产品名称：AflatoxinB1 ELISA Kit(AFB1)

Because the standards are concentrated 10x, they have to be diluted by the enclosed standard/sample diluent 1:10 (e.g. 50 μL standard + 450 μL diluent), before using them in the assay procedure.

1. Prepare samples as described above.
   2. Pipet 100 μL diluted (1:10) standards or prepared samples in duplicate into the appropriate wells of the microtiter plate. Immediately add 50 μL aflatoxin B1 antibody into each well.
   3. Cover the microtiter plate with a plastic foil and incubate for 60 minutes at room temperature on a microtiter plate shaker (or 90 minutes without shaker).
   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-rabbit-IgG-HRP) into each well.
   6. Cover the microtiter plate with a plastic foil and incubate for 60 minutes at room temperature on a microtiter plate shaker (or 90 minutes without shaker).
   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.

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 pg/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 aflatoxin B1 in pg/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 (50 for the above described extraction). The factor is dependent on the sample preparation procedure 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 0 pg/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. Aflotoxin B1 (pg/mL) ( % binding of 0 ng/mL) 0 100 10 90 40 85 100 70 400 40 1000 25 PERFORMANCE