Abscisic Acid ELISA 试剂盒

• Assay plate
• Standard (10 x concentrate)
• Antibody (100 x concentrate)
• HRP-conjugate (100 x concentrate)
• Antibody Diluent
• Sample Diluent
• HRP-conjugate Diluent
• Sample Extraction Buffer (25 x concentrate)
• Wash Buffer (25 x concentrate)
• TMB Substrate
• Stop Solution
• Adhesive Strip

1. Prepare reagents, samples and standards as instructed.
2. Set a Blank well without any solution.
3. Add 50 µL standard or sample to each well.
4. Add 50μl of Antibody (1x) to each well (not to Blank well)
5. Incubate for 30 minutes at 37°C
6. Aspirate and wash 3 times.
7. Add 50 µL HRP-conjugate (1x) to each well (Not to Blank well).
8. Incubate 1 hour at 37 °C
9. Aspirate and wash 3 times.
10. Add 90 μL of TMB Substrate to each well. Incubate for 20 minutes at 37 °C. Protect from light.
11. Add 50 µL Stop Solution to each well. Read at 450 nm within 10 minutes.

Note:

• Kindly use graduated containers to prepare the reagent.
• Bring all reagents to room temperature (18-25 °C) before use for 30 min.
• Prepare fresh standard for each assay. Use within 4 hours and discard after use.
• Making serial dilution in the wells directly is not permitted.
• To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10 μL for once pipetting.
• Distilled water is recommended to be used to make the preparation for reagents. Contaminated water or container for reagent preparation will influence the detection result.
• Antibody (1x) - Centrifuge the vial before opening. Antibody requires a 100-fold dilution. A suggested 100-fold dilution is 10 μL of Antibody + 990 μL of Antibody Diluent.
• HRP-conjugate (1x) - Centrifuge the vial before opening. HRP- conjugate requires a 100-fold dilution. A suggested 100-fold dilution is 10 μL of HRP- conjugate + 990 μL of HRP- conjugate Diluent.
• Sample Extraction Buffer(1x)- If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Sample Extraction Buffer Concentrate (25 x) into deionized or distilled water to prepare 500 mL of Sample Extraction Buffer(1x).
• Wash Buffer(1x)- If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate (25 x) into deionized or distilled water to prepare 500 mL of Wash Buffer (1 x).
• Standard Centrifuge the standard vial at 6000-10000rpm for 30s before opening. Dilute the Standard(10x) with Sample Diluent. A suggested 10-fold dilution is 50 μL of Standard(10x) + 450 μL of Sample Diluent. This diluted Standard (S7) serves as the high standard (10 μg/mL). Do not substitute other diluents. Mix the standard to ensure complete dilution and allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. Pipette 250 μL of Sample Diluent into each tube (S0-S6). Use the diluted Standard (S7) solution to produce a 2-fold dilution series (below). Mix each tube thoroughly before the next transfer. Sample Diluent serves as the zero standard (0 μg/mL).

• It is recommended to use fresh samples without long storage, otherwise protein degradation and denaturation may occur in these samples, leading to false results. Samples should therefore be stored for a short period at 2 - 8 °C or aliquoted at -20 °C (≤1 month) or -80 °C (≤ 3 months). Repeated freeze-thaw cycles should be avoided. Prior to assay, the frozen samples should be slowly thawed and centrifuged to remove precipitates.
• If the sample type is not specified in the instructions, a preliminary test is necessary to determine compatibility with the kit.
• If a lysis buffer is used to prepare tissue homogenates or cell culture supernatant, there is a possibility of causing a deviation due to the introduced chemical substance. The recommended dilution factor is for reference only.
• Please estimate the concentration of the samples before performing the test. If the values are not in the range of the standard curve, the optimal sample dilution for the particular experiment has to be determined.

Bring all reagents and samples to room temperature before use. Centrifuge the sample again after thawing before the assay. It is recommended that all samples and standards be assayed in duplicate.

1. Prepare all reagents, working standards, and samples as directed in the previous sections.
2. Determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the ziploc, store unused wells at 4°C.
3. Set a Blank well without any solution. Add 50 μL of standard and sample per well.
4. Add 50μl of Antibody (1x) to each well (not to Blank well). Mix well and then incubate for 30 minutes at 37°C.
5. Aspirate each well and wash, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (200 μL) using a squirt bottle, multi-channel pipette, manifold dispenser, or autowasher, and let it stand for 10 seconds, complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add 100μl of HRP-conjugate (1x) to each well (not to Blankwell). Mix well and then incubate for 30 minutes at 37°C.
7. Repeat the aspiration/wash process for five times as in step5.
8. Add 90μl of TMB Substrate to each well, mix well. Incubate for 20 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark.
9. Add 50 μL of Stop Solution to each well, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 10 minutes, using a microplate reader set to 450 nm.

1. The final experimental results will be closely related to validity of the products, operation skills of the end users and the experimental environments.
2. Samples or reagents addition: Please use the freshly prepared Standard. Please carefully add samples to wells and mix gently to avoid foaming. Do not touch the well wall as possible. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and specimens, although not required, is recommended. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
3. Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation time and temperature must be observed.
4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate. Insufficient washing will result in poor precision and falsely elevated absorbance reading. When using an automated plate washer, adding a 1 minute soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision.
5. Controlling of reaction time: Observe the change of color after adding TMB Substrate (e.g. observation once every 10 minutes), TMB Substrate should change from colorless or light blue to gradations of blue. If the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading.
6. TMB Substrate is easily contaminated. TMB Substrate should remain colorless or light blue until added to the plate. Please protect it from light.
7. Stop Solution should be added to the plate in the same order as the TMB Substrate. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the TMB Substrate.