OMB First to Final Action Recommendation Checklist 11-12-18

(b) Result reporting .—Results are reported in mg/kg for solid samples or mg/L for liquid samples. J. Criteria for Acceptance of the Standard Curve The shape of the standard curve is shown in the quality assurance certificate enclosed in the test kit. Absorbances may vary between different runs (e.g., due to different temperatures or analysts). However, the shape of the standard curve should be similar to the one given in the quality assurance certificate. Minimum requirements are as follows: (1) OD at 450 nm for standard 1 higher than 0.8. (2) OD values for standards should continuously decrease with higher concentrations, especially when comparing standard 1 (0 ng/mL) and standard 2 (20 ng/mL). (3) An OD value for standard 1 that is much higher than the OD value stated in the certificate could be an indication of errors during pipetting or incubation. References: (1) Osman, A.A., Uhlig, H.H., Valdes, I., Amin,

(b) ELISA testing .—( 1 ) Insert a sufficient number of wells into the microwell holder for all standards and samples to be run in duplicate. Record standard and sample positions. (2) Add 50 µL of each standard solution or prepared sample, G ( b ), to separate wells in duplicate. (3) Add 50 µL of diluted enzyme conjugate, F ( d ), mix gently by shaking the plate manually, and incubate for 30 min at room temperature (20–25°C/68–77°F). (4) Pour the liquid out of the wells and tap the microwellholder upside down vigorously (three times in a row) against absorbent paper to ensure complete removal of liquid from the wells. Fill all wells with 250 µL washing buffer F ( e ), and pour out the liquid again. Repeat two more times. (5) Add 100 µL Red Chromogen Pro (substrate/chromogen solution; brown cap) to each well. Mix gently by shaking the plate manually and incubate for 10 min at room temperature (20– 25°C/68–77°F) in the dark. (6) Add 100 µL stop solution to each well. Mix gently by shaking the plate manually and measure the absorbance at 450 nm against an air blank. Read within 10 min after addition of stop solution. I. Calculation Interpretation and Test Result Report) (a) Result calculation .—Special software RIDA ® SOFT Win (Part. No. Z9999) is available and strongly recommended for evaluation of the RIDASCREEN ® product line. The calculation should be done using a cubic spline function. Extrapolation is not recommended. The prolamin concentration in an extracted sample is read from the calibration curve and given as ng/mL.To calculate the concentration of prolamins or gluten in a sample, the following equations should be used. (1) Solid samples.— Gluten, mg/kg = gluten concentration in extract, ng/mL × 500/1000

M., Mendez, E., & Mothes, T. (2001) Eur. J. Gastroenterol. Hepatol . 13 , 1189–1193. http:// dx.doi.org/10.1097/00042737-200110000- 00011 (2) Kahlenberg, F., Sanchez, D., Lachmann, I., Tuckova, L., Tlaskalova, H., Méndez, E., & Mothes, T. (2005) Eur. Food Res. Technol . 13 , 1189–1193 (3) Tye-Din, J., Stewart, J., Dromey, J., Beissbarth, T., van Heel, D., Tatham, A., Henderson, K., Mannering, S., Gianfrani, C., Jewell, D., Hill, A., McCluskey, J., Rossjohn, J., &Anderson, R. (2010) Sci. Transl. Med . 2 , 41–51. http://dx.doi. org/10.1126/scitranslmed.3001012 (4) Gessendorfer, B., Koehler, P., & Wieser, H. (2009) Anal. Bioanal. Chem. 395 , 1721–1728. http://dx.doi.org/10.1007/s00216-009-3080-6

(2) Liquid samples.—

Gluten, mg/L = gluten concentration in extract, ng/mL × 500/1000.

J. AOAC Int . 98 , 1346(2015) DOI: 10.5740/jaoacint.CS2015.15

Alternatively, a second order polynomial curve fitting could be used.

Posted: October 13, 2015

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