATPIMOL,‘* CHOCKPISIT CHANNUNTAPIPAT,’ PITTAYA LIEWSAREE,Z THANIT PEWNIM,’ KAZUO ITO, MASARU IIZUKA,3 AND NOSH1 MINAMIURA3 Faculty of Science,’ Faculty of Industrial Technology,2 Silpakorn University, Nakorn Pathom 73000, Thailand and Faculty of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-0022,3 Japan Received 6 January 199WAccepted 27 April 1998 Indirect enzyme-linked immunosorbent assay (ELISA) and double antibody sandwich (DAS) ELISA using polyclonal antibodies (PAb), egg yolk immunoglobulin G (IgY), rabbit antisera (IgG) or monoclonal antibodies (MAb) alone, or any two in combination, were compared for the detection of Cymbidium mosaic virus (CyMV) in orchids. An indirect ELBA using IgG, IgY or MAh was able to detect SO-100 ng of purified virus and 28-fold dilution of infected leaf extract. Detection of purified CyMV by DAS-ELISA using IgY as the detecting antibody gave 4-16 times higher sensitivity than that using IgG on either MAb or PAh coated microplates. The DAS-ELISA using MAb as the coating antibody and IgY as the detecting antibody was shown to be the most sensitive, being able to detect 6.25 ng of purified virus and 1024-fold dilution of an infected leaf extract. [Key words:
cymbidium mosaic virus, orchid virus, serological diagnosis]
Cymbidium mosaic virus (CyMV) has been reported to cause frequent infections in a wide range of orchid genera in many countries (l-3). This RNA virus belongs to the group of flexuous rod-shaped potexviruses that are approximately 475-490nm in length (2, 4). Orchid cultivars infected with CyMV often show reduced flower quality, necrosis and flower disfigurement as well as foliage symptoms, resulting in great economic loss (5, 6). The most important orchids from a commercial standpoint are cut-flower orchids that Thailand has been the largest producer with a total export volume of around $35 million (3). In order to produce high-quality orchids and to prevent the virus from being spread through propagation by tissue culture, the starting tissue should be tested. CyMV infections in orchids can be identified by a host plant bioassay procedure as well as electron microscopy (7). Various serological techniques based on polyclonal antisera are frequently employed but some limitations still exist such as lack of high-quality antisera as well as a readily available supply of highly specific antisera (8). Many researchers have described the advantages of using hens to produce antibodies against plant viruses (9-11). The advantages included production of large amounts of antibody concentrated in egg yolk after injection of relatively small amounts of virus (12, 13). The structural differences between egg yolk IgY and rabbit IgG have been described (14) and IgY derived from hens can replace IgG produced by conventional methods in mammals (15). Hybridoma technology provides a means to produce a potentially unlimited supply of highly specific uniform antibody (8). The monoclonal antibody is useful in the detection of some plant viruses and could solve the problems associated with conventional antibody production in rabbits (16-18). Although detection of CyMV by polymerase chain reaction was reported to be highly sensitive (19, 20), ELISA tests are still frequently used for routine viral screening by orchid growers and the industry because of their specificity, speed, the scope * Corresponding
provided for standardization and inexpensive assay facilities. Some of the serological techniques that have been used for detecting CyMV in infected orchids are immunodiffusion (21) and immunosorbent electron microdot blot immunoassay and ELBA. However, scopy, ELISA was reported to be the least sensitive for detecting the virus in plant sap (22). In this paper, indirect ELBA and double antibody sandwich ELISA using IgY, IgG or MAb alone or any two in combination, were compared for the detection of CyMV in orchid. MATERIALS
AND
METHODS
Virus was isolated from Virus and purification chlorotic streak leaf of Dendrobium Pompadour and inoculated onto leaves of the indexing host, Cassia occidentafis. Local lesions on CyMV-infected Cassia were mechanically transferred to Datura stramonium for multiplication and purification as previously described (23). Dilution of purified virus was prepared based on the assumption that 1 mg/ml concentration of the virus had an absorbance of 3 at 260nm (4). Purified odontoglossum ringspot virus (ORSV), tomato mosaic virus (ToMV), passion fruit woodiness virus (PWV), soybean mosaic virus (SMV) and potato virus X (PVX) were used in the specificity test. Purified virus was stored by freezing at ~20°C. One gram of leaf from Preparation of leaf extract either healthy plant or CyMV-infected orchid was minced in 2 ml coating buffer (0.05 M carbonate buffer, pH 9.6). The sap was briefly clarified by centrifugation at 5OOOxg for 10min and further dilutions were made before using in ELISA. IgG for Antibody production and purification CyMV detection was obtained from a white New Zealand rabbit. The rabbit was given three intramuscular injections at four-day intervals, each containing I mg of purified CyMV emulsified with an equal volume of Freund’s adjuvant and boosted at 9 weeks after the first immunization. The rabbit was bled once a week starting
author. 65
66
VEJARATPIMOL
J. FERMENT.BIOENG.,
ET AL.
at day 22. Egg yolk IgY was obtained from eggs of hens immunized intramuscularly with an emulsion of 1 mg of purified virus in adjuvant and boosted twice at two-week intervals. IgY was purified using polyethylene glycol (PEG 6,000), as described by Poison et al. (12), followed by dissolving the pellet to the original volume of yolk using 0.1 M NaCl, in 10 mM sodium phosphate (pH 7.4). The residual PEG was removed by adding cool ethanol to obtain a final concentration of 40% (v/v). IgY was collected by centrifugation at 4000 x g for 10 min. The pellet was dried in a dessicator before dissolving in PBS (10mM sodium potassium phosphate, pH 7.4), then applied to Bio-Gel A-l.5 column. MAb (IgG,) was obtained from ascitic fluid of BALB/c mice injected intraperitoneally with a suspension of 5 x lo6 hybridoma cells (SU 393) as previously described (24). Rabbit antisera and ascitic fluid were purified by Protein A Sepharose 4 B (Zymed) column chromatography according to the manufacturer’s instructions. The antibody fraction was detected by the indirect ELISA method, collected and stored at -20°C. The purity of immunoglobulins was determined by SDS-polyacrylamide gel electrophoresis. Antibody concentrations were determined based on the assumption that 1 mg/ml soIution of antibody had an absorbance of 1.5 at 280nm (25). Indirect ELISA We compared the immunoreactivities of MAb, IgY and IgG with the purified virus and with the virus in infected leaf extracts. Flat-bottomed immunoplates (4-42404, NUNC) were coated and incubated overnight at 4°C with either 50~1 of purified virus or leaf extract in coating buffer and then rinsed three times with PBS-Tween (PBST). The remaining binding sites were saturated with 1% bovine serum albumin (BSA) in PBST for 10min at room temperature. The blocking buffer was also used for diluting antibodies or conjugated antibodies in the test. The plates were washed again with PBST and then 50$ of antibody, either MAb, IgY or IgG, was added to the plate wells and incubated at 37°C for 1 h. After washing the plates with PBST, a total volume of 50 1’1 of a 1 : 4000 dilution of alkaline phosphatase-conjugated antibodies (Zymed), either goat anti-mouse IgG (GAM) or goat anti-chicken IgG (GAC) or goat anti-rabbit IgG (GAR) labelled with the enzyme, respectively, was added. The plates were incubated for one hour at 37°C and washed with buffer. Then, 200 /*I of a substrate solution containing p-nitrophenyl phospH 9.0, was phate at 1 mg/ml in lo,0O/ diethanolamine, added. The enzyme reaction was stopped after incubating at 37°C for 1.5 h by adding 50 1’1of 3 M NaOH and the absorbance at 405 nm was measured using a Lab System Uniskan II. Normal sera and immune sera from corresponding animals were used at 1 : 1500 dilution as negative and positive controls. Indirect double antibody sandwich ELBA (DASSix formats of DAS-ELISA were conducted ELISA) according to Clark and Adams (26) using the same conditions as the indirect ELISA described above. Plates were coated with 50~1 of coating antibodies, either MAb or PAb (IgY or IgG), and PAb or MAb was used as the detecting antibody to reveal trapped viruses. To determine the optimum conditions for ELISA, plates initially coated with various concentrations of antibody and virus were probed with different dilutions of antibody. The ten isolates of CyMV listed in Table 1 were used to test their reaction with antibody by indirect ELISA and DAS-ELISA. The specificity test was moni-
TABLE
1.
