Information on standard material:
The formulation of the standard is 0.01 M PBS. The standard contains additives (1 % BSA).

Information on reagents:
Reagents include 1 M SO₂. Azide, thimerosal, 2-mercaptoethanol (2-ME) or any other poisonous materials are not used.

Information on antibodies:
The provided antibodies and their host vary in different kits. All antibodies are affinity purified

The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.

Bring all reagents to room temperature (18-25 °C) before use.
Wash Buffer: Dilute 30 mL of Concentrated Wash Buffer into 750 mL of Wash Buffer with deionized or distilled water. Put unused solution back at 5 °C. If crystals have formed in the concentrate, you can warm it with 45 °C water bath (Heating temperature should not exceed 55 °C) and mix it gently until the crystals have completely dissolved. The solution should be cooled to room temperature before use.
Standard: Prepare standard within 15 minutes before use. Reconstitute the Standard with 1.0 mL of Sample Diluent, let it stand for 10 minutes until it dissolved fully. This reconstitution produces a stock solution. Then make serial dilutions as needed (Making serial dilution in the wells directly is not permitted). The Sample Diluent serves as the zero (0).
Biotinylated Detection Ab: Calculate the required amount before experiment (100 μL /well). In actual preparation, you should prepare 100~200 μL more. Centrifuge the stock tube before use, dilute the concentrated Biotinylated Detection Ab to the working concentration using Diluent for Biotinylated Detection Ab (1:100).
Concentrated HRP Conjugate: Calculate the required amount before experiment (100 μL /well). In actual preparation you should prepare 100~200 μL more. Dilute the Concentrated HRP Conjugate to the working concentration using Diluent for Concentrated HRP Conjugate (1:100).
Substrate Reagent: As it is sensitive to light and contaminants, so you shouldn't open the vial until you need it! The needed dosage of the reagent can be aspirated with sterilized tips and the unused residual reagent shouldn't be dumped back into the vial again.
Note: please don't prepare the reagent directly in the Diluent vials provided in the kit. Contaminated water or container for reagent preparation will influence the result.

Bring all reagents and samples to room temperature before use. Centrifuge the sample again after thawing before the assay. All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. It's recommended that all samples and standards be assayed in duplicate.
1. Add Sample: Add 100 μL of Standard, Blank, or Sample per well. The blank well is added with Reference Standard & Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 90?minutes at 37?°C.
2. Biotinylated Detection Ab: Remove the liquid of each well, don'tn'tn't wash. Immediately add 100 μL of Biotinylated Detection Ab working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37?°C.
3. Wash: Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 350 μL) (a squirt bottle, multi-channel pipette, manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, remove remained Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4. HRP Conjugate: Add 100 μL of HRP Conjugate working solution to each well. Cover with the Plate sealer. Incubate for 30?minutes at 37?°C.
5. Wash: Repeat the wash process for five times as conducted in step 3.
6. Substrate: Add 90 μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for about 15?minutes at 37?°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30?minutes. When apparent gradient appeared in standard wells, user should terminate the reaction.
7. Stop: Add 50 μLof Stop Solution to each well. Then, the color turns to yellow immediately. The order to add stop solution should be the same as the substrate solution.
8. OD Measurement: Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9. After experiment, put all the unused reagents back into the refrigerator according to the specified storage temperature respectively until their expiry

Average the duplicate readings for each standard and samples, then subtract the average zero standard optical density. Create a standard curve by plotting the mean OD value for each standard on the y-axisagainst the concentration on the x-axis and draw a best fit curve through the points on the graph. It is recommended to use some professional software to do this calculation, such as curve expert 1.3 or 1.4. In the software interface, a best fitting equation of standard curve will be calculated using OD values and concentrations of standard sample. The software will calculate the concentration of samples after entering the OD value of samples. If samples have been diluted, the concentration calculated from the standard curve must be multiplied by the dilution factor. If the OD of the sample surpasses the upper limit of the standard curve, you should re-test it after appropriate dilution. The actual concentration is the calculated concentration multiplied dilution factor.