ELISA Reagents
DAS ELISA, alkaline phosphatase conjugate

Content List

ELISA Reagents
DAS ELISA, alkaline phosphatase conjugate

Safety and Storage

Always wash hands thoroughly after using this product. Prevent direct skin and eye contact with, or ingestion of, product components. Obtain medical attention in case of accidental ingestion of reagent components.

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.

                    Please contact AC Diagnostics, Inc. if you have any questions about safety and storage of this product.

                    Check 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.

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.

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.

Incubate the plate in a humid box for overnight in the refrigerator (4 °C) or 4 hours at room temperature (21-24 °C).

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.

Washing Plate

Wash the plate when the incubation is complete. Use a quick flipping motion to empty the wells into a sink or waste container.

Wash the plate by filling the wells with PBST, then quickly emptying them again. Repeat 4 to 6 times.

To remove drops of PBST from the wells after washing, hold the frame upside down and tap firmly on a folded paper towel.

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).

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.

                    Dispense 100 µl of prepared enzyme conjugate per well.

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.

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 full dissolving in the buffer.

                    Wash the plate 6 to 8 times with PBST as above.

Incubation With Substrate

                    Dispense 100 µl of PNP substrate solution per well.

                    Test results can be examined by eye, or measured on a plate reader at 405 nm.

Buffer Formulations

        Coating Buffer
                Sodium carbonate (anhydrous)                      1.60 g
                Sodium bicarbonate                                      2.92 g
                Sodium azide                                                0.2 g
                Dissolve in distilled water to 1000 ml. Adjust pH to 9.6. Store at 4° C.

        PBST Buffer
                Sodium phosphate, dibasic (anhydrous)                      1.15 g
                Potassium phosphate, monobasic (anhydrous)             0.2 g
                Sodium chloride                                                          8.0 g
                Potassium chloride                                                      0.2 g
                Tween-20                                                                   0.5 g
                Dissolve in distilled water to 1000 ml. Adjust pH to 7.3.