Cymbidium
Isolate designation
mosaic
Original
virus isolates
CyMV DO CyMV D2 CyMV D4
Dendrobium sp. Dendrobium Pompadour Dendrobium
CyMV CyMV CyMV CyMV CyMV CyMV CyMV
Dendrobium Walter Candy Cattleya sp. Cattleya Queen Sirikit Cattleya Lisa Ann Oncidium Goldiana Cymbidium Showgirl Aranda Christine
Jacqalyne D14 Cat1 Cat3 Cat5 0 1 CyM2 Al
Thomas
and their original Symptoms
host
Mosaic Chlorotic Mosaic
host
of host streak
White
_
Mottle Symptomless Color breaking Flower necrosis Mosaic Necrotic streak Mosaic
tored with five other plant viruses. RESULTS Determination of the optimum concentrations of antibodies In order to determine the optimum concentrations of antibodies for the indirect ELISA, plates were initially coated with 1 : 500 dilution of purified CyMV D2 (1 mg/ml) before adding detecting antibodies at various concentrations. The chosen concentrations of antibody that gave absorbance readings higher than 0.25 at 405 nm were 0.05 (2.5 ng of antibody per well), 0.20 and 0.50 /‘g/ml for MAb, IgY and IgG, respectively (Fig. 1). The optimum concentrations of antibodies to be used in the six formats of DAS-ELISA were determined. The combinations of each dilution series for coating antibodies and detecting antibodies were compared for the detection of CyMV D2 (1 : 500 dilution of 1 mg/ml). The chosen concentrations were the same as those of the above indirect ELISA. The chosen concentrations of coating antibody for the DAS-ELISA format I were 0.0125 /‘g/ml (0.625 ng) for MAb and 0.25 /.‘g/ml (12.5 ng) for IgY-detecting antibody (Fig. 2A). The coating and detecting antibody concentrations chosen in DASELISA formats II, III, IV, V and VI are shown in Figs. 2B-2F and 3. Sensitivity of indirect ELISA The relative sensitivities for CyMV D2 detection by indirect ELISA using MAb, IgY and IgG as detecting antibodies were compared. The virus was incubated in carbonate buffer in uncoated plates at concentrations ranging from 0.125 to 8 /(g/ml. A reading was considered positive if the absor0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
0.05 0.07 0.10 0.20 0.30 0.40 0.50 0.60 0.70 1.00 Antibody concentration
(pg/ml)
FIG. 1. Reactions of MAb, IgY and IgG against 1 : 500 dilution of CyMV D2 (1 mg/ml) in an indirect ELISA. Symbols: n , MAb (SU393); 0, egg yolk antibody (IgY); A, rabbit IgG.
VOL. 86, 1998
DETECTION
OF CYMBIDIUM
MOSAIC
VIRUS
IN ORCHIDS
67
0.6
0.050 1.0
0.100 IgY-detecting
0.250 antibody
0.500 (pg/ml)
1
,
0.025
1.000
0.050 IgG-detecting
0.100 antibody
0.250 (pg/ml)
0.500
2.5
0.8 2 0.7 5
0.6
t
4 0.0 0.025
0.050 Mab-detecting
0.075 antibody
0.100 (pg/ml)
0.025
0.200
0.050 Mab-detecting
0.075 antibody
0.100
0.200
(pglml)
2.5
0.3
1F
E
0.050
4
0.100 IgG-detecting
0.250 antibody
0.500
1 .ooo
(pglml)
0.050
0.100 IgY-detecting
0.250 antibody
0.500
1.000
(pg/ml)
FIG. 2. Comparison of reactions of coating antibody and detecting antibody dilutions with 1 : 500dilution of CyMV D2 (1 mg/ml) DAS-ELISA formats. The coating antibodies were MAb (A and B), IgY (C and E) and IgG (D and F). The coating antibody concentrations l 0.0125, 0 0.0250, 0 0.0500, 4 0.0750, n 0.1, 0 0.2500, A0.50OOand 0 l.OOOOyg/ml.
bance value at 405 nm was higher than the average absorbance value of the negative control plus four standard deviations. The indirect ELISA was sufficiently sensitive for detecting purified CyMV 02 at a concentration of 2.0 pg/ml (100ng of virus per well) when using MAb (0.05 /Ig/ml) or IgY (0.2 pg/ml) as detecting antibodies (Table 2). The method could also detect the virus at 1.0 lfg/rnl (50 ng of virus per well) when using IgG (0.5 (Ig/ml) as detecting antibody. The absorbance values at 405 nm for MAb, IgY and IgG indirect ELBA reactions were 0.297t0.057, 0.251kO.044 and 0.165+-0.015, respectively. The reactions of MAb, IgY and IgG to CyMV isolates
in six were
and to other plant viruses by indirect ELBA are presented in Table 3. All ten isolates of CyMV reacted with MAb, demonstrating that those isolates are serologically related even though they have different hosts and symptoms. However, there were differences in the degree of reaction among CyMV isolates to MAb. All CyMV isoIates also reacted to PAb. No reactions were observed with other plant viruses and plant proteins from healthy Spathoglofhis and Datura. Sensitivity of indirect double antibody sandwich ELBA (DAS-ELISA) The relative sensitivities for CyMV D2 detection using six DAS-ELISA formats were compared as shown in Fig. 3. The absorbance values obtained
68
VEJARATPIMOL
J. FERMENT.BIOENG.,
ET AL.
DAS-ELISA
FORMAT
Specific antibody
absorbed
to plate @g/ml)
I
II
MAb
MAb
(0.0125)
(0.0500)
III
IV
V
VI
I0
I@
IgY
IgG
(0.1000)
(0.0125)
(0.5000)
(0.0250)
1
4 “C
, overnight
wash & block Add purified virus or sap
1 h,37“C
1 wash
Add detecting antibody (@ml)
IgY
IgG
MAb
MAb
I&
IgY
(0.2500)
(0.5000)
(0.0750)
(0.0250)
(0.5000)
(0.2500)
1 h ,37 “C
1 wash Add enzyme-labelled hmnunoglobulin
GAC
GAR
GAM
GAM
(1:4000)
GAR
GAC
1 h,37”C
1 wash
Add substrate
1
3 M NaOH (50 ml)
Stop enzyme activity
color intensity (00
Measure ELISA activity
FIG. 3. Schematic representation six formats used in the indirect double antibody goat anti-mouse IgG; GAR, goat anti-rabbit IgG labelled with alkaline phosphatase.
from each format are presented in Table 4. The results indicated that all formats could easily detect CyMV D2, even though the sensitivity levels varied between formats. The DAS-ELISA formats I, II, III, IV, V and VI were capable of detecting CyMV D2 at concentrations as low as 0.125 (6.25ng of virus), 2.0, 0.5, 0.5, 1.0 and 0.25 pg/ml of virus, respectively. Detection of the virus TABLE
2.
Sensitivity
of indirect ELISA CyMV D2 Absorbance
Purified CyMV D2 (/fg/ml) 8.000 4.000 2.000 1.000 0.500 0.250 0.125 NC NC+4 S.D.
Detecting
antibody
1.5 h, 37 ‘=C
for the detection
th,
of
nmP
concentration
MAb (0.05 pg/ml)
IgY (0.20 pg/ml)
0.724kO.067 0.497+-0.081 0.297 +0.057b 0.077 f0.009 0.047~0.014 0.037+0.015 0.015~0.012 0.041 f0.023 0.133
0.667 iO.035 0.458+0.031 0.251 +0.044b 0.128iO.012 0.056+0.037 0.029+0.019 0.029+0.017 0.050t0.020 0.130
I& (0.50 /‘g/ml) 0.636f0.027 0.402?0.037 0.296kO.039 0.165+0.015b 0.090f0.007 0.026?0.012 0.011 F0.006 0.045i0.021 0.129
a Data are the mean f standard deviation (S.D.) of the &OS values of five replicates. An absorbance value higher than the negative control (NC) plus four standard deviations is assessed as positive. b First positive results.
sandwich
405)
ELISA.
