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Proper washing of the plates between steps is important. The standard procedure is to wash the plate three times between each step with phosphate-buffered saline (PBS) containing 0.5 percent Tween 20 (PBST). Sodium azide is frequently included in PBST solutions as a preservative. However, it is highly poisonous and may form an explosive complex with some metals. It is unnecessary to use sodium azide in ELISA wash solutions and it should be omitted. The two most critical wash steps are after sample incubation when cross-contamination must be avoided between wells containing different samples (omit the first wash immediately if carry-over between wells occurs) and after the conjugate incubation step. If even a minor residue of unattached conjugate remains, high background readings may occur (add another wash at this point when in doubt). Various plate washers are available which can promote consistent washing operations, but a plastic squeeze bottle will work well for small volumes of plates (Figure 154). Solutions in the plate wells can be removed by aspiration to avoid contamination, but usually the plate is inverted rapidly with a quick shake of the hand and tapped firmly on clean blotting paper or paper towels.
A wide variety of reactant concentrations and incubation times and conditions have been reported for ELISA(Clark and Bar-Joseph,1984; Clark, Lister and Bar-Joseph,1988; McLaughlin et al.,1981). The choice of conditions depends to some extent on the basic goals. By using high concentrations of reactants, short incubation times can be used and, if necessary, the entire ELISA procedure can be completed within two hours. Increasing the incubation time while decreasing concentration (especially of the conjugate) will conserve reactants. Reactions occur most rapidly at 30-37°C, but room temperatures (20-28°C) will give satisfactory results. Gentle shaking during incubation may improve efficiency. Many workers find it convenient to do the sample-incubation step overnight and often do this at 4-6°C.
Some experimentation will be necessary to determine optimum conditions for each situation. Schedules 1-3 give examples which should provide a good starting point. Moderate changes in times and conditions are unlikely to cause a test failure, and changes can often be made to render the schedule more convenient for the user with no loss of information. New users should certainly experiment with different schedules to find the optimum for their purpose.
One of the major variables in ELISA to be evaluated is the concentration of conjugate to use. Commercially prepared enzyme-labelled antibodies normally have a recommended working dilution (frequently between 1/1 000 and 1/2 000). Conjugates which are prepared experimentally may differ markedly and published values for other systems are of little help. Optimum dilutions of 1/100 to 1/20000 of the stock preparation (approximately 1 mg/ml) have been reported. The effective dilution will depend on the basic affinity of the antibody, the titre of specific and non-specific (host) antibodies, the source and activity of the enzyme used and the effectiveness of the conjugation procedure. When starting with a new or unknown batch of conjugate, test three tenfold dilutions starting at a 1/100 dilution to determine the approximate activity. Using these results, make a second test in the appropriate dilution range indicated. Normally, the objective is to obtain a strong positive reaction to a good positive sample within 20 to 60 minutes and little or no reaction to healthy extracts. If the conjugate concentration is so high that a reaction is instantly visible, a background reaction is often also observed with healthy extracts (and even with buffer controls). Reduce conjugate concentration and, if a nonspecific reaction persists, adsorb the antiserum against a concentrated extract of healthy plant tissue to remove antibodies against healthy antigens. If possible, do this before purifying the IgG. Adding healthy tissue extract to the buffer used to dilute the conjugate may also reduce nonspecific reactions (Clark, Lister and Bar-Joseph,1988).
The use of appropriate controls is essential. Each plate should have at least one healthy and one known positive sample as controls. A buffer control is also useful to determine the level of background reaction to healthy extracts. Frequently, a slightly higher reading will be observed for the buffer control than for the healthy extract because proteins in the extract block exposed protein binding sites on the plastic, which may later non-specifically bind conjugate molecules. Each sample should be tested in at least two wells. A random loading pattern can be used. but paired wells are normally used for routine work. Special applications may require additional replication and random selection.
Edge effects in the plates were frequently noted when ELISA first became popular and outer wells were avoided. Plates have steadily improved and normally all wells can be used. Uniformity in new lots of plates can be checked by loading a uniform sample in all wells.
One of the major tasks of any indexing procedure is to identify samples properly during the testing process and to record results in a usable format.
