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ELISA Reagents
DAS ELISA, Horseradish Peroxidase (HRP) conjugate

Content List

Lot Number

Item

500wells

1000 wells

5000 wells

Storage

Coating antibody

0.25 ml

0.50 ml

2x1.25 ml

4 oC

Detecting conjugate, HRP

0.25 ml

0.50 ml

2x1.25 ml

4 oC

96-well ELISA plates

5

10

50

Room temperature

Instruction

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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.

 

Preparing For The Test
Check all the components in the package of ELISA Reagents.

Prepare all of buffer solutions according to the attached buffer formulations.

Make sure all laboratory equipments and facilities required for the test are ready.

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.

 

Coating Plate With Antibody
Lay out all items that will be required for the plate coating step before starting. Prepare coating antibody in a container made of glass, polyethylene or a material that does not readily bind coating antibody. It is recommended that the plate be coated 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; 100 l 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.

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 l of coating antibody into each well.

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

 

Preparing Samples
Select symptomatic and/or infective tissues for the test. Leaf tissue is often used in ELISA testing. Other plant tissues such as stem, sprout, seed, tuber, and root can also be tested.

We suggest that each test well be used for a single 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.

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.

Grind sample with a mortar and pestle, or other grinding devices. If you are using a mortar and pestle, wash and rinse it thoroughly between samples.

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).

If you have any questions about sampling, sample preparation, or the appropriate extraction buffer for your samples, please contact AC Diagnostics, Inc..

 

Plate Washing
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 time.

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
About 100 l 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 l 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 l of prepared sample into sample wells. Dispense 100 l of positive control into positive control wells, and dispense 100 l of negative control or extraction buffer into negative control wells.

Put the plate inside the humid box and incubate overnight in the refrigerator (4 C) or for 2.5 hours at room temperature (21-24 C).

 

Preparing Enzyme Conjugate
Always make enzyme conjugate solution within 10 minutes before use. Prepare the enzyme conjugate, using s ECB3 buffer and a cleaning container.

The volume of ECB3 buffer required depends on the number of test wells used, with 100 l 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 ECB3 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 ECB3 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.

 

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 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 Incubation
Dispense 100 l of prepared enzyme conjugate per well for all test wells.

Incubate the plate in the humid box for 2.5 hours at room temperature (21-24 C).

 

Preparing Substrate Solution
Concentration of O-phenylenediamine (OPD) in substrate is 0.6 mg/ml. An amount of 6.0 mg of OPD powder will make 10 ml of OPD substrate solution which is enough for 96 test wells or twelve 8-well strips.

Do not touch the OPD powder or expose the OPD solution to strong light. Light or contamination could cause background color in negative wells.

Prepare OPD substrate about 10-15 minutes before the end of the above incubation step. Measure out the amount of OPD powder you will need for your test. Measure 10 ml of OPD buffer for each 6.0 mg OPD, then add the OPD powder to the buffer. Mix by vortexing or stirring to allow the OPD powder to fully dissolve in the buffer. OPD is a carcinogen. Do not touch OPD powder or OPD substrate solution.

 

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

 

Incubation With Substrate
Dispense 100 l of OPD substrate solution per well.

Incubate the plate for 20 to 30 minutes in a humid box at room temperature (21-24 C).

To stop reaction, add 50 l of 3M sulfuric acid to each well. Then, the plate can be interpreted visually or with a plate reader.

 

Evaluating Results
Results can be examined by eye, or measured on a plate reader at 490 nm.

Development of 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 30 minutes of incubation as long as negative control wells remain virtually clear.

 

Buffer Formulations

Coating Buffer: 
        Sodium carbonate (anhydrous)                             1.60 g
   
     Sodium bicarbonate                                               2.92 g
   
     Sodium azide                                                          0.20g
   
Dissolve in distilled water and make 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 and make to 1000 ml. Adjust pH to 7.3.

SB1 Buffer
   
     Powdered egg (chicken) albumin, Grade II             2.0 g
   
     Polyvinylpyrrolidone (PVP) MW, 24-40,000         10.0 g
   
     Sodium sulfite (anhydrous)                                       1.3 g
   
     Sodium azide                                                             0.2 g
   
     Tween-20                                                                 10.0 g
   
Dissolve in 1000 ml of 1X PBST. Adjust pH to 7.3. Store at 4 C.

ECB3 Buffer
   
     Bovine serum albumin (BSA)                                 2.0 g
   
     Polyvinylpyrrolidone (PVP) MW 24-40,000         10.0 g
   
     Thimerosal (Optional)                                             0.1 g
   
Dissolve in 1000 ml of 1X PBST. Adjust pH to 7.3. Store at 4 C.

OPD Buffer
   
     Citric acid (anhydrous)                                           5.11 g
   
     Sodium phosphate, dibasic (anhydrous)                7.32 g
   
     Hydrogen peroxide (30%)                                     0.4 ml
   
Dissolve in 800 ml distilled water and Adjust pH to 5.0 Adjust final volume to 1000 ml 
    with distilled water. Store at 4 C.


RECORDING SHEET OF ELISA

 

TEST:                                                         DATE:                                      BY:                                              

TIMING:  Coating                          Sample                           EC                           Substrate                         

KEY POINTS:                                                                                                                                                 

           Coating Antibody:                                 ul,  Coating Buffer:                               ml,

                     Enzyme Conjugate:                               ul,  ECB3:                                      ml

                     PNP Substrate:                                       ml

 

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