GAC,
Goat anti-chicken
IgG; GAM,
by DAS-ELISA using IgY as detecting antibody gave higher sensitivity compared to that using IgG on either MAb (format I) or IgG (format VI) coated plates. Detection of the virus by DAS-ELISA using IgG as the detecting antibody was possible at 1 .O pg/ml of virus on IgY coated plate (format V) and at 2.0!1g/ml of virus on MAb coated plate (format II). When plates were coated with PAb, both IgY (format III) and IgG (format IV) gave equal sensitivities at 0.5 pg/ml when MAb was used as the detecting antibody. Of the six DAS-ELISA formats evaluated, format I was determined to be the most sensitive for the detection of the virus while formats II and V were the least sensitive. Therefore, these two formats were not used for the detection of CyMV in orchid tissue. The reactions of antibodies in various combinations to CyMV isolates and other plant viruses in six DASELBA formats are presented in Table 5. The results indicated that antibodies used in all formats were able to detect all isolates of CyMV tested but no reactions were observed with other plant viruses and plant proteins from healthy Spathoglothis and Datura. However, there were differences in the degree of reaction of different CyMV isolates in each format. Formats I and VI using IgY as the detecting antibody yielded similar results while formats II and V gave lower reactivities. Format V in particular showed negative reactivity to CyMV isolates Cat1
DETECTION OF CYMBIDIUM MOSAIC VIRUS IN ORCHIDS
VOL. 86, 1998 TABLE 3.
CyMV CyMV CyMV CyMV CyMV CyMV CyMV CyMV CyMV CyMV ORSV PVX ToMV PWV SMV
2
Reactions of antibodies with CyMV isolates and selected viruses in an indirect ELISA
DO D2 D4 D14 Cat1 Cat3 Cat5 01 CyM2 Al
Spathoglothisb Daturab NC NC+4 S.D.
i
Absorbance (Am5 nrnp Detecting antibody concentration MAb IgY IgG (0.05 &ml) (0.25 &ml) (0.50 &ml)
Purified virus (2 &ml)
1.000*0.051 0.482kO.074 0.373f0.031 2.072f0.040 0.609+0.095 0.874kO.064 0.546kO.054 0.236kO.022 1.185kO.010 1.028kO.113 0.027-tO.017 0.041 kO.025 -0.048+0.129 0.059f0.030 ~0.062+0.122
0.57k3*0.017 0.347kO.085 0.346kO.018 0.984kO.059 0.829kO.024 0.785 kO.018 0.56OkO.046 0.232kO.031 0.574t0.072 1.053zkO.018 0.039zkO.027 0.034-tO.023 0.053kO.026 0.047f0.031 0.036+0.020
0.245kO.023 0.295t0.031 0.268kO.042 0.386kO.027 0.237kO.033 0.392kO.051 0.277kO.042 0.255 kO.021 0.331 to.027 0.452kO.031 0.025t0.083 0.018*0.061 0.039-t0.079 0.008kO.045 -0.028f0.142
0.028kO.024 -0.121 to.158 0.042kO.025 0.142
0.091+0.006 0.051 kO.024 0.053 kO.020 0.133
-0.014+-0.079 0.023 f0.067 0.048+0.021 0.132
r“ 3
Purified CyMV D2 (pg/ml) 4.000 2.000 1 .ooo 0.500 0.250 0.125 0.062 NC NC+4 a plus b c
S.D.
of six formats
of indirect
0.707-+0.037 0.457*0.045 0.273fO.016 0.202+0.003 0.137+0.006 0.116k0.004c 0.108~0.001 0.039kO.018 0.111
0.277kO.005 0.245f0.028C 0.122~0.022 0.082f0.014 0.059kO.020 O.O42i-0.021 0.027kO.018 0.044+0.017 0.112
3 2
c1
%
Fi
z;’
;;
We have evaluated and compared three indirect ELISA formats and six DAS-ELISA formats for the detection of purified CyMV D2. The results indicated that DASELISA formats I, III, IV and VI gave higher sensitivity than indirect ELISA for the detection of purified virus. The highest sensitivity was obtained from DAS-ELISA format I, in which plates were coated with MAb and virus samples were probed with IgY. The sensitivity for detecting purified CyMV of format I was 8 times that of IgG indirect ELISA, and 16 times that of both MAb indirect ELISA and IgY indirect ELISA. In the case of detecting CyMV in crude sap of infected orchid, the sensitivity of DAS-ELISA format I was about 512 times that of either MAb indirect ELISA or IgG indirect ELISA. The sensitivity was 128 times that of IgY indirect ELISA. DAS-ELISA, however, requires an additional step during the assay. The usefulness of IgY as detecting antibody to MAb-trapped barley yellow dwarf virus isolates was reported (10). In contrast to our results, Culver and Sherwood found that MAb indirect ELISA had higher absolute sensitivity than the MAb sandwich test for the detection of peanut strip virus in peanut
DAS-ELISA
for detection
of Cymbidium
mosaic
virus (CyMV D2)
(Am5 nm)
DAS-ELISA 11 __
z
4
DISCUSSION
Absorbance 1
3
Dilution of leaf extract
produced absorbance values higher than those of homologous reactions at the same concentration. Detection of CyMV in orchid Determination of the sensitivities of three indirect ELISA and four DASELISA formats in detecting virus from sap of CyMVinfected Dendrobium sp. (at 2-fold dilution) was conducted. The results indicated that the indirect ELISA was able to detect virus in an orchid leaf sap diluted 2-, 8- and 2-folds when using MAb, IgY and IgG as detecting antibody, respectively. DAS-ELISA formats I, III, IV and VI were able to detect virus in sap diluted 1024-, 512-, .512- and 512-folds, respectively (Fig. 4). Sensitivity
00
FIG. 4. Detection of Cymbidium mosaic virus in leaf sap by indirect ELISA and DAS-ELISA. One hundred microliters of twofold serial dilution of CyMV-infected Dendrobium sp. was used per well. Indirect ELISA: + MAb, w IgG, A IgY, DAS-ELISA format: 0 format I, A format III, 0 format IV and i format VI.
and Cat3. Format V, therefore, would not be suitable for accurate detection of virus. CyMV isolates 01 and Cat3 had lower reactivities than the homologous antigen to antibodies in DAS-ELISA formats. Unexpectedly, CyMV isolates DO, D4, D14, Catl, Cats, CyM2 and Al
4.
1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 02 ‘0
3 ;;I 8 9 e $ $
a Data are the mean + standard deviation of the AdO5values of five replicates. An absorbance value higher than the negative control (NC) plus four standard deviations (S.D.) is assessed as positive. b Extract from Spathoglothis and Datura leaves with dilution of 1 : 10 was used as negative control of plant protein.
TABLE
69
TIT ___ 0.569kO.025 0.467kO.014 0.341 kO.042 0.162k0.046c 0.019~0.015 0.018kO.007 -0.029~0.013 0.039~0.019 0.115
Data are the mean k standard deviation (S.D.) of the A405 values of five replicates. four standard deviations is assessed as positive. Formats are described in Fig. 3. First positive result.
formatb
IV _ 0.446kO.054 0.279+0.015 0.222*0.001 0.161~0.031’ 0.113kO.038 0.013f0.002 -0.067kO.018 0.045 -to.017 0.113 An absorbance
V 0.273 kO.023 0.245 kO.033 0.190+0.060c 0.072zkO.078 0.069+0.004 0.036kO.047 0.006-c0.003 0.031 kO.020 0.111
VI 0.679iO.077 0.49OiO.081 0.406iO.086 0.254iO.017 0.184i0.025c 0.112io.022 0.105t0.021 0.055+-0.018 0.127
value higher than the negative
control
(NC)
VEJARATPIMOL
70
J. FERMENT.BIOENG.,
ET AL..
TABLE
5.
