Creative Diagnostics - Model DEIAPV1 - ELISA Cymbidium Mosaic Virus Kit
From Elisa Kits - Plant Pathogen Kits
Cymbidium mosaic virus (CymMV) and the Odontoglossum ringspot virus (ORSV) are two of the most prevalent and economically important viruses affecting cultivated orchids worldwide. Infected plants can reduce plant vigor and have less desirable flowers or other problems, causing significant financial losses to orchid growers. The virus has not often been reported in wild orchid populations. The virus is spread throughout commercial orchid collections by contaminated tools and pots during division of plants and harvest of flowers.
Product Details
Size
1000T
Sample
orchid plants
Intended Use
The test can be used to detect CymMV in infected and asymptomatic orchid plants
Contents of Kit
- 500 wells
- Coating antibody: 0.250 ml, 4°C
- Detecting conjugate, alkaline phosphatase: 0.250 ml, 4°C
- 96-well ELISA plates: 5, Room temperature
- Instruction: 1
1000 wells
- Coating antibody: 0.50 ml, 4°C
- Detecting conjugate, alkaline phosphatase: 0.50 ml, 4°C
- 96-well ELISA plates: 10, Room temperature
- Instruction: 1
5000 wells
- Coating antibody: 2*1.25 ml, 4°C
- Detecting conjugate, alkaline phosphatase: 2*1.25 ml, 4°C
- 96-well ELISA plates: 50, Room temperature
- Instruction: 1
Storage
All reagent components should be stored at the recommended temperature to assure their full shelf life. Do not store prepared working solution from day to day.
Sensitivity
Sensitivity of the ELISA is very high. The virus can be consistently detected in infected plant tissues diluted at 1:2430.
Principles of Testing
The enzyme-linked immunosorbent assay (ELISA) is a serological solid-phase method for identification of diseases based on antibodies and color change in the assay. In this method, specific antibodies are used to coat the microtitre plate, which then trap the target epitopes (antigens) from the viruses, bacteria and fungi. An enzyme-labelled specific antibody conjugate is then used for the detection. The detection can be visualized or measured in a computer controlled plate reader based on color changes resulting from the interaction between the substrate and the immobilized enzyme.
Reagents And Materials Provided
500 wells
- Coating antibody: 0.250 ml, 4°C
- Detecting conjugate, alkaline phosphatase: 0.250 ml, 4°C
- 96-well ELISA plates: 5, Room temperature
- Instruction: 1
1000 wells
- Coating antibody: 0.50 ml, 4°C
- Detecting conjugate, alkaline phosphatase: 0.50 ml, 4°C
- 96-well ELISA plates: 10, Room temperature
- Instruction: 1
5000 wells
- Coating antibody: 2*1.25 ml, 4°C
- Detecting conjugate, alkaline phosphatase: 2*1.25 ml, 4°C
- 96-well ELISA plates: 50, Room temperature
Instruction: 1
Assay Procedure
Preparing For The Test
1. Check all the components in the package of ELISA Reagents.
2. Prepare all buffer solutions according to the attached buffer formulations.
3. Make sure all laboratory equipments and facilities required for the test are ready.
4. Prepare a humid box for incubation steps.
5. Make a copy of the attached recording sheet and create a loading diagram by recording the locations of your samples, controls, and other reagents needed.
Coating Plate With Antibody
1. Lay out all items that will be required for the plate coating step before beginning. Prepare coating antibody in a container made of glass, polyethylene or any material that does not readily bind coating antibody.
2. Coat the plate immediately after preparing the coating antibody. Some coating antibody can be lost if too much time elapses between diluting the coating antibody and coating the plate.
3. The volume of coating buffer required depends on the number of test wells used; 100 µl is needed per test well. One way to estimate the volume needed is to prepare 1 ml of coating buffer for each 8-well strip used, or 10 ml for each 96-well plate.
4. Dilute the concentrated coating antibody into coating buffer at the dilution given on the label. Mix well.
5. Always prepare coating antibody immediately before use.
6. Pipette 100 µl of coating antibody into each well.
7. Incubate the plate in a humid box for overnight in the refrigerator (4°C) or 4 hours at room temperature (21-24°C).
Preparing Samples
1. Select symptomatic and/or infective tissues for the test. Leaf tissue is often used in ELISA testing. Plant tissues such as stem, sprout, seed, tuber, root and others can also be used.
2. We suggest that each test well be used for only one sample. In some cases, composites of up to ten leaves per test well can be used to make testing more economical. However, too many plant samples per well can reduce the sensitivity of the test.
3. CD's SB1 buffer can be used as extraction buffer for most of the plant samples. However, other buffers are also recommended for some plant species.
4. Grind sample with a mortar and pestle, or other grinding device. If you are using a mortar and pestle, wash and rinse it thoroughly between samples.
5. If you extract plant sap, dilute the sap into sample extraction buffer at a ratio of 1:10 (sap volume: buffer volume). Or you can grind plant tissue in extraction buffer at a 1:10 ratio (tissue weight: Extraction Buffer volume).
6. If you have any questions about sampling, sample preparation, or the appropriate extraction buffer for your samples, please contact CD, Inc.
Plate Washing
1. Wash the plate when the incubation is complete. Use a quick flipping motion to empty the wells into a sink or waste container.