The identity of the sample must be maintained through the multiple steps of collection, processing, extraction and testing. It is usually convenient to give each sample a code number at the time of collection and to use this code during the test process. If samples are collected directly in the grinding vessel (usually a glass or plastic tube), labelling steps can be reduced. If the sample is collected in a container other than the grinding vessel, a transferable label is often convenient. Many ELISA plates have a coding system on the plate margins to identify individual wells, but there is no space to mark individual wells on the plate. Most workers develop a data sheet similar to the one shown as Figure 156, which is used to record the loading sequence of test samples and other pertinent data for that test, such as reactant concentration and incubation conditions. Each plate and data sheet should have a corresponding number recorded in a logbook to facilitate retrieval of information.
It is important to mark the loading pattern for each plate prior to loading samples and to arrange the samples to be loaded in the appropriate sequence. Note changes or errors that may occur during loading, and store tubes and samples under refrigeration until the testing process is complete.
Visual readings of the plate can be recorded directly on the plate data sheet. Printouts from a plate reader (Figure 47) can also be attached to the original data form. Use of computers to store and analyse data is increasing rapidly and is convenient for long-term storage of large amounts of data. Data from the reader may also be converted into another format for further analysis and spread-sheet presentation on the computer. Permanent visual records of important tests can also be obtained by photographing individual plates on a light box or over a white background. A 35-mm transparency film is economical, and a standard exposure can be obtained if a constant light source is used.
EVALUATION OF RESULTS
Evaluation of ELISA results often presents some problems to the novice user. With highly specific antisera and samples with good antigen titre, results are normally very clear. When the antisera used are weak or contain some antibodies to host proteins and/or the samples have a very low antigen titre, determination of a positive result can be more difficult. Reactions can be evaluated visually with some precision if background readings for healthy controls are low. Normally, the eye can discern differences in OD405 of 0.05 to 0.1 above a low background. A graded scale with three to five levels is useful to report the relative degree of reaction. Where greater accuracy is required, the degree of reaction can be measured by testing a diluted sample in a spectrophotometer or by reading the plate in an ELISA plate reader(Figure 147). A wide variety of plate readers are available, from simple manual models suitable for modest numbers of plates, to highly automated models capable of various levels of data analysis and storage.
Recently, emphasis has increased on measuring reaction rate rather than a single final optical density value. This eliminates some sources of error where accurate quantitative data are needed, and also allows more accurate comparison of samples with large differences in antigen concentration. Rate calculation requires several measurements of the same plate at a measured time interval, and a plate reader is essential. Some plate readers do rate calculations automatically. Expensive plate readers are not necessary until a definite need for them is identified.
The limits of reliable detection are correlated to the precision used and the number of replications. Confidence levels can be calculated statistically when in doubt. Most workers establish an arbitrary threshold value relative to the healthy control for a positive reaction, such as a reading twice the healthy control or the healthy reading plus 0.1 OD. Where reaction to healthy extracts is low (<0.05), the eye can usually consistently detect reactions 0.1 OD or higher and this becomes the effective limit for visual recording. It is best to establish a conservative threshold rating and retest all questionable samples. Some experimentation with different dilutions of known samples will help.
PURIFICATION OF IMMUNOGLOBULINS
Numerous procedures are now available for purification of immunoglobulins (IgG) from polyclonal antisera or for purification of monoclonal antibodies from culture fluid or ascites fluid. The easiest method is to use a commercial kit or system containing detailed instructions. Many of the kits are based on separation of the IgG component by protein A bound to a solid substrate. The IgG is subsequently eluted from the protein A by changing buffers and collected. An alternative, which is slower but inexpensive, is to precipitate the IgG from solution by use of ammonium sulphate, and then fractionate the dialysed resuspended pellet by column chromatography on a DEAE cellulose column (Figure 155).
Purified IgG solutions are normally adjusted to a concentration of 1 mg/ml (equal to an OD280 of 1.4 when measured spectrophotometrically) and stored at 4°C in PBS containing at least 0.02 percent sodium azide. IgG preparations can also be stored in 50 percent glycerol at -20°C or freeze-dried. More detailed instructions can be found in Clark, Lister and Bar-Joseph (1988).