Reactions
of antibodies
to CyMV
isolates and selected viruses in DAS-ELISA
Absorbance Purified virus (2 /‘g/ml) CyMV DO CyMV D2 CyMV D4 CyMV D14 CvMV Cat 1 C;MV Cat3 CvMV Cat5
DAS-ELISA
I 0.863 f0.090 0.626+0.043 0.906kO.033 0.733f0.067 0.659-+0.051 0.499+0.055 1.081+0.145 0.390f0.072 1.127f0.134 0.805 f0.041 0.072kO.044 0.02O-tO.070 0.062f0.287 0.043 io.014 0.053 kO.026 0.034*0.013 0.045 iO.024 0.042iO.021 0.126
11 __ 0.285kO.043 0.226kO.016 0.295 i-O.041 0.30210.058 0.258t0.034 0.252kO.035 0.33 1 kO.039 0.216kO.001 0.337kO.053 0.252-+0.047 0.03310.035 0.006iO.004 0.033io.034 0.022+-0.023 0.011 io.047 0.062kO.018 0.026+0.014 0.052kO.021 0.132
III 0.693 kO.064 0.554*0.077 0.862f0.085 0.852f0.076 0.645 to.035 0.455 kO.061 1.087iO.140 0.393 iO.038 0.998kO.146 0.554iO.069 0.035*0.015 0.010*0.001 0.062t0.019 0.032-cO.030 0.057iO.024 0.064iO.018 0.014i0.001 0.041 io.001 0.129
(AJos nm) forma@ IV
V
VI
0.471 kO.080 0.412iO.063 0.879kO.079 0.704*0.071 0.600i0.800 0.409*0.057 1.236t0.162 0.377~0.070 0.961 kO.081 0.565 iO.038 0.061 iO.029 0.011 to.007 0.051 io.020 0.034+0.028 0.052f0.010 0.039~0.017 0.066rt0.018 0.053f0.019 0.129
0.221 io.020 0.171 kO.033 0.28OkO.030 0.317kO.026 0.083f0.023 0.071 f0.030 0.838kO.026 0.307kO.024 0.624kO.023 0.655 f0.030 -0.121 f0.221 0.054f0.001 -0.092+-0.228 -0.248+-0.257 -0.136kO.255 -0.129f0.026 -0.567+0.192 0.040*0.019 0.116
0.672kO.214 0.5821-0.044 0.916t0.058 0.613kO.027 0.499*0.009 0.409t0.002 0.865kO.012 0.402t0.026 0.741 f0.013 0.58OkO.010 0.043 io.040 0.051 iO.024 0.036kO.019 0.056 k 0.028 0.056iO.018 0.034kO.025 0.076kO.012 0.059kO.02 0.139
Data are the mean i- standard deviation (S.D.) of the AJus values of five replicates. An absorbance value higher than the negative control four standard deviations is assessed as positive. Formats are described in Fig. 3. Extract from Spafhoglothis and Datura leaves with dilution of 1 : 10 was used as negative control.
seed when 1 /(g/ml of rabbit IgG was used as coating antibody (27). Lommel et al. also reported that IgG indirect ELISA gave higher sensitivity for detecting purified virus in carnations than DAS-ELISA, in which rabbit IgG was used as both coating and detecting antibodies. In the presence of host protein, the detection of virus by DA’SELISA was unaffected whereas a decrease in absorbance was obtained by IgG indirect ELISA (28). These differences in reactivity might be due to the concentration and type of antibody used in the test. Although DAS-ELISA format II, in which plates were coated with MAb and viruses probed with IgG, exhibited the lowest sensitivity among the six formats, its sensitivity was about the same as that of MAb indirect ELISA and IgY indirect ELISA. However, DAS-ELISA in which the plates were coated with PAb, IgY (format III) or IgG (format IV) having MAb as the detecting antibody was able to detect purified viruses. These formats had higher sensitivity than the polyclonal DAS-ELISA format V, in which plates were coated with IgY and virus samples probed with IgG. These results are similar to those of Sherwood et al. (29) in which they detected peanut mottle virus using plates that were either coated with rabbit IgG and the sample probed with MAb or coated with MAb and the sample probed with polyclonal rabbit antibody. Similar observations were reported by Dore et al. in the detection of odontoglossum ringsport virus and the virus from plant sap using IgY-coated plates with MAb as the detecting antibody (11). Three indirect ELISA formats were sufficient for the detection of purified CyMV; however, the detection was limited to about l-2 /rg/ml (50-100ng) of virus. Since the most critical step in the detection of CyMV by an indirect ELISA is the absorption of virus onto polystyrene plates, it is necessary to investigate this step further if one aims at achieving higher sensitivity. In the case of detecting CyMV in orchid sap, the results indicated that the three indirect ELISA formats were not as sensitive
(NC)
when compared with that of the four DAS-ELISA formats. The lower sensitivity was probably due to the interference of nonviral proteins in the coating step. It was previously reported by Lommel et al. (27) that indirect ELISA activities of pure carnation mottle virus with specific antibody decreased when BSA was present. At 200-fold excess, the absorbance value was found to reduce by 520; compared with that of pure carnation mottle virus alone. It should be noted that Hsu et al. found the sensitivity of MAb indirect ELISA for the detection of CyMV in orchid tissue to be the lowest in comparison to that of immunosorbent electron microscopy and dot blot immunoassay and the dilution endpoint for those three detection methods was about 1 : 400, 1 : 800 and 1 : 3200, respectively (22). The serological relationships between CyMV isolates were observed in our study. The differences in degree of reaction of the CyMV isolates to MAb may indicate some slight serological difference among the CyMV isolates. The separation of CyMV isolates into various groups by serological data is not possible at this stage but preparation of MAb from hybridoma clones that recognize different epitopes on CyMV will make the reciprocal tests possible. Our results indicated that of the indirect ELISA formats evaluated, the DAS-ELISA format I was the most sensitive detection format for pure cymbidium mosaic virus as well as for the virus in orchid tissue. We investigated the potential use of IgY as detecting antibody in DAS-ELISA and found that it was better detecting antibody than MAb and IgG. This difference might be due in part to the more efficient detection by IgY. A number of previous studies on advantages of using hens to produce antibodies against plant viruses and other antigens have appeared. This is due to the remarkable ability of IgY to rapidly and efficiently generate an abundant supply of high antibody titer (9-11). It is, therefore, proposed that the final step involved in the indirect
DETECTION
VOL. 86, 1998
DAS-ELISA can be shortened ing IgY to a reporter enzyme.
by conjugating
the detect-
ACKNOWLEDGMENTS This work was supported programme, Science and Thailand (CPT88B-l-20-114). Silpakorn University.
by NRCT-JSPS scientific cooperation Technology Development Board of IDP from Australia and RDI of
REFERENCES purification and some properties of 1. Francki, R. I. B.: Isolation, two viruses from cultivated cymbidium orchids. Aust. J. Biol. Sci., 19, 555-564 (1966). 2. Lawson, R. H. and Brannigan, M.: Virus diseases of orchid, p. 2-49. In Handbook on orchid pests and diseases, Revised (ed.), American Orchid Society, USA (1986). 3. Zettler, F. W., Ko, N. J., Wisler, G. C., Elliot, M. S., and and their control. Plant Wong, S. M.: Viruses of orchids Disease, 74, 621-626 (1990). 4. Frowd, J. A. and Tremaioe, J. H.: Physical, chemical and serological properties of cymbidium mosaic virus. Phytopathology, 67, 43-49 (1977). 5. Lawson, R. H.: Etiology of flower necrosis in Cuttlera orchids. Phytopathology, 60, 36-40 (1970). R. J., Hammond, J., Lawson, R. H., and Pridg6. Griesbach, eon, A.M.: New methods may aid quest for virus-resistant orchids. Am. Orchid Sot. Bull., 58, 271-273 (1989). 7. Lawson, R. H.: Orchid viruses and their control, p. 66-101. In Pridgeon, A. M. and Tillman, L. L. (ed.), Handbook on orchid pests and diseases. American Orchid Society, USA (1990). antibodies in 8. Halk, E. L. and de Boerd, S. H.: Monoclonal plant-disease research. Ann. Rev. Phytopathology, 23, 321-350 (1985). M. and Malkinson, M.: Hen egg yolk as a source 9. Bar-Joseph, of antiviral antibodies in the enzyme-linked immunosorbent assay (ELISA): a comparison of two plant viruses. J. Virol. Methods, 1, 179-183 (1980). and 10. Hu, J. S., Rochow, W. F., and Dietert, R. R.: Production use of antibodies from hen eggs for the SGV isolate of barley yellow dwarf virus. Phytopathology, 75, 914-919 (1985). E. L., Porta, C., and van Regenmortel, 11. Dare, I., Dekker, M. H. V.: Detection by ELISA of two obomoviruses in orchids using monoclonal antibodies. J. Phytopathol., 20, 317-326 (1987). 12. Poison, A., von Wecbmar, M. B., and Regenmortel, M. H. V.: Isolation of viral IgY antibodies from yolks of immunized hens. Immunol. Commun., 9, 475-493 (1980). 13. Hatta, H.. Tsuda, K., Akachi, S.. Kim, M., and Yamamoto, T.: Productivity and some properties of egg yolk antibody (IgY) against human rotavirus compared with rabbit IgG. Biosci. Biotech. Biochem., 57, 450-454 (1993). 14. Shimizu, M., Nagashima, H., Sano, K., Hashimoto, K., Ozeki, M., Tsuda, K., and Hatta, H.: Molecular stability of chicken and rabbit immunoglobulin G. Biosci. Biotech. Bio-
