M . QUALITY ASSURANCE MEASURES FOR VALID ANALYSIS
• In order to qualify as a valid calibration curve suitable for accurate quantification
of DA in samples, the requirements listed in Table 2 must be fulfilled.
• Sample concentrations should only be calculated from datapoints that are
within the valid working range of the calibration curve as defined by the Excel macro.
• The estimated curve fit (%CV of prediction) for the calibration curve should be
<20%, as indicated in the “Results” sheet of the Excel Macro EMA31.
• The concentration difference should not be more 15% between two duplicate
wells for a given sample.
TABLE2 : QUALITY ASSURANCE REQUIREMENTS FOR VALID CALIBRATION CURVE
N . REFERENCES
• Garthwaite I, Ross KM, Miles CO, Briggs L, Towers N, Borell T, Busby P. (2001) J. AOAC Int.
84, 1643-1648.
• Garthwaite I, Ross KM, Miles CO, Hansen RP, Foster D, Wilkins, AL, Towers N. (1998) Nat.
Toxins 6, 93-104.
• Kleivdal H, Holland P, McNabb P. (2003) Biosense Laboratories AS report No. 2003:6.
• Kleivdal H. (2004) Biosense Laboratories AS report No. 2004:1.2.
• Scholin CA, Gulland F, Douchette GJ et al. (2000) Nature 403, 80-84.
• Wright JLC, Boyd RK, DeFreitas ASW, Falk M, Foxall RA, Jamieson WD, Laycock MV,
McCulloch AW, McInnes AG, Odense P, Pathak VP, Quilliam MA, Ragan MA, Sim PG,
Thibault P, Walter JA, Richard DJA, Dwar D. (1989) Can. J. Chem. 67, 481-490.
O . QUICKGUIDE
1. Prepare dilutions of standard and samples.
2. Pre-soak the wells for 5-10 minutes with 300 μl Washing buffer. Empty before
use.
3. Add 50 μL Standard/Sample buffer to the Amax and Blank wells.
4. Add 50 μl Antibody-HRP buffer to the Blank wells.
5. Transfer 50 μL of diluted standards and samples (in duplicate) to the plate.
6. Dilute the concentrated antibody-HRP conjugate and add 50 μL to all wells
except the Blank wells.
7. Seal the plate and incubate at room temperature for 1 hour (keep dark).
8. Wash the wells.
9. Add 100 μL TMB peroxidase substrate to all wells.
10. Incubate at room temperature for 15 minutes (keep dark).
11. Add 100 μL of 0.3 M H2SO4 to all wells to stop the reaction.
12. Read the absorbance at 450 nm.
13. Calculate the concentrations using the Excel Macro EMA31.
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