das-elisa das-elisa (double antibody sandwich enzyme-linked inmunosorbent assay) is an...
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For the detection of Ralstonia solanacearum
INTERNATIONAL POTATO CENTER (CIP) 2001
INSTRUCTIONS FOR USE
DAS-ELISA kit For the detection of Ralstonia solanacearum in soil
INSTRUCTIONS FOR USE
Dr Sylvie Priou International Potato Center (CIP) 2001
© 2001 Centro Internacional de la Papa, Lima, Peru. All rights reserved
Sample preparation 8
Serological test 11
Sources/suppliers of the products included in the kit 15
List of supplies included in the kit 16
Appendices Appendix 1. Preparation of the enrichment broth (1x) 18 Appendix 2. Protocol for sample collection 18 Appendix 3. Sample size 19
Coat the wells of a microtitration plate with ( )-specific
rabbit immunoglobulins (IgG) Ralstonia solanacearum Rs
Rs-specific bbit immunoglobulins (IgG)
Rs in soil solution
Enzyme-labeled - specific rabbit IgG (conjugated IgG)
Add soil solution; in the soil will bind to the IgGRs
Add enzyme-labeled -specific rabbit IgG; these will bind to the -IgG complex to
form the double antibody sandwich
Add the enzyme substrate; if is present a color will developRs
ProcedureFigure 1: The four step ps of the DAS-ELISA
DAS-ELISA (double antibody sandwich enzyme-linked inmunosorbent assay) is an immuno- enzymatic assay that uses a microtitration plate as a support for the samples and reagents.
The procedure for the detecting Ralstonia solanacearum in soil, summarized in Figure 1, consists of:
1. Coating a microtitration plate with Ralstonia solanacearum (Rs)-specific rabbit immunoglobulins (IgG)
2. Adding solutions prepared from soil samples to the wells of the microtitration plate 3. Adding enzyme-labeled Rs-specific rabbit immunoglobulins (conjugated IgG) 4. Adding the enzyme substrate; if Rs is present in the soil solution this produces a color
reaction, the intensity of which is proportional to the bacterial concentration
The bacteria may be present in soil at only low concentrations, so before performing the DAS-ELISA an enrichment procedure must be carried out to allow the bacteria to multiply to detectable levels. This is done by incubating the soil solution in an enrichment broth at 30°C for 48 hours with constant agitation. By this procedure the method is capable of detecting original bacterial concentrations as low as 20 bacteria per gram of soil (instead of 107
bacteria/g without enrichment).
All races, biovars and serotypes of Rs can be detected with the Rs-specific immunoglobulins provided in the kit. Some saprophytic bacteria may cross-react with the antibodies produced against Rs when in pure culture at concentrations equal to or higher than 108 bacteria/ml. However, after enrichment of soil solutions containing these saprophytic bacteria, no cross- reactions have been detected in DAS-ELISA.
This kit can be used to monitor Rs in bare soil after harvest as well as in the rhizosphere, rhizoplane and roots of potato or other hosts, to study the epidemiology of the disease, to evaluate the effectiveness of a control method in reducing soil populations of the pathogen, and to determine the suitability of a field for seed production.
Before using the DAS-ELISA kit, read the following instructions carefully and be sure that you have all materials ready.
As soon as you receive the kit, wrap it in a plastic bag and store it in a refrigerator at 4°C. The products are stable for six months. The supplies included in the kit are sufficient for testing six microtitration plates of approximately 40 samples each (for sample size see Appendix 3 on Page 19).
The supplies included in the kit are listed on page 16. The following equipment and materials are also needed for the assay but are not included in the kit (everything must be very clean):
To collect soil samples and prepare soil solutions
• Small hand hoe • Sodium hypochloride • Polyethylene bags for collecting soil samples • A bunsen burner or alcohol to disinfect lab tools • A sterile 250-ml erlenmeyer for each soil solution • A hammer for disintegrating soil clumps • A marker or wax pencil to mark the sample bags and plates • A spatula • One 1000-ml graduated cylinder • Two 1000-ml and one 2000-ml flasks (bottles or erlenmeyers) to prepare and store buffers • Fifty liters of distilled water (alternative: clean boiled rain water) • A pH meter • Test tubes • Vortex
For the enrichment procedure (all must be sterile)
• One sterile 1000-ml flask or erlenmeyer • One sterile 1000-ml graduated cylinder
• Three liters of sterile distilled water to dilute the enrichment broth • An incubator shaker at 30°C • Five sterile 25- or 50-ml erlenmeyers for each sample • One sterile 10-ml pipette
To perform the ELISA
• One 25- or 50-ml beaker • One 100-ml graduated cylinder • Two-and-half liters of sterile distilled water • An adjustable 100–1000-µl micropipetor or 50–300-µl multipipetor and sterile
corresponding tips, or Pasteur pipettes • A refrigerator • An incubator at 37°C • One 1000-ml erlenmeyer or bottle • One 500-ml erlenmeyer or bottle • Three 125-ml containers or bottles • One 250-ml wash bottle
The procedure for soil extraction and sample enrichment is summarized in Figure 2.