OF CYMBIDIUM
MOSAIC
VIRUS
IN ORCHIDS
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them., 56, 270-274 (1992). in diagno15. Gross, M. and Speck, J.: Avain vitelline antibodies sis and research. Deutsche Tierarztliche Wochenschrift, 103, 417-422 (1996). 16. Halk, E. L., Hsu, H. T., Aebig, J., and Franke, J.: Production of monoclonal antibodies against three ilarviruses and alfalfa mosaic virus and their use in serotyping. Phytopathology, 74, 367-372 (1984). J. L., Sanborn, M. R., Keyser, G. C., and Myers, 17. Sherwood, L. D.: Use of monoclonal antibodies in detection of tomato spotted virus. Phytopathology, 79, 61-64 (1989). J., Jordan, R. L., Larsen, R. C., and Moyer, 18. Hammond, J. W.: Use of polyclonal antisera and monoclonal antibodies to examine serological relationships among three filamentous viruses of sweetpotato. Phytopathology, 82, 713-717 (1992). 19. Lim, S. T., Wong, S. M., Yeong, C. Y., Lee, S. C., and Gob, C. J.: Rapid detection of cymbidium mosaic virus by the polymerase chain reaction (PCR). J. Virol. Methods, 41, 37-46 (1993). 20. Srifah, P., Loprasert, S., and Rungroj, N.: Use of reverse transcription polymerase chain reaction for cloning of coat protein encoding genes of cymbidium mosaic virus. Gene, 179, 105107 (1996). 21. Wisler, G. C., Zettler, F. W., and Purcifull, D. E.: A serodiagnostic technique for cymbidium mosaic and odontoglossum ringspot viruses. Phytopathology, 72, 835-837 (1982). Vongsasitorn, D., and Lawson, R. H.: An im22. Hsu, H.T., proved method for serological detection of cymbidium mosaic potexvirus infection in orchids. Phytopathology, 82, 491-495 (1992). C., Vejaratpimol, R., and Pevnim, T.: Detec23. Chanuuntapipat, tion and collection of patially purified cymbidium mosaic virus isolates from orchids. Silpakorn University J., 17, 227-249 (1997). R., Channuntapipat, C., Lewsaree, P., Pewnim, 24. Vejaratpimol, N.: Characterization of T., Sithisarn, P., and Minamiura, monoclonal antibodies against cymbidium mosaic virus in orchid. Silpakorn University J., 13, 65-76 (1992). K., Hamaguchi, Y., Okawa, S., Ishikawa, E., 25. Kato, Kobayashi, K., and Katsunuma, N.: Use of rabbit antibody for the sandwich enzyme immunoassay of macromolecular antigens. J. Biochem., 81, 1557-1566 (1977). M. F. and Adams, A. N.: Characteristics of the 26. Clark, microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol., 34, 475-483 (1977). J. L.: Detection of peanut strip 27. Culver, J. N. and Sherwood, virus in peanut seed by an indirect enzyme-linked immunosorbent assay using a monoclonal antibody. Plant Dis., 72, 676679 (1988). 28. Lommel, S. A., McCain, A. H., and Morris, T. J.: Evaluation of indirect enzyme-linked immunosorbent assay for the detection of plant viruses. Phytopathology, 72, 1018-1022 (1982). 29. Sherwood, J. L., Sanborn, M. R., and Keyser, G. C.: Production of monoclonal antibodies to peanut mottle virus and their use in enzyme-linked immunosorbent assay and dot immunobinding assay. Phytopathology, 77, 1158-1161 (1987).
Evaluation of Enzyme-Linked Immunosorbent Assays for the Detection of Cymbidium Mosaic Virus in Orchids RENU VEJARATPIMOL,‘* CHOCKPISIT CHANNUNTAPIPAT,’ PITTAYA LIEWSAREE,Z THANIT PEWNIM,’ KAZUO ITO, MASARU IIZUKA,3 AND NOSH1 MINAMIURA3 Faculty of Science,’ Faculty of Industrial Technology,2 Silpakorn University, Nakorn Pathom 73000, Thailand and Faculty of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-0022,3 Japan Received 6 January 199WAccepted 27 April 1998 Indirect enzyme-linked immunosorbent assay (ELISA) and double antibody sandwich (DAS) ELISA using polyclonal antibodies (PAb), egg yolk immunoglobulin G (IgY), rabbit antisera (IgG) or monoclonal antibodies (MAb) alone, or any two in combination, were compared for the detection of Cymbidium mosaic virus (CyMV) in orchids. An indirect ELBA using IgG, IgY or MAh was able to detect SO-100 ng of purified virus and 28-fold dilution of infected leaf extract. Detection of purified CyMV by DAS-ELISA using IgY as the detecting antibody gave 4-16 times higher sensitivity than that using IgG on either MAb or PAh coated microplates. The DAS-ELISA using MAb as the coating antibody and IgY as the detecting antibody was shown to be the most sensitive, being able to detect 6.25 ng of purified virus and 1024-fold dilution of an infected leaf extract. [Key words:
cymbidium mosaic virus, orchid virus, serological diagnosis]
Cymbidium mosaic virus (CyMV) has been reported to cause frequent infections in a wide range of orchid genera in many countries (l-3). This RNA virus belongs to the group of flexuous rod-shaped potexviruses that are approximately 475-490nm in length (2, 4). Orchid cultivars infected with CyMV often show reduced flower quality, necrosis and flower disfigurement as well as foliage symptoms, resulting in great economic loss (5, 6). The most important orchids from a commercial standpoint are cut-flower orchids that Thailand has been the largest producer with a total export volume of around $35 million (3). In order to produce high-quality orchids and to prevent the virus from being spread through propagation by tissue culture, the starting tissue should be tested. CyMV infections in orchids can be identified by a host plant bioassay procedure as well as electron microscopy (7). Various serological techniques based on polyclonal antisera are frequently employed but some limitations still exist such as lack of high-quality antisera as well as a readily available supply of highly specific antisera (8). Many researchers have described the advantages of using hens to produce antibodies against plant viruses (9-11). The advantages included production of large amounts of antibody concentrated in egg yolk after injection of relatively small amounts of virus (12, 13). The structural differences between egg yolk IgY and rabbit IgG have been described (14) and IgY derived from hens can replace IgG produced by conventional methods in mammals (15). Hybridoma technology provides a means to produce a potentially unlimited supply of highly specific uniform antibody (8). The monoclonal antibody is useful in the detection of some plant viruses and could solve the problems associated with conventional antibody production in rabbits (16-18). Although detection of CyMV by polymerase chain reaction was reported to be highly sensitive (19, 20), ELISA tests are still frequently used for routine viral screening by orchid growers and the industry because of their specificity, speed, the scope * Corresponding
provided for standardization and inexpensive assay facilities. Some of the serological techniques that have been used for detecting CyMV in infected orchids are immunodiffusion (21) and immunosorbent electron microdot blot immunoassay and ELBA. However, scopy, ELISA was reported to be the least sensitive for detecting the virus in plant sap (22). In this paper, indirect ELBA and double antibody sandwich ELISA using IgY, IgG or MAb alone or any two in combination, were compared for the detection of CyMV in orchid. MATERIALS
AND
METHODS
Virus was isolated from Virus and purification chlorotic streak leaf of Dendrobium Pompadour and inoculated onto leaves of the indexing host, Cassia occidentafis. Local lesions on CyMV-infected Cassia were mechanically transferred to Datura stramonium for multiplication and purification as previously described (23). Dilution of purified virus was prepared based on the assumption that 1 mg/ml concentration of the virus had an absorbance of 3 at 260nm (4). Purified odontoglossum ringspot virus (ORSV), tomato mosaic virus (ToMV), passion fruit woodiness virus (PWV), soybean mosaic virus (SMV) and potato virus X (PVX) were used in the specificity test. Purified virus was stored by freezing at ~20°C. One gram of leaf from Preparation of leaf extract either healthy plant or CyMV-infected orchid was minced in 2 ml coating buffer (0.05 M carbonate buffer, pH 9.6). The sap was briefly clarified by centrifugation at 5OOOxg for 10min and further dilutions were made before using in ELISA. IgG for Antibody production and purification CyMV detection was obtained from a white New Zealand rabbit. The rabbit was given three intramuscular injections at four-day intervals, each containing I mg of purified CyMV emulsified with an equal volume of Freund’s adjuvant and boosted at 9 weeks after the first immunization. The rabbit was bled once a week starting
author. 65
66
VEJARATPIMOL
J. FERMENT.BIOENG.,
ET AL.
