产品名称：Histamine(HIS) ELISA Kit

Before application in the test the standards have to be derivatized as follows: To 500 μepsilon of each standard 25 μepsilon reaction solution is pipetted, mixed thoroughly and incubated for 20 minutes at room temperature in the dark. 100 μepsilon neutralizing solution is pipetted to the respective standards, mixed thoroughly and incubated for 20 minutes at room temperature. The standards can now be inserted into the test directly.

1. Prepare samples as described above.
   2. Pipette 100 μepsilon derivatized standards or prepared samples in duplicate into the appropriate wells of the microtiter plate. Immediately add 50 μepsilon histamine antibody into each well.
   3. Cover the microtiter plate with a plastic foil and incubate for 30 minutes at room temperature on a microtiter plate shaker (or 45 minutes without shaker).
   4. Wash the plate three times as follows: Discard the contents of the wells (dump or aspirate). Pipette 300 μepsilon 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. Pipette 100 μL of conjugate (anti-mouse-IgG-HRP) into each well.
   6. Cover the microtiter plate with a plastic foil and incubate for 30 minutes at room temperature on a microtiter plate shaker (or 45 minutes without shaker).
   7. Wash the plate as outlined in 4.
   8. Pipette 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.

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 ng/mL on semi-log graph paper with the optical density on the vertical (y) axis and the concentration on the horizontal (x) axis.
   
   3. Using the mean optical density value for each sample, determine the corresponding concentration of histamine in ng/mL from the standard curve. Depending on experience and/or the availability of computer capability, other methods of data reduction may be employed.
   4. The diluted samples must be further converted by the appropriate dilution factor. The dilution factor is 2 for wine and 10 for cheese and fish extraction according to the sample preparation procedure as described above.
   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 0 ng/mL standard. These values are only an example and should not be used instead of the standard curve which has to be measured in every new test. Histamine (ng/mL) ( % binding of 0 ng/mL) 0 100 2 90 4 84 10 51 40 15 100 7