产品名称：Walnut ELISA Kit

The procedure is according to the following scheme:
1. Prepare samples as described above.
2. Pipet 100 μL ready-to-use standards or prepared samples in duplicate into the appropriate wells of the microtiter plate.
3. Incubate for 20 minutes at room temperature.
4. Wash the plate three times as follows: Discard the contents of the wells (dump or aspirate). Pipet 300 μL of diluted washing solution into each well. After the third repetition empty the wells again and remove residual liquid by striking the plate against a paper towel. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbencies.
5. Pipet 100 μL of conjugate (anti-walnut-peroxidase) into each well.
6. Incubate for 20 minutes at room temperature.
7. Wash the plate as outlined in 4.
8. Pipet 100 μL of substrate solution into each well.
9. Allow the reaction to develop in the dark (e.g. cupboard or drawer, the chromogen is light-sensitive) for 20 minutes at room temperature. 10.Stop enzyme reaction by adding 100 μL of stop solution (0.5 M H2SO4) into each well. The blue colour will turn yellow upon addition. 11.After thorough mixing, measure absorbance at 450 nm (reference wavelength 620 nm), using an ELISA reader. The colour is stable for 30 minutes.

The ready-to-use standards are prepared for a direct determination of sample concentrations. The dilution of samples in the extraction process as described in the above stated sample preparation procedure is already considered. Additional dilution due to high sample concentration has to be accounted for.
1. Calculate the average optical density (OD 450 nm) for each set of reference standards or samples.

2. Construct a standard curve by plotting the mean optical density obtained for each reference standard against its concentration in ppm on semi-log graph paper with the optical density on the vertical (y) axis and the concentration on the horizontal (x) axis. Alternatively the evaluation can be carried out by software. In this case the 4-parameter method should be preferred.

3. Using the mean optical density value for each sample, determine the corresponding concentration of walnut in ppm from the standard curve. Depending on experience and/or the availability of computer capability, other methods of data reduction may be employed.
TYPICAL STANDARD VALUES
The following table contains an example for a typical standard curve. The binding is calculated as percent of the absorption of the 60 ppm standard. These values are only an example and should not be used instead of the standard curve which has to be measured in each new test. Walnut (ppm) % binding of 0 ng/mL) 60 100 20 80 6 55 2 32 0 12