at day 22. Egg yolk IgY was obtained from eggs of hens immunized intramuscularly with an emulsion of 1 mg of purified virus in adjuvant and boosted twice at two-week intervals. IgY was purified using polyethylene glycol (PEG 6,000), as described by Poison et al. (12), followed by dissolving the pellet to the original volume of yolk using 0.1 M NaCl, in 10 mM sodium phosphate (pH 7.4). The residual PEG was removed by adding cool ethanol to obtain a final concentration of 40% (v/v). IgY was collected by centrifugation at 4000 x g for 10 min. The pellet was dried in a dessicator before dissolving in PBS (10mM sodium potassium phosphate, pH 7.4), then applied to Bio-Gel A-l.5 column. MAb (IgG,) was obtained from ascitic fluid of BALB/c mice injected intraperitoneally with a suspension of 5 x lo6 hybridoma cells (SU 393) as previously described (24). Rabbit antisera and ascitic fluid were purified by Protein A Sepharose 4 B (Zymed) column chromatography according to the manufacturer’s instructions. The antibody fraction was detected by the indirect ELISA method, collected and stored at -20°C. The purity of immunoglobulins was determined by SDS-polyacrylamide gel electrophoresis. Antibody concentrations were determined based on the assumption that 1 mg/ml soIution of antibody had an absorbance of 1.5 at 280nm (25). Indirect ELISA We compared the immunoreactivities of MAb, IgY and IgG with the purified virus and with the virus in infected leaf extracts. Flat-bottomed immunoplates (4-42404, NUNC) were coated and incubated overnight at 4°C with either 50~1 of purified virus or leaf extract in coating buffer and then rinsed three times with PBS-Tween (PBST). The remaining binding sites were saturated with 1% bovine serum albumin (BSA) in PBST for 10min at room temperature. The blocking buffer was also used for diluting antibodies or conjugated antibodies in the test. The plates were washed again with PBST and then 50$ of antibody, either MAb, IgY or IgG, was added to the plate wells and incubated at 37°C for 1 h. After washing the plates with PBST, a total volume of 50 1’1 of a 1 : 4000 dilution of alkaline phosphatase-conjugated antibodies (Zymed), either goat anti-mouse IgG (GAM) or goat anti-chicken IgG (GAC) or goat anti-rabbit IgG (GAR) labelled with the enzyme, respectively, was added. The plates were incubated for one hour at 37°C and washed with buffer. Then, 200 /*I of a substrate solution containing p-nitrophenyl phospH 9.0, was phate at 1 mg/ml in lo,0O/ diethanolamine, added. The enzyme reaction was stopped after incubating at 37°C for 1.5 h by adding 50 1’1of 3 M NaOH and the absorbance at 405 nm was measured using a Lab System Uniskan II. Normal sera and immune sera from corresponding animals were used at 1 : 1500 dilution as negative and positive controls. Indirect double antibody sandwich ELBA (DASSix formats of DAS-ELISA were conducted ELISA) according to Clark and Adams (26) using the same conditions as the indirect ELISA described above. Plates were coated with 50~1 of coating antibodies, either MAb or PAb (IgY or IgG), and PAb or MAb was used as the detecting antibody to reveal trapped viruses. To determine the optimum conditions for ELISA, plates initially coated with various concentrations of antibody and virus were probed with different dilutions of antibody. The ten isolates of CyMV listed in Table 1 were used to test their reaction with antibody by indirect ELISA and DAS-ELISA. The specificity test was moni-
TABLE
1.
Cymbidium
Isolate designation
mosaic
Original
virus isolates
CyMV DO CyMV D2 CyMV D4
Dendrobium sp. Dendrobium Pompadour Dendrobium
CyMV CyMV CyMV CyMV CyMV CyMV CyMV
Dendrobium Walter Candy Cattleya sp. Cattleya Queen Sirikit Cattleya Lisa Ann Oncidium Goldiana Cymbidium Showgirl Aranda Christine
Jacqalyne D14 Cat1 Cat3 Cat5 0 1 CyM2 Al
Thomas
and their original Symptoms
host
Mosaic Chlorotic Mosaic
host
of host streak
White
_
Mottle Symptomless Color breaking Flower necrosis Mosaic Necrotic streak Mosaic
tored with five other plant viruses. RESULTS Determination of the optimum concentrations of antibodies In order to determine the optimum concentrations of antibodies for the indirect ELISA, plates were initially coated with 1 : 500 dilution of purified CyMV D2 (1 mg/ml) before adding detecting antibodies at various concentrations. The chosen concentrations of antibody that gave absorbance readings higher than 0.25 at 405 nm were 0.05 (2.5 ng of antibody per well), 0.20 and 0.50 /‘g/ml for MAb, IgY and IgG, respectively (Fig. 1). The optimum concentrations of antibodies to be used in the six formats of DAS-ELISA were determined. The combinations of each dilution series for coating antibodies and detecting antibodies were compared for the detection of CyMV D2 (1 : 500 dilution of 1 mg/ml). The chosen concentrations were the same as those of the above indirect ELISA. The chosen concentrations of coating antibody for the DAS-ELISA format I were 0.0125 /‘g/ml (0.625 ng) for MAb and 0.25 /.‘g/ml (12.5 ng) for IgY-detecting antibody (Fig. 2A). The coating and detecting antibody concentrations chosen in DASELISA formats II, III, IV, V and VI are shown in Figs. 2B-2F and 3. Sensitivity of indirect ELISA The relative sensitivities for CyMV D2 detection by indirect ELISA using MAb, IgY and IgG as detecting antibodies were compared. The virus was incubated in carbonate buffer in uncoated plates at concentrations ranging from 0.125 to 8 /(g/ml. A reading was considered positive if the absor0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
0.05 0.07 0.10 0.20 0.30 0.40 0.50 0.60 0.70 1.00 Antibody concentration
(pg/ml)
FIG. 1. Reactions of MAb, IgY and IgG against 1 : 500 dilution of CyMV D2 (1 mg/ml) in an indirect ELISA. Symbols: n , MAb (SU393); 0, egg yolk antibody (IgY); A, rabbit IgG.
VOL. 86, 1998
DETECTION
OF CYMBIDIUM
MOSAIC
VIRUS
IN ORCHIDS
67
0.6
0.050 1.0
0.100 IgY-detecting
0.250 antibody
0.500 (pg/ml)
1
,
0.025
1.000
0.050 IgG-detecting
0.100 antibody
0.250 (pg/ml)
0.500
2.5
0.8 2 0.7 5
0.6
t
4 0.0 0.025
0.050 Mab-detecting
0.075 antibody
0.100 (pg/ml)
0.025
0.200
0.050 Mab-detecting
0.075 antibody
0.100
0.200
(pglml)
2.5
0.3
1F
E
0.050
4
0.100 IgG-detecting
0.250 antibody
0.500
1 .ooo
(pglml)
0.050
0.100 IgY-detecting
0.250 antibody
0.500
1.000
(pg/ml)
FIG. 2. Comparison of reactions of coating antibody and detecting antibody dilutions with 1 : 500dilution of CyMV D2 (1 mg/ml) DAS-ELISA formats. The coating antibodies were MAb (A and B), IgY (C and E) and IgG (D and F). The coating antibody concentrations l 0.0125, 0 0.0250, 0 0.0500, 4 0.0750, n 0.1, 0 0.2500, A0.50OOand 0 l.OOOOyg/ml.
bance value at 405 nm was higher than the average absorbance value of the negative control plus four standard deviations. The indirect ELISA was sufficiently sensitive for detecting purified CyMV 02 at a concentration of 2.0 pg/ml (100ng of virus per well) when using MAb (0.05 /Ig/ml) or IgY (0.2 pg/ml) as detecting antibodies (Table 2). The method could also detect the virus at 1.0 lfg/rnl (50 ng of virus per well) when using IgG (0.5 (Ig/ml) as detecting antibody. The absorbance values at 405 nm for MAb, IgY and IgG indirect ELBA reactions were 0.297t0.057, 0.251kO.044 and 0.165+-0.015, respectively. The reactions of MAb, IgY and IgG to CyMV isolates
in six were
and to other plant viruses by indirect ELBA are presented in Table 3. All ten isolates of CyMV reacted with MAb, demonstrating that those isolates are serologically related even though they have different hosts and symptoms. However, there were differences in the degree of reaction among CyMV isolates to MAb. All CyMV isoIates also reacted to PAb. No reactions were observed with other plant viruses and plant proteins from healthy Spathoglofhis and Datura. Sensitivity of indirect double antibody sandwich ELBA (DAS-ELISA) The relative sensitivities for CyMV D2 detection using six DAS-ELISA formats were compared as shown in Fig. 3. The absorbance values obtained
68
VEJARATPIMOL
J. FERMENT.BIOENG.,
ET AL.