PREPARATION OF ENZYMELABELLED ANTIBODIES
Conjugated molecules of antibody and enzyme can be prepared in several ways (Bar-Joseph and Garnsey, 1981; Clark and Adams, 1977; Clark and Bar-Joseph, 1984; Clark, Lister and Bar-Joseph, 1988; Engvalland Pesce, 1978). Alkaline phosphatase is the most widely used enzyme, and the single step glutaraldehyde method is commonly used to prepare alkaline phosphatase conjugates. Enzymelabelled antibodies are formed by conjugating enzyme and antibody molecules. One way is to mix the enzyme preparation (which is purchased commercially) with purified IgG and then add glutaraldehyde to a concentration of 0.05 percent. The mixture is incubated for several hours and then dialysed. The enzyme normally comes as a salt precipitate which is centrifuged from solution and resuspended in the IgG solution. Dialysis is also done before adding glutaraldehyde. Test this system first before experimenting with other procedures. Alkaline phosphatase is usually received as an enzyme precipitate in a salt solution. The precipitate is recovered from solution by centrifugation and about 5 mg is dissolved in 2 ml of a 1 mg/ml solution of IgG. The mixture is dialysed thoroughly to remove excess salts and then fresh glutaraldehyde is added to a final concentration of 0.05 percent. After 4-h incubation, the conjugate is dialysed three times to remove the glutaraldehyde, and stored at 4°C in PBS containing 0.04 percent sodium azide and 5 mg/ml bovine serum albumen. Use an enzyme source with a quality suitable for ELISA. Careful dialysis is very important for good results. New lots of conjugate must be tested to determine optimum dilutions for use (Sanchez-Vizcaino and Cambra Alvarez,1987). Good-quality conjugates can normally be used at least a 1/500 dilution and dilutions as great as 1 /5 000 or more may be possible. See Clark, Lister and Bar-Joseph (1988) for further details and for preparation of horseradish peroxidase conjugates.
Conjugates are stable for long periods at 4°C. Freezing or freeze-drying are not recommended unless preliminary testing indicates it is possible. If freezing is necessary, add 50 percent glycerol.
BASIC SUPPLIES AND EQUIPMENT
Sources of supplies and equipment change rapidly and new equipment continues to appear. It is not possible to list all suppliers in every country. To be of help, we have indicated some possible sources for some essential supplies. It is not essential to use these specific sources, and the user may, in fact, find a more convenient and economical local source than those listed. The Laboratory buyer's guide, available from International Scientific Communications, Inc., PO Box 870, Shelton, CT 06484, United States of America, is a useful directory of manufacturers. Basic equipment and supplies are emphasized rather than some of the more sophisticated and expensive equipment also available for large-scale clinical work. It is assumed that most users of the latter already know the sources of supply.
A source of antiserum to the pathogen you wish to detect is essential. Both polyclonal and monoclonal antisera have been produced to a number of citrus pathogens, and more are being developed. Polyclonal antisera are frequently preferable for general detection work where discrimination of a particular isolate is either unnecessary or undesirable. Unless the antigen used to produce the antiserum was well purified, polyclonal antisera frequently contain some antibodies to plant proteins as well as to the specific pathogen. It is advisable to check the specificity of the antiserum to be used in the initial stages to ensure acceptability. Monoclonal antibodies are generally more specific because, if properly prepared, only a single epitope of the antigen is involved. To do DAS-I assays, antibodies are needed from two animal species unless the F(ab')2 procedure is followed.
Production of high-quality antisera is frequently a time-consuming and difficult task, especially for people without experience in this process. Inexperienced users of ELISA should ordinarily try to obtain a small amount of antisera from existing sources for their initial work. Frequently, a modest supply of antiserum can be obtained from a colleague working on citrus pathogens. Most scientists working on virus like pathogens of citrus are members of the International Organization of Citrus Virologists (IOCV) and can be identified by writing to the Secretary-Treasurer of IOCV, c/o Department of Plant Pathology, University of California, Riverside, CA92521, United States of America. Small amounts of purified IgG or enzymelabelled conjugate may also be available for limited experimental tests.
Increasing access to antisera from commercial sources and the American Type Culture Collection can be expected. Current commercial outlets of ELISA supplies for citrus pathogens include: Agdia Inc., 30380 County Road 6, Elkhart, IN 46514, United States of America; Ingenasa, Hermanos Garcia, Noblejas 41,28037 Madrid, Spain; and Sanofi Santé Animale, Z.I. de la Ballastière, BP 126, 33500 Libourne, France.