Prepare and store buffers in clean flasks. Autoclave (20 minutes at 120°C) the buffers after preparation. Store in a refrigerator.
The first time you prepare a buffer solution measure its pH with a pH meter, and then add HCl (bottle #15) or NaOH (bottle #16) as necessary to achieve the desired pH. Write the volumes of HCl or NaOH added in your manual for faster future applications (if the same water quality is used, there is no need to check the pH each time).
Preparation of the extraction buffer pH 7.4 (2000 ml)
Dissolve the contents of one packet #4 in 2000 ml distilled water while agitating. Adjust pH to 7.4 if necessary. Autoclave (20 minutes at 120°C) and store.
Preparation of the citrate buffer pH 5.6 (1000 ml)
Dissolve the contents of one packet #14A and one packet #14B in 1000 ml distilled water while agitating. Adjust pH to 5.6 if necessary. Autoclave (20 minutes at 120°C) and store.
Preparation of the soil solution
Sample collection is described in Appendix 2, page 18, and sample size in Appendix 3, page 19.
• Mix 10 g of soil with 90 ml of extraction buffer • Agitate at 180 rpm for 30 minutes at room temperature • Allow the soil suspension to settle for 40 seconds • For a semi-quantification of populations of Rs in soil, make serial 10-fold dilutions up to
10–5 by mixing 1 ml of the supernatant (soil solution) with 9 ml of citrate buffer • For qualitative evaluation of soil inoculum (presence or absence of Rs), make dilutions
to 10–1 and 10–2 by mixing 1 ml of the supernatant (soil solution) with 9 ml of citrate buffer
Figure 2: Soil extraction and enrichment process for detection of in soil by DAS-ELISARalstonia solanacearum
SERIAL 10-FOLD DILUTIONS IN CITRATE BUFFER
Incubation at 30º C for 48 hours under constant agitation
Agitation 30 mi Settling 40 seconds
90 ml PBS bu +
10 g soil
9 ml of CIP enrichment broth
10-1 10-2 10-5
1 ml of diluted soil extract
Enrichment procedure (48 hours)
• Dilute 50 ml of the 10x enrichment broth (one bottle #11) with 450 ml of sterile distilled water
• Dispense 9 ml of the diluted (1x) enrichment broth in each of five sterile 25- or 50-ml erlenmeyers
• Add 1 ml of a different dilution of the soil solution to each erlenmeyer of enrichment broth
• Incubate 48 hours at 30°C with constant agitation (180 rpm) in an incubator shaker • At the end of the incubation time, continue with the ELISA test, or store 1 ml of enriched
soil solution in an eppendorf tube at –20°C for later use
The composition of the enrichment broth (1x) is given in Appendix 1, page 18.
All volumes and quantities indicated (except for the phosphate buffer saline (PBS)) are for a test using one microtitration plate. For each plate you will need exactly 250 ml of PBS buffer.
1. Coating the microtitration plate with Rs-specific rabbit immunoglobulins (IgG)
1.1. Preparation of the coating buffer pH 9.6 (100 ml)
Dissolve the contents of packet #1 in 100 ml of distilled water.
1.2. Preparation of the coating solution (12.5 ml)
To 12.5 ml of the coating buffer add 100 µl (0.1 ml) of Rs-specific rabbit IgG (from eppendorf tube #7). Mix well, but gently, avoiding the formation of a foam.
1.3. Incubation with the coating solution
Add 125 µl (0.125 ml) of the coating solution to each well of the microtitration plate; ensure that all wells are filled. Cover the plate with a piece of parafilm to prevent evaporation and incubate at 37°C for 4 hours. This process will all