Biotin ELISA 试剂盒

• Pre-coated, ready to use 96-well strip plate
• Plate sealer for 96 wells
• Reference Standard
• Standard Diluent
• Detection Reagent A
• Assay Diluent A
• TMB Substrate
• Stop Solution
• Wash Buffer (30 x concentrate)
• Instruction manual

Information on standard material:
The standard might be recombinant protein or natural protein, that will depend on the specific kit. Moreover, the expression system is E.coli or yeast or mammal cell. There is 0.05% proclin 300 in the standard as preservative.

Information on reagents:
The stop solution used in the kit is sulfuric acid with concentration of 1 mol/L. And the wash solution is TBS. The standard diluent contains 0.02 % sodium azide and assay diluent A contain 0.01% sodium azide. Some kits can contain is BSA in them.

Information on antibodies:
The provided antibodies and their host vary in different kits.

1. Prepare all reagents, samples and standards,
2. Add 50μL standard or sample to each well.
   Then add 50μL prepared Detection Reagent A immediately.
   Shake and mix. Incubate 1 hour at 37 °C,
3. Aspirate and wash 5 times,
4. Add 90μL Substrate Solution. Incubate 10-20 minutes at 37 °C,
5. Add 50μL Stop Solution. Read at 450 nm immediately.

1. Bring all kit components and samples to room temperature (18-25 °C) before use. If the kit will not be used up in one time, please only take out strips and reagents for present experiment, and leave the remaining strips and reagents in required condition.
2. Standard - Reconstitute the Standard with 3.0 mL of Standard Diluent, kept for 10 minutes at room temperature, shake gently(not to foam). The concentration of the standard in the stock solution is 20,000pg/mL. Please prepare 5 tubes containing 0.4 mL Standard Diluent and produce a quintuple dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer. Set up 5 points of diluted standard such as 20,000pg/mL, 4,000pg/mL, 800pg/mL, 160pg/mL, 32pg/mL,and the last EP tubes with Standard Diluent is the blank as 0pg/mL.
3. Detection Reagent A - Briefly spin or centrifuge the stock Detection A before use. Dilute them to the working concentration 100-fold with Assay Diluent A.
4. Wash Solution - Dilute 20 mL of Wash Solution concentrate (30x) with 580 mL of deionized or distilled water to prepare 600 mL of Wash Solution (1x).
5. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.

Note:

1. Making serial dilution in the wells directly is not permitted.
2. Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37 °C directly.
3. Detection Reagent A are sticky solutions, therefore, slowly pipette them to reduce the volume errors.
4. Please carefully reconstitute Standards or working Detection Reagent A according to the instruction, and avoid foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10μL for once pipetting.
5. The reconstituted Standards and Detection Reagent A can be used only once.
6. If crystals have formed in the Wash Solution concentrate (30x), warm to room temperature and mix gently until the crystals are completely dissolved.
7. Contaminated water or container for reagent preparation will influence the detection result.

• 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.

1. Determine wells for diluted standard, blank and sample. Add 50 µL each of standard dilutions (read Reagent Preparation), blank, and sample to the appropriate wells. Immediately add 50 µL of Detection Reagent A to each well. Shake the plate gently (use of a microplate shaker is recommended). Cover the plate with a Plate Sealer. Incubate at 37 °C for 1 hour.
2. Aspirate the solution and add 350 µL 1x Wash Solution into each well using a squirt bottle, multichannel pipette, manifold dispenser, or automated washer and allow to soak for 1-2 minutes. Completely remove the remaining liquid from all wells by tapping the plate on absorbent paper. Repeat the process 3 times. After the last wash, remove all remaining Wash Buffer by aspirating or decanting. Turn the plate over and blot it against absorbent paper.
3. Add 90 µL of Substrate Solution to each well. Cover with a new Plate Sealer. Incubate for 10 - 20 minutes at 37 °C (no longer than 30 minutes). Protect from light. The liquid will turn blue with the addition.
4. Add 50 µL of Stop Solution to each well. The liquid will turn yellow. Mix the liquid by tapping the side of the plate. If the color change does not appear even, gently tap the plate to ensure thorough mixing.
5. Remove any water droplets or fingerprints from the bottom of the plate and make sure there are no bubbles on the surface of the liquid. Then start the microplate reader and immediately take a measurement at 450 nm.

1. For unopened kit: All reagents should be stored according to the labels on the vials. The Standard, Detection Reagent A and 96-well Strip Plate should be stored at -20 °C upon receipt, while the other reagents should be stored at 4 °C.
2. For opened kits: the remaining reagents must be stored according to the above storage conditions. In addition, please return the unused wells to the foil pouch containing the desiccant and seal the foil pouch with the zipper.