DAS-ELISA
FORMAT
Specific antibody
absorbed
to plate @g/ml)
I
II
MAb
MAb
(0.0125)
(0.0500)
III
IV
V
VI
I0
I@
IgY
IgG
(0.1000)
(0.0125)
(0.5000)
(0.0250)
1
4 “C
, overnight
wash & block Add purified virus or sap
1 h,37“C
1 wash
Add detecting antibody (@ml)
IgY
IgG
MAb
MAb
I&
IgY
(0.2500)
(0.5000)
(0.0750)
(0.0250)
(0.5000)
(0.2500)
1 h ,37 “C
1 wash Add enzyme-labelled hmnunoglobulin
GAC
GAR
GAM
GAM
(1:4000)
GAR
GAC
1 h,37”C
1 wash
Add substrate
1
3 M NaOH (50 ml)
Stop enzyme activity
color intensity (00
Measure ELISA activity
FIG. 3. Schematic representation six formats used in the indirect double antibody goat anti-mouse IgG; GAR, goat anti-rabbit IgG labelled with alkaline phosphatase.
from each format are presented in Table 4. The results indicated that all formats could easily detect CyMV D2, even though the sensitivity levels varied between formats. The DAS-ELISA formats I, II, III, IV, V and VI were capable of detecting CyMV D2 at concentrations as low as 0.125 (6.25ng of virus), 2.0, 0.5, 0.5, 1.0 and 0.25 pg/ml of virus, respectively. Detection of the virus TABLE
2.
Sensitivity
of indirect ELISA CyMV D2 Absorbance
Purified CyMV D2 (/fg/ml) 8.000 4.000 2.000 1.000 0.500 0.250 0.125 NC NC+4 S.D.
Detecting
antibody
1.5 h, 37 ‘=C
for the detection
th,
of
nmP
concentration
MAb (0.05 pg/ml)
IgY (0.20 pg/ml)
0.724kO.067 0.497+-0.081 0.297 +0.057b 0.077 f0.009 0.047~0.014 0.037+0.015 0.015~0.012 0.041 f0.023 0.133
0.667 iO.035 0.458+0.031 0.251 +0.044b 0.128iO.012 0.056+0.037 0.029+0.019 0.029+0.017 0.050t0.020 0.130
I& (0.50 /‘g/ml) 0.636f0.027 0.402?0.037 0.296kO.039 0.165+0.015b 0.090f0.007 0.026?0.012 0.011 F0.006 0.045i0.021 0.129
a Data are the mean f standard deviation (S.D.) of the &OS values of five replicates. An absorbance value higher than the negative control (NC) plus four standard deviations is assessed as positive. b First positive results.
sandwich
405)
ELISA.
GAC,
Goat anti-chicken
IgG; GAM,
by DAS-ELISA using IgY as detecting antibody gave higher sensitivity compared to that using IgG on either MAb (format I) or IgG (format VI) coated plates. Detection of the virus by DAS-ELISA using IgG as the detecting antibody was possible at 1 .O pg/ml of virus on IgY coated plate (format V) and at 2.0!1g/ml of virus on MAb coated plate (format II). When plates were coated with PAb, both IgY (format III) and IgG (format IV) gave equal sensitivities at 0.5 pg/ml when MAb was used as the detecting antibody. Of the six DAS-ELISA formats evaluated, format I was determined to be the most sensitive for the detection of the virus while formats II and V were the least sensitive. Therefore, these two formats were not used for the detection of CyMV in orchid tissue. The reactions of antibodies in various combinations to CyMV isolates and other plant viruses in six DASELBA formats are presented in Table 5. The results indicated that antibodies used in all formats were able to detect all isolates of CyMV tested but no reactions were observed with other plant viruses and plant proteins from healthy Spathoglothis and Datura. However, there were differences in the degree of reaction of different CyMV isolates in each format. Formats I and VI using IgY as the detecting antibody yielded similar results while formats II and V gave lower reactivities. Format V in particular showed negative reactivity to CyMV isolates Cat1
DETECTION OF CYMBIDIUM MOSAIC VIRUS IN ORCHIDS
VOL. 86, 1998 TABLE 3.
CyMV CyMV CyMV CyMV CyMV CyMV CyMV CyMV CyMV CyMV ORSV PVX ToMV PWV SMV
2
Reactions of antibodies with CyMV isolates and selected viruses in an indirect ELISA
DO D2 D4 D14 Cat1 Cat3 Cat5 01 CyM2 Al
Spathoglothisb Daturab NC NC+4 S.D.
i
Absorbance (Am5 nrnp Detecting antibody concentration MAb IgY IgG (0.05 &ml) (0.25 &ml) (0.50 &ml)
Purified virus (2 &ml)
1.000*0.051 0.482kO.074 0.373f0.031 2.072f0.040 0.609+0.095 0.874kO.064 0.546kO.054 0.236kO.022 1.185kO.010 1.028kO.113 0.027-tO.017 0.041 kO.025 -0.048+0.129 0.059f0.030 ~0.062+0.122
0.57k3*0.017 0.347kO.085 0.346kO.018 0.984kO.059 0.829kO.024 0.785 kO.018 0.56OkO.046 0.232kO.031 0.574t0.072 1.053zkO.018 0.039zkO.027 0.034-tO.023 0.053kO.026 0.047f0.031 0.036+0.020
0.245kO.023 0.295t0.031 0.268kO.042 0.386kO.027 0.237kO.033 0.392kO.051 0.277kO.042 0.255 kO.021 0.331 to.027 0.452kO.031 0.025t0.083 0.018*0.061 0.039-t0.079 0.008kO.045 -0.028f0.142
0.028kO.024 -0.121 to.158 0.042kO.025 0.142
0.091+0.006 0.051 kO.024 0.053 kO.020 0.133
-0.014+-0.079 0.023 f0.067 0.048+0.021 0.132
r“ 3
Purified CyMV D2 (pg/ml) 4.000 2.000 1 .ooo 0.500 0.250 0.125 0.062 NC NC+4 a plus b c
S.D.
of six formats
of indirect
0.707-+0.037 0.457*0.045 0.273fO.016 0.202+0.003 0.137+0.006 0.116k0.004c 0.108~0.001 0.039kO.018 0.111
0.277kO.005 0.245f0.028C 0.122~0.022 0.082f0.014 0.059kO.020 O.O42i-0.021 0.027kO.018 0.044+0.017 0.112
3 2
c1
%
Fi
z;’
;;
We have evaluated and compared three indirect ELISA formats and six DAS-ELISA formats for the detection of purified CyMV D2. The results indicated that DASELISA formats I, III, IV and VI gave higher sensitivity than indirect ELISA for the detection of purified virus. The highest sensitivity was obtained from DAS-ELISA format I, in which plates were coated with MAb and virus samples were probed with IgY. The sensitivity for detecting purified CyMV of format I was 8 times that of IgG indirect ELISA, and 16 times that of both MAb indirect ELISA and IgY indirect ELISA. In the case of detecting CyMV in crude sap of infected orchid, the sensitivity of DAS-ELISA format I was about 512 times that of either MAb indirect ELISA or IgG indirect ELISA. The sensitivity was 128 times that of IgY indirect ELISA. DAS-ELISA, however, requires an additional step during the assay. The usefulness of IgY as detecting antibody to MAb-trapped barley yellow dwarf virus isolates was reported (10). In contrast to our results, Culver and Sherwood found that MAb indirect ELISA had higher absolute sensitivity than the MAb sandwich test for the detection of peanut strip virus in peanut
DAS-ELISA
for detection
of Cymbidium
mosaic
virus (CyMV D2)
(Am5 nm)
DAS-ELISA 11 __
z
4
DISCUSSION
Absorbance 1
3
Dilution of leaf extract
produced absorbance values higher than those of homologous reactions at the same concentration. Detection of CyMV in orchid Determination of the sensitivities of three indirect ELISA and four DASELISA formats in detecting virus from sap of CyMVinfected Dendrobium sp. (at 2-fold dilution) was conducted. The results indicated that the indirect ELISA was able to detect virus in an orchid leaf sap diluted 2-, 8- and 2-folds when using MAb, IgY and IgG as detecting antibody, respectively. DAS-ELISA formats I, III, IV and VI were able to detect virus in sap diluted 1024-, 512-, .512- and 512-folds, respectively (Fig. 4). Sensitivity
00
FIG. 4. Detection of Cymbidium mosaic virus in leaf sap by indirect ELISA and DAS-ELISA. One hundred microliters of twofold serial dilution of CyMV-infected Dendrobium sp. was used per well. Indirect ELISA: + MAb, w IgG, A IgY, DAS-ELISA format: 0 format I, A format III, 0 format IV and i format VI.
and Cat3. Format V, therefore, would not be suitable for accurate detection of virus. CyMV isolates 01 and Cat3 had lower reactivities than the homologous antigen to antibodies in DAS-ELISA formats. Unexpectedly, CyMV isolates DO, D4, D14, Catl, Cats, CyM2 and Al
4.