If a prepared ELISA kit or purified sources of IgG and labelled conjugates are available, ELISA tests can be done in a very simple laboratory equipped with a balance, a simple pH meter, basic glassware, a refrigerator and a supply of deionized water. The other essential items are ELISA plates, several repeating pipettes and the chemicals to prepare the necessary buffers (see below). To purify IgG and to prepare antibodyenzyme conjugates require access to a low speed centrifuge, a UV spectrophotometer and some column chromatography equipment.
ELISA plates are available from numerous suppliers including Dynatech Laboratories, 14340 Sullyfield Circle, Chantilly, VA 22021 (United States of America), and Nunc Inc.,2000 N. Aurora Road, Naperville, IL 60540 (United States of America). Plate quality can vary. Plates that work well for sandwich assays may work less well for plate-trapped antigen assays. If possible, find a reputable local supplier and test several types of plate before buying a large supply.
Several repeating pipettes, as shown in Figures 141 and 142, are more or less essential. Fixed volume models are economical and will suffice, but adjustable models are much more convenient and can be used for many other tasks. The minimum requirement is one pipette that will measure accurately in the 1 -20 ml range (to make dilutions of IgG and conjugates) and one that will operate in the 100-1 000 ml range (or 2001 000 ml). Pipettes for the ranges of 20-200 ml and 1-5 ml are also extremely useful. Multichannel pipettes (Figures 141 and 143) that can simultaneously dispense the same volume into four to 12 wells are very useful when many plates must be loaded. All repeating pipettes use disposable plastic tips. If possible, select pipettes that can use interchangeable tips. There are numerous manufacturers of pipettes and numerous models. Consult a laboratory supply company or manufacturers such as Flow Laboratories S.A., Lugano (Switzerland) or Rainin Instruments Co., Woburn, MA 01801 (United States of America) for current information.
Plates can be read visually, but if a large number of plates are to be done on a regular basis, a plate reader greatly speeds reading and makes evaluation of results easier. Numerous models with varying degrees of automation are available and details change rapidly. It is not necessary, and probably not advisable, to buy a reader until the user has some initial experience and knows the system will work. Before purchase, ask for a demonstration and also consult users in other laboratories for their recommendations. Large-scale users should consider readers that are computer-compatible.
See Part IV for additional information on laboratory equipment.
ELISA BUFFERS AND SOLUTIONS
A limited number of chemicals are required to make the buffers and solutions needed for ELISA, and these are shown in Table 6. Many of these should be readily available in most biological laboratories. Specific sources and catalogue numbers have not been listed, but suggestions can be obtained from other ELISA users. Sigma Chemical Co., St Louis, MO 63178 (United States of America), Boehringer Mannheim Biochemicals, Indianapolis, IN 46250 (United States of America), and Pharmacia LKB Biotechnology AB, Uppsala (Sweden), or Piscataway, NJ 08854 (United States of America), are useful general sources for chemicals, enzymes and antibodies mentioned in this section if satisfactory local suppliers cannot be located. See also the Laboratory buyer's guide mentioned above.
The materials listed are for the alkaline phosphatase system. If horseradish peroxidase or another enzyme is used, make appropriate changes in substrate and substrate buffer.
The formulae for buffers and substrate solutions needed are shown in Table 7. Use glass-distilled or high-quality deionized water to prepare buffers and solutions. The formulae given in Table 7 are for one-litre quantities. Larger quantities of the wash buffer (PBST) than other solutions are used. The dry salts needed for PBS can be weighed in advance in units to make a convenient volume, mixed dry, and stored in sealed plastic bags until needed. A new supply of PBS can be obtained rapidly, as needed, by adding the required volume of distilled water to the weighed salts.
Use standard buffer solutions with pH values near 7.0 and 10.0 to calibrate pH meters. Store buffers (except PBST) at 4°C if possible.
Schedules are shown here for types of ELISA illustrated in Figures 139a, 139b and 139d, to provide some specific examples of reactant concentration and incubation time and conditions. The details shown are those typically used for CTV with an alkaline phosphatase enzyme-label system. No schedule is shown for the amplified form of DAS-I illustrated in Figure 139c. The preliminary steps are the same as used for DAS-I and the schedule for the enhancement steps will vary with the enhancement protocol used. Specific instructions are generally provided with the enhancement materials when these are purchased in kit form.
Normally, 200 ml of solution are added per well, but smaller volumes can be used. If NaOH is used to stop the reaction, 50 ml are added to the wells already containing substrate.
