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© 2015 AOAC INTERNATIONAL
(
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 microwell holder
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
(
2
)
Liquid samples.—
Gluten, mg/L = gluten concentration in extract,
ng/mL × 500/1000.
Alternatively, a second order polynomial curve fitting could be
used.
(
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,
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 J. AOAC Int . 98 , 1346(2015)DOI: 10.5740/jaoacint.CS2015.15
Posted: October 13, 2015
Candidates for 2016 Method of the Year
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