2. Wash the plate by filling the wells with PBST, then quickly emptying them again. Repeat 4 to 6 times.
3. To remove drops of PBST from the wells after washing, hold the frame upside down and tap firmly on a folded paper towel.
Sample Dispensing and Incubation
1. About 100 µl of diluted sample extract is needed per test well. Always have an additional amount to assure easy dispensing. A convenient way to prepare this diluted sample is to measure 100 µl of undiluted sap into a small test tube, then add 1 ml of extraction buffer.
2. Following your loading diagram on your recording sheet, dispense 100 µl of prepared sample into sample wells. Dispense 100 µl of positive control into positive control wells, and dispense 100 µl of negative control or extraction buffer into negative control wells.
3. Put the plate inside the humid box and incubate for 2.5 hours at room temperature (21-24°C) or overnight in the refrigerator (4°C).
Preparing Enzyme Conjugate
1. Always make enzyme conjugate solution within 10 minutes before use. Prepare the enzyme conjugate, using CD's ECB1 buffer and a cleaning container.
2. The volume of ECB1 buffer required depends on the number of test wells used; 100 µl are needed per test well. To estimate the volume needed, prepare 1 ml for each 8-well strip used, or 10 ml for each 96-well plate.
3. The volume of enzyme conjugate required for each test is calculated based on the volume of ECB1 buffer used and on the dilutions given on the bottles. Use a new, sterile pipette tip and change the tip for each pipetting to prevent contamination.
4. First dispense appropriate volume of ECB1 buffer into a cleaning container, then add enzyme conjugate according to the dilution given on the label. Mix the conjugate solution thoroughly. It is important to mix the enzyme conjugate well for a consistent test result.
5. Prepare enzyme conjugate just before use. Keep the prepared enzyme conjugate at a safe place and use it after washing the plate.
Washing Plate
1. Wash the plate when the incubation is complete. Use a quick flipping motion to empty the wells into a sink or waste container without mixing the contents.
2. Wash the plate by filling the wells with PBST, then quickly emptying them again. Repeat 6 to 8 times.
3. To remove drops of PBST from the wells after washing, hold the frame upside down and tap firmly on a folded paper towel.
Enzyme Conjugate Incubation
1. Dispense 100 µl of prepared enzyme conjugate per well for all test wells.
2. Incubate the plate in the humid box for 2.5 hours at room temperature (21-24°C).
Preparing Substrate Solution
1. Concentration of PNP in substrate is 1 mg/ml. Each PNP tablet will make 5 ml of PNP solution, which is enough for 48 test wells or five 8-well strips.
2. Do not touch the PNP tablets or expose the PNP solution to strong light. Light or contamination could cause background color in negative wells.
3. Prepare PNP substrate about 10-15 minutes before the end of the above incubation step. Measure 5 ml of PNP buffer for each tablet, then add the PNP tablets to the buffer. Mix by vortexing or stirring to let the PNP tablet fully dissolve in the buffer.
Washing plate
Wash the plate 6 to 8 times with PBST as instructed above.
Incubation With Substrate
1. Dispense 100 µl of PNP substrate solution per well.
2. Incubate the plate for 30 to 60 minutes in a humid box at room temperature (21-24°C).
3. To stop reaction, add 50 µl of 3M sodium hydroxide to each well. This step is optional. The plate can be interpreted visually or with a plate reader without adding the stop solution.
Quality Control
Negative controls are made from a healthy host plant and are tested for the absence of the respective pathogen. They are lyophilized and sufficient for 10 test wells. Positive controls are made from infected plant material or bacterial cultures, if not stated otherwise. They are lyophilized and sufficient for 10 test wells. Inactivated positive controls are additionally tested for the absence of infectivity. Unspecific non-pathogenic 'method' controls are sold for some quarantine pathogens.
Interpretation Of Results
Test results can be examined by eye, or measured on a plate reader at 405 nm.
Development of yellow color in test wells indicate positive results. Wells in which there is no significant color development indicate negative results. Test results are valid only if positive control wells give a positive result and negative control wells remain clear.
Results may be interpreted after more than 60 minutes of incubation as long as negative control wells remain virtually clear.
Precautions
1. Always wash hands thoroughly after using this product.
2. Prevent direct skin and eye contact with, or ingestion of, product components. Obtain medical attention in case of accidental ingestion of reagent components.
Our products are suitable for a wide variety of ap...
Our products are suitable for a wide variety of applications include plant pathological research, plant quarantine, early detection of plant pathogens, tracking of pathogen expansion and emergence in agriculture, detecting pathogens in seeds and other propagative plant materials, and recognizing pathogens in the field for rational and timely management of the diseases, at fair prices without laboratory equipment and specialist knowledge.
More and Customized Kits
The diagnostics products for more plant pathogens are in development and will be soon available. We also offer or guide the production of customer-specific ELISA kits and immunological strips on request, based on our state-of-the-art technology, well-trained staff and strict quality control measures. Please feel free to contact us for the reagents or test strips against non-listed pathogens.
Creative Diagnostics also offers high-quality plant pathogens antibodies, which are suitable for use in ELISA or related immunoassays and can be ordered in bulk quantity. Please click the link above for more information.
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