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Safety and Storage
Please contact AC Diagnostics, Inc. if you have any questions about safety and storage of this product.
Preparing For The TestCheck all the components in the package of ELISA Reagents.
Prepare all buffer solutions according to the attached buffer formulations.
Make sure all laboratory equipments and facilities required are ready for the test.
Prepare a humid box for incubation steps.
Coating Plate With Antibody
Lay out all items that will be required for the plate coating step before beginning. Prepare coating antibody in a container made of a material such as glass or polyethylene that does not readily bind coating antibody. Coat the plate immediately after preparing coating antibody. Some coating antibody can be lost if too much time elapses between diluting the coating antibody and coating the plate.
The volume of coating buffer required depends on the number of test wells used, with 100 µl 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.
Dilute the concentrated coating antibody into coating buffer at the dilution given on the label. Mix well. Always prepare coating antibody immediately before use.
Pipette 100 µl of coating antibody into each well.
Select symptomatic and/or infective tissues for the test. Leaf tissue is often used in ELISA testing. However, plant tissues such as stem, sprout, seed, tuber, root and others can also be tested.
Single sample is suggested to be used in each test well. 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.
Sample Dispensing and Incubation
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.
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.
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
Always make enzyme conjugate solution within 10 minutes before use. Prepare the enzyme conjugate, using AC Diagnostics’s ECB1 buffer and a cleaning container.
The volume of ECB1 buffer required depends on the number of test wells used, with 100 µl 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.
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.
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.
Prepare enzyme conjugate just before use. Keep the prepared enzyme conjugate at safe place and use it after washing the plate.
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.
Wash the plate by filling the wells with PBST, then quickly emptying them again. Repeat 6 to 8 times.
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 IncubationDispense 100 µl of prepared enzyme conjugate per well.
Incubate the plate in the humid box for 2.5 hours at room temperature (21-24 °C).
Preparing Substrate Solution
Concentration of PNP in substrate is 1 mg/ml. Each PNP tablet will make 5 ml of PNP solution, which is enough for five 8-well strips.
Do not touch the PNP tablets or expose the PNP solution to strong light. Light or contamination could cause background color in negative wells.
Washing plateWash the plate 6 to 8 times with PBST as above.
Incubation With Substrate
Dispense 100 µl of PNP substrate solution per well.
Incubate the plate for 30 to 60 minutes in a humid box at room temperature (21-24 °C).
Evaluating ResultsTest results can be examined by eye, or measured on a plate reader at 405 nm.
RECORDING SHEET OF ELISA
TEST: DATE: BY:
TIMING: Coating Sample EC Substrate
Enzyme Conjugate: ul, ECB1: ml
PNP Substrate: ml