1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 02 ‘0
3 ;;I 8 9 e $ $
a Data are the mean + standard deviation of the AdO5values of five replicates. An absorbance value higher than the negative control (NC) plus four standard deviations (S.D.) is assessed as positive. b Extract from Spathoglothis and Datura leaves with dilution of 1 : 10 was used as negative control of plant protein.
TABLE
69
TIT ___ 0.569kO.025 0.467kO.014 0.341 kO.042 0.162k0.046c 0.019~0.015 0.018kO.007 -0.029~0.013 0.039~0.019 0.115
Data are the mean k standard deviation (S.D.) of the A405 values of five replicates. four standard deviations is assessed as positive. Formats are described in Fig. 3. First positive result.
formatb
IV _ 0.446kO.054 0.279+0.015 0.222*0.001 0.161~0.031’ 0.113kO.038 0.013f0.002 -0.067kO.018 0.045 -to.017 0.113 An absorbance
V 0.273 kO.023 0.245 kO.033 0.190+0.060c 0.072zkO.078 0.069+0.004 0.036kO.047 0.006-c0.003 0.031 kO.020 0.111
VI 0.679iO.077 0.49OiO.081 0.406iO.086 0.254iO.017 0.184i0.025c 0.112io.022 0.105t0.021 0.055+-0.018 0.127
value higher than the negative
control
(NC)
VEJARATPIMOL
70
J. FERMENT.BIOENG.,
ET AL..
TABLE
5.
Reactions
of antibodies
to CyMV
isolates and selected viruses in DAS-ELISA
Absorbance Purified virus (2 /‘g/ml) CyMV DO CyMV D2 CyMV D4 CyMV D14 CvMV Cat 1 C;MV Cat3 CvMV Cat5
DAS-ELISA
I 0.863 f0.090 0.626+0.043 0.906kO.033 0.733f0.067 0.659-+0.051 0.499+0.055 1.081+0.145 0.390f0.072 1.127f0.134 0.805 f0.041 0.072kO.044 0.02O-tO.070 0.062f0.287 0.043 io.014 0.053 kO.026 0.034*0.013 0.045 iO.024 0.042iO.021 0.126
11 __ 0.285kO.043 0.226kO.016 0.295 i-O.041 0.30210.058 0.258t0.034 0.252kO.035 0.33 1 kO.039 0.216kO.001 0.337kO.053 0.252-+0.047 0.03310.035 0.006iO.004 0.033io.034 0.022+-0.023 0.011 io.047 0.062kO.018 0.026+0.014 0.052kO.021 0.132
III 0.693 kO.064 0.554*0.077 0.862f0.085 0.852f0.076 0.645 to.035 0.455 kO.061 1.087iO.140 0.393 iO.038 0.998kO.146 0.554iO.069 0.035*0.015 0.010*0.001 0.062t0.019 0.032-cO.030 0.057iO.024 0.064iO.018 0.014i0.001 0.041 io.001 0.129
(AJos nm) forma@ IV
V
VI
0.471 kO.080 0.412iO.063 0.879kO.079 0.704*0.071 0.600i0.800 0.409*0.057 1.236t0.162 0.377~0.070 0.961 kO.081 0.565 iO.038 0.061 iO.029 0.011 to.007 0.051 io.020 0.034+0.028 0.052f0.010 0.039~0.017 0.066rt0.018 0.053f0.019 0.129
0.221 io.020 0.171 kO.033 0.28OkO.030 0.317kO.026 0.083f0.023 0.071 f0.030 0.838kO.026 0.307kO.024 0.624kO.023 0.655 f0.030 -0.121 f0.221 0.054f0.001 -0.092+-0.228 -0.248+-0.257 -0.136kO.255 -0.129f0.026 -0.567+0.192 0.040*0.019 0.116
0.672kO.214 0.5821-0.044 0.916t0.058 0.613kO.027 0.499*0.009 0.409t0.002 0.865kO.012 0.402t0.026 0.741 f0.013 0.58OkO.010 0.043 io.040 0.051 iO.024 0.036kO.019 0.056 k 0.028 0.056iO.018 0.034kO.025 0.076kO.012 0.059kO.02 0.139
Data are the mean i- standard deviation (S.D.) of the AJus values of five replicates. An absorbance value higher than the negative control four standard deviations is assessed as positive. Formats are described in Fig. 3. Extract from Spafhoglothis and Datura leaves with dilution of 1 : 10 was used as negative control.
seed when 1 /(g/ml of rabbit IgG was used as coating antibody (27). Lommel et al. also reported that IgG indirect ELISA gave higher sensitivity for detecting purified virus in carnations than DAS-ELISA, in which rabbit IgG was used as both coating and detecting antibodies. In the presence of host protein, the detection of virus by DA’SELISA was unaffected whereas a decrease in absorbance was obtained by IgG indirect ELISA (28). These differences in reactivity might be due to the concentration and type of antibody used in the test. Although DAS-ELISA format II, in which plates were coated with MAb and viruses probed with IgG, exhibited the lowest sensitivity among the six formats, its sensitivity was about the same as that of MAb indirect ELISA and IgY indirect ELISA. However, DAS-ELISA in which the plates were coated with PAb, IgY (format III) or IgG (format IV) having MAb as the detecting antibody was able to detect purified viruses. These formats had higher sensitivity than the polyclonal DAS-ELISA format V, in which plates were coated with IgY and virus samples probed with IgG. These results are similar to those of Sherwood et al. (29) in which they detected peanut mottle virus using plates that were either coated with rabbit IgG and the sample probed with MAb or coated with MAb and the sample probed with polyclonal rabbit antibody. Similar observations were reported by Dore et al. in the detection of odontoglossum ringsport virus and the virus from plant sap using IgY-coated plates with MAb as the detecting antibody (11). Three indirect ELISA formats were sufficient for the detection of purified CyMV; however, the detection was limited to about l-2 /rg/ml (50-100ng) of virus. Since the most critical step in the detection of CyMV by an indirect ELISA is the absorption of virus onto polystyrene plates, it is necessary to investigate this step further if one aims at achieving higher sensitivity. In the case of detecting CyMV in orchid sap, the results indicated that the three indirect ELISA formats were not as sensitive
(NC)
when compared with that of the four DAS-ELISA formats. The lower sensitivity was probably due to the interference of nonviral proteins in the coating step. It was previously reported by Lommel et al. (27) that indirect ELISA activities of pure carnation mottle virus with specific antibody decreased when BSA was present. At 200-fold excess, the absorbance value was found to reduce by 520; compared with that of pure carnation mottle virus alone. It should be noted that Hsu et al. found the sensitivity of MAb indirect ELISA for the detection of CyMV in orchid tissue to be the lowest in comparison to that of immunosorbent electron microscopy and dot blot immunoassay and the dilution endpoint for those three detection methods was about 1 : 400, 1 : 800 and 1 : 3200, respectively (22). The serological relationships between CyMV isolates were observed in our study. The differences in degree of reaction of the CyMV isolates to MAb may indicate some slight serological difference among the CyMV isolates. The separation of CyMV isolates into various groups by serological data is not possible at this stage but preparation of MAb from hybridoma clones that recognize different epitopes on CyMV will make the reciprocal tests possible. Our results indicated that of the indirect ELISA formats evaluated, the DAS-ELISA format I was the most sensitive detection format for pure cymbidium mosaic virus as well as for the virus in orchid tissue. We investigated the potential use of IgY as detecting antibody in DAS-ELISA and found that it was better detecting antibody than MAb and IgG. This difference might be due in part to the more efficient detection by IgY. A number of previous studies on advantages of using hens to produce antibodies against plant viruses and other antigens have appeared. This is due to the remarkable ability of IgY to rapidly and efficiently generate an abundant supply of high antibody titer (9-11). It is, therefore, proposed that the final step involved in the indirect
DETECTION
VOL. 86, 1998
DAS-ELISA can be shortened ing IgY to a reporter enzyme.
by conjugating
the detect-
ACKNOWLEDGMENTS This work was supported programme, Science and Thailand (CPT88B-l-20-114). Silpakorn University.
by NRCT-JSPS scientific cooperation Technology Development Board of IDP from Australia and RDI of
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