Double antibody sandwich ELISA
1.Coat ELISA plates (Figure 141) with antibodies (IgG) diluted to 1-2 xg/ml in carbonate coating buffer. Incubate for 1-4 h at 25-30°C and wash three times with PBST (Figure 154).
2. Add sample extracts (Figure 142) prepared at a 1/10 to 1/20 dilution in extraction buffer in duplicate or triplicate wells. Incubate for 2-4 h at 30-37°C or overnight at 4-6°C. Wash thoroughly three times with PBST. Avoid cross contamination of samples when washing.
3. Add enzyme-antibody conjugate diluted in conjugate buffer to an optimum concentration (normally between 1/500 and 1/5 000) (Figure 143). Incubate 2-4 h at 37°C. Wash at least three times with PBST to remove unbound conjugate from the wells.
4. Add substrate freshly prepared at a concentration of 0.6 to 1 mg/ml in substrate buffer (10 percent diethanolamine, pH 9.8) (Figure 144). Incubate until strong colour change develops in positive controls (normally 30-60 min) (Figure 145) and read plates (Figure 147). Plates may be read at several intervals without stopping the reaction, so rate of reaction can be calculated, or the reaction can be stopped at an appropriate time by addition of 3M NaOH, and a single reading made. If plates are read visually, score the estimated relative strength of reaction. If read on a spectrophotometer or with a plate reader (Figure 147), record the OD405 values.
TABLE 6.List of chemicals for ELISA
|1. Alkaline phosphatase type VlI|
|2. Bovine serum albumen||BSA|
|3. DEAE cellulose|
|6. Hydrochloric acid||HCl|
|8. 4-nitrophenyl phosphate|
|9. Polyvinyl pyrrolidone MW 40 000|
|10. Potassium chloride||KCl|
|11. Potassium phosphate||KH2PO4|
|12. Sodium azide||NaN3|
|13. Sodium bicarbonate||NaHCO3|
|14. Sodium carbonate||Na2CO3|
|15. Sodium chloride||NaCl|
|16. Sodium hydroxide||NaOH|
|17. Sodium phosphate (dibasic)||Na2HPO4|
|18. Tris(hydroxymethyl)aminomethane HCl||Tris-HCl|
|19. Tween 20|
Double antibody sandwich-indirect ELISA
1. Coat ELISA plates (Figure 141) with antibodies (IgG) specific to the antigen to be tested. The IgG concentration should be 1-2 mg/ml in carbonate coating buffer. Incubate for 1-4 h at 25-30°C and wash three times with PBST (Figure 154).
TABLE 7.ELISA buffers and solutions
1. Coating buffer
1.59 9 Na2CO3
2.93 9 NaHCO3
0.20 9 NaN3
(pH should be 9.6)
2. Phosphate buffered saline (PBS)
8.00 9 NaCl
0.20 9 KH2PO4
2.90 g Na2HP04-12 H2O
(1.15 9 anhydrous)
0.20 9 KCl
(pH should be 7.2 to 7.4)1
3. Washing buffer
1.0 litre PBS
0.5 ml Tween 20
4. Extraction buffer
1.0 litre PBST
20 9 polyvinyl pyrrolidone, MW 40 0002
(Option -15.7 9 Tris-HCl, adjust to pH 7.8 with NaOH)
5. Conjugate buffer
1.0 litre PBST
20.0 g polyvinyl pyrrolidone, MW 40 0002
2.0 g ovalbumen
0.20 g NaN3
6. Substrate buffer
97 ml diethanolamine
0.2 g NaN3
(adjust pH to 9.8 by adding HCl)
7. Reaction stopping solution
120 g NaOH
1pH should be close to value,. adjust slightly if necessary.
2Polyvinyl pyrrolidone is not essential for extraction or conjugate buffers.
2. Add sample extracts (Figure 142) prepared at a 1/10 to 1/20 dilution in extraction buffer. Load duplicate or triplicate wells with each sample. Incubate for 2-4 h at 30-37°C or overnight at 46°C. Wash thoroughly three times with PBST avoiding cross-contamination of samples.
3. Add unlabelled intermediate antibody at an appropriate dilution, normally 0.25 xg/ml or less. Incubate for 30-60 min at 30-37°C, and wash plate three times with PBST.
4. Add enzyme-labelled antibody specific to the intermediate antibody, diluted according to the instructions supplied. Incubate 1-2 h at 3037°C and wash plate carefully at least three times with PBST.
