SAH ELISA 2

1. Sample collection must be carried out at 4°C. Remove precipitation by centrifugation when necessary, and test sample as

soon as possible or store at -20°C or below. Avoid repeated freeze-thaw cycles.

2. Avoid NaN3 in samples, since it will inactivate Horseradish peroxidase (HRP).

3. Some unknown factors may have effects on the assay (matrix effects). Dilute samples with sample diluent or prepare standards/samples with the same matrix as the actual sample matrix, which should avoid or reduce the mat fix effects.

Data processing: Always clear the background by subtracting the absorbance at 450 nm (0D450) of blank well from that of the test well. 
The binging rate of each well (standard or sample) is equal to A/As,, with A being the average absorbance of the standard wells or the sample wells, A80 being the average absorbance of the SO standard wells. 
Creation of standard curve: Construct a standard curve by plotting the binding rate of each standard on the y-axis against the logarithm of its concentration on x-axis. Use quadratic polynomial curve to fit the data (r>0.99). The SAH level of the sample can be calculated by substituting its binding rate into the standard curve equation. Then multiply the extent of dilution if the sample has been diluted.

1. Using reagents and samples without rewarming or ambient temperature is less than 20° C may lead to reduced 0D450 values.

2. Extra drying post washing may have negative effect on the results, such as poor standard curve and repeatabity. To avoid this, operate the next step immediately after washing.

3. Mix solution well and wash completely, as these procedures will have influence on the assay

4. Seal the plate with micro-plate sealer and avoid light when incubating the plate.

5. Duplicate wells for standards and samples are recommended, and quadratic polynomial is suggested for standard curve fitting (r>0.99). The detected concentration of quality control vial should be in the detection range.

6. The absorbance of sample well may be higher than that of SO standard well due to matrix effects. Dilute the sample at least ten­fold under this circumstance. If SAH level cannot be detected after 10-fold dilution, use the same matrix as the sample to be measured to prepare a new set of standards and the measure again.

7. The concentrated wash solution may crystallize. Warm it up to allow salts dissolved completely before diluting.

8. The micro-plate sealers should be disposable in order to avoid cross contamination.

9. The Substrate should be kept out of light

10. The Stop Solution is diluted sulfuric acid. Avoid direct contact with skin and other things.