5. Add substrate freshly prepared at a 0.6 to 1 mg/ml concentration in substrate buffer (10 percent diethanolamine, pH 9.8). Incubate until strong colour change develops in positive controls (normally 30-60 min) and read plates. Plates may be read at several intervals without stopping the reaction, so rate of reaction can be calculated, or the reaction stopped at an appropriate time by addition of 3 M NaOH and a single reading made. If plates are read visually, score estimated relative strength of reaction. If read on a spectrophotometer or plate reader, record the OD405 values.
Plate-trapped indirect ELISA
1. Add antigen extracts to uncoated ELISA plates and incubate 1-4 h at 25-30°C or overnight at 4-6°C. (Note, samples prepared for PTA should not have Tween 20 in the extraction buffer used.) Wash plates three times with PBST and avoid contamination while washing.
2. Add unlabelled antibody specific to the antigen at an appropriate dilution (normally 1mg/ml or less). Unpurified polyclonal antisera or ascites fluid can be used. Incubate 1-2 h at 30-37°C and wash three times with PBST.
3. Add enzyme-label led antibody specific to the unlabelled antibody, at the specified dilution (normally about 1/1 000), and incubate 1-2 h at 30-37°C. Wash carefully at least three times with PBST.
4. Add substrate prepared at a concentration of 0.6 to 1 mg/ml in substrate buffer (10 percent diethanolamine, pH 9.8). Incubate until a strong colour change develops in positive controls (normally 30-60 min) and read plates. Plates may be read at several intervals without stopping the reaction, so rate of reaction can be calculated, or the reaction can be stopped at an appropriate time by addition of 3 M NaOH and a single reading made. If plates are read visually, score estimated relative strength of reaction. If read on a spectrophotometer or plate reader, record the OD405 values.
Several types of problem may be encountered with ELISA. Some understanding of the operating principles of ELISA helps in doing some systematic trouble-shooting to identify and correct the problem. Several of the most common situations are covered here. If the suggestions given do not solve the problem encountered, seek the help of someone who has extensive experience with ELISA.
No reaction or reaction is very slow
The common causes are: (a) use of an incorrect buffer in one or more steps; (b) antigen was inactivated during processing or storage; (c) loss of enzyme activity in the conjugate (commonly occurs if conjugate is accidentally frozen); (d) antibody used has low affinity for test antigens or lost affinity when it was conjugated; (e) inactive substrate; or (f) a gross miscalculation when making dilutions.
Recommendations. Test conjugate and substrate by mixing a small amount of dilute conjugate with fresh substrate in a small beaker. If no reaction occurs, test each separately again and replace the faulty component. Test reactivity of the antibody by an alternative procedure such as immunodiffusion or microprecipitation. Check calculation of dilutions and test a freshly prepared positive control. Test other extraction buffers. Run a different virus system with the same buffers and protocols.
Colour development is non-specific
When all wells, including buffer and healthy controls, show a strong reaction, it could indicate: (a) the antibody source for either coating or conjugate phase antigens is giving nonspecific reaction (common with antisera to SDS-degraded antigens); (b) the washing was incomplete, especially after the conjugate step; (c) the coating of the plate was incomplete, or Tween 20 was left out of the PBST buffer and enzyme-labelled antibody is being adsorbed nonspecifically to the plate; or (d) the substrate is contaminated or faulty.
If the buffer control is negative, but the healthy control shows a positive reaction, the antiserum used probably has a high concentration of antibodies to healthy plant antigens. The alternatives are either to absorb the antiserum with healthy plant proteins to remove the antibodies in the serum specific to the healthy plant proteins, or to prepare other antisera.
Colour development is erratic
The common causes of erratic reaction within a plate are: (a) defective plates; (b) careless performance of one or more steps, especially washing; (c) failure to mix thoroughly diluted IgG and conjugate solutions; or (d) contamination between wells.
Recommendations. Check another source of plates and review the care used in the operating procedure.
Reaction very rapid, some reaction also in healthy samples
This normally indicates that the conjugate concentration is much too high. Try several tenfold dilutions. If differentiation still fails to occur between healthy and positive samples with normal incubation periods, see recommendations above.
Hundreds of references are available on the ELISA technique and its application to numerous plant viruses. We have listed a few here. Many of these citations contain additional literature citations.
Adams, A.N. & Barbara, D.J.1982. The use of F(ab')2-based ELISA to detect serological relationships among carla-viruses. Ann. Appl. Biol., 101: 495-500.
Bar-Joseph, M. & Garnsey, S.M.1981. Enzymelinked immunosorbent assay (ELISA): principles and application for diagnosis of plant viruses. In Maramorosch, K. & Harris, K.F., eds. Plant diseases and vectors: ecology and epidemiology, p. 35-59. New York, Academic Press.
Bar-Joseph, M., Garnsey, S.M., Gonsalves, D., Moscovitz, M., Purcifull, D.E., Clark, M.F. & Loebenstein, G.1979. The use of enzymelinked immunosorbent assay for detection of citrus tristeza virus. Phytopathol., 69:190-194.
Clark, M.F.1981. Immunosorbent assays in plant pathology. Ann. R. Phyto.,19: 83-106.
Clark, M.F. & Adams, A.N.1977. Characteristics of the micro-plate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol., 34: 475-483.
Clark, M.F. & Bar-Joseph, M.1984. Enzyme immunosorbent assays in plant virology. In Maramorosch, K. & Koprowski, H., eds. Methods Virol., 7: 51 -85. New York, Academic Press.
Clark, M.F., Lister, R.M. & Bar-Joseph, M.1988. ELISA techniques. In Weisbach, A. & Weisbach, H., eds. Methods for plant molecular biology, p. 507-530. New York, Academic Press.
Engvall, E. & Pesce, A.J.1978. Quantitative enzyme immunoassay. London, Blackwell Scientific Publications. 129 pp.
Jones, R.A.C. & Torrance, L. 1986. Developments and applications in virus testing. Wellesbourne, War., UK, AAB. 300 pp.
Koenig, R. & Paul, H.L.1982. Variants of ELISA in plant virus diagnosis. J. Virol. Methods, 5: 113-125.
Maggio, E.T. 1980. Enzyme-immunoassay. Boca Raton, Florida, CRC Press. 295 pp.
McLaughlin, M.R., Barnett, O.W., Burrows, P.M. & Baum, R.H.1981. Improved ELISA conditions for detection of plant viruses. J. Virol. Methods, 3: 1325.
Permar, T.A., Garnsey, S.M., Gumpf, D.J. & Lee, R.F.1988. A monoclonal antibody which discriminates strains of citrus tristeza virus. Phytopathol., 78: 1559.
Sanchez-Vizcaino, J.M. & Cambra Alvarez, M.1987. Enzyme immunoassay techniques, ELISA, in animal and plant diseases. Tech. series No.7 2nd ed. Paris, Office international des epizooties. 54 pp. (Available in English, French and Spanish)
Van Regenmortel, M.H.V.1982. Serology and immunochemistry of plant viruses. New York, Academic Press. 302 pp.
Vela, C., Cambra, M., Cortes, E., Moreno, P., Miguet, S.G., Perez de San Roman, C. & Sanz, A.1986. Production and characterization of monoclonal antibodies specific for citrus tristeza virus and their use in diagnosis. J. Gen. Viral., 67: 91-96.
FIGURE 139 Diagram of the components of four popular types of ELISA
Diagram of the components of four popular types of ELISA: a) Double antibody sandwich ELISA (DAS). The most widely used form of ELISA for plant pathogens. The wells of the ELISA plate (the solid phase or immunosorbent surface) are coated with an unlabelled antibody specific to the pathogen, which becomes the trapping antibody (TA). The antigen (V) is captured by the trapping antibody and detected by the enzyme-labelled antibodies (LA) which are normally from the same polyclonal antiserum used for trapping and detection; b) Double antibody sandwich-indirect ELISA (DAS-I). The intermediate antibody (IA) is unlabelled and must be from a different animal species to that of the coating antibody. The LA is an antibody specific for the IA. If the F(ab')2 antibody component is used for coating, the whole unlabelled antibody from the same animal can be used as the IA and is detected with protein A conjugated to an enzyme; c) Enhanced DAS-I. This is similar to DAS-I, but the enzyme concentration on the LA is amplified by an additional treatment to increase sensitivity. Frequently, the LA is biotinylated to react to avidin-enzyme conjugates; d) Plate trapped antigen-indirect ELISA. The antigen (V) is trapped directly on the plate surface and detected by using an unlabelled antibody specific to the antigen (IA) plus an enzyme-labelled antibody (LA) specific to the IA. Enhancement as shown for DAS-I is also possible
FIGURE 140 ELISA is normally done using plastic microtitre plates specially formulated for that purpose. Plates with 96 wells are most common, but other configurations exist. Plastic beads or strips can also be used as the solid phase (immunosorbent surface) and are convenient for small numbers of samples
FIGURE 141 The first step in DAS or DAS-I ELISA is to coat the plate with the trapping antibody (TA in Figure 139). The trapping antibody is prepared at a concentration of 1 2 g/ml in carbonate buffer and 100-200 ml are placed in each well (note use of an eight-channel pipette for fast loading) and incubated for 1-4 h. Unbound antibody is removed by washing the plate with PBST (Figure 154)
FIGURE 142 The second step in DAS and DAS-I is to load the test samples which have been previously prepared (Figures 148-153), coded and arranged in sequence for easy loading. Each sample is placed in at least two wells, and each plate contains a positive and negative control sample for reference. Each plate is marked for identification and orientation. The plates are incubated for 2-4 h at 30-37°C, or overnight at 4-6°C, and then washed with PBST to remove materials not bound specifically to the trapping antibody
FIGURE 143 The third step in DAS is to add the enzymelabelled conjugate diluted to a predetermined optimum concentration in PBST (in DAS-I this step is preceded by application of the intermediate antibody). Incubate 2-4 h at 37°C. Wash at least three times with PBST to remove unbound conjugate from the wells. Thorough washing is important prior to substrate addition to avoid non-specific reactions
FIGURE 144 The fourth step is preparation and addition of the substrate solution to the test plate. Substrates should be freshly made. Colour change in the substrate will be proportional to the number of enzyme-labelled antibodies bound to the antigen present
FIGURE 145 Once an appropriate level of reaction is reached, the reaction can be stopped by addition of 50 Ill of 3M NaOH and then the plate is evaluated. Plates may be read several times during the Incubation period to determine the rate of reaction. Plates may also be frozen for future evaluation
FIGURE 146 The reaction is evaluated by determining the degree of colour change which occurred in the substrate. This may be done visually by scoring the degree of reaction (e.g. negative, weak, moderate or strong). Comparisons are made with both the healthy and positive control samples
FIGURE 147 The degree of reaction may be determined by measuring the colour change photometrically (at 405 nm for alkaline phosphatase) by use of a plate reader, which measures and reports the absorbance in each well. By reading the plate at several timed intervals, the rate of reaction can also be calculated
FIGURE 148 Proper selection of tissue to test is highly important for success in ELISA. Virus concentration is normally highest in new flushes of growth and these should be used wherever possible
FIGURE 149 CTV, greening and stubborn are all phloemlimited, and the sample must include phloem tissue for these pathogens. Young twig bark, feat midribs and the button area of young fruit are excellent tissue sources for CTU If possible, collect from several sites on the test plant and prepare a composite sample for testing
FIGURE 150 Sample tissue is finely diced to avoid clogging the homogenizer and placed in the grinding vessel at a ratio of 1 part tissue to 10-20 parts of buffer. Samples may be stored frozen in buffer if immediate testing is not convenient
FIGURE 151 A dispersion homogenizer is very convenient for grinding tough fibrous tissue samples such as citrus bark and leaf midrib tissue. Samples can also be ground by other methods including a pestle and mortar (adding an abrasive makes it easier)
FIGURE 152 The shaft of the homogenizer must be rinsed in clean water between samples to avoid contamination
FIGURE 153 Extracts of citrus tissue usually contain debris which makes pipetting difficult. One way to avoid clogging the tip is to pipette through a filter. The extract can also be centrifuged or the tip of the pipette cut off to create a larger bore
FIGURE 154 Washing is a very important part of ELISA. Each plate is washed at least three times between each step. The plate is emptied by rapidly inverting it over a sink, blotted on a clean towel, filled with wash solution (normally PBST) by a squeeze bottle or other dispenser, incubated with gentle agitation for several minutes and the cycle repeated. The final wash solution may be left in the plate until the next step is initiated
FIGURE 155 Antibodies are immunoglobulins (IgG) and can be purified from whole antisera by use of commercially prepared kits, or by precipitation of the serum with ammonium sulphate and chromatography of the dialysed precipitate on a small column of DEAE cellulose prepared for that purpose
FIGURE 156. Data sheet for recording ELISA results
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