AOAC-RI ERP Book - Gluten.pdf

F. Sample and Test Portion Preparation

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and mix vigorously on a vortex. Visually check for clumps, and

continue mixing until samples are well dispersed in the extraction

solution.

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rotator, check the vials visually if all sample material has suspended

in the liquid. If clumps have formed, vortex and let the vials rotate

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to obtain a clear

aqueous layer between the particulate sediment and supernatant.

Note, in some cases, a thin fatty layer creaming on top of the

supernatant. Collect the aqueous supernatant (extract) and transfer

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with prediluted sample dilution buffer (depending on the expected

prolamin content of the sample). If prediluted samples are not

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G. Determination (Assay)

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before use.

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Run standards and diluted sample extracts in duplicate. Place an

appropriate number of antibody-coated microwells in a microwell

strip holder. Record standard and sample positions.

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standard or prepared sample into the appropriate well. Use a fresh

pipet tip for each standard or sample. Make sure the pipet tip has

been completely emptied.

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emptying the buffer from the microwell strips. Repeat this step four

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from the holder during the wash procedure. Lay several layers of

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bottom of the microwells with a dry cloth or towel.

Measure the required amount of conjugate from the green-capped

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container (e.g., reagent boat when using the eight-channel pipettor).

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each well.

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emptying the buffer from the microwell strips. Repeat this step four

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from the holder during the wash procedure. Lay several layers of

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bottom of the microwells with a dry cloth or towel.

Measure the required amount of substrate from the blue-capped

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separate container (e.g., reagent boat for an eight-channel pipettor).

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Measure the required amount of stop solution from the

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into a separate container (e.g., reagent boat for an eight-channel

pipet).

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channel pipettor. The color should change from blue to yellow.

H. Reading

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affect analytical results.

Read the absorbance of wells with a microwell reader using a

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I. Calculations

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gluten). Gluten concentration given for the standards already

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protocol. Gluten concentrations of samples can be calculated by

interpolation from this standard curve using a point-to-point

calculation.

If a sample contains gluten levels higher than the highest

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with dilution buffer such that the diluted sample results are in the

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J. Criteria for Acceptance of Standard Curve

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repeated analysis is recommended. The additional dilution factor

must be taken into consideration during calculation.

Any coloration of the substrate solution prior to the analysis or

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may indicate instability or deterioration of reagents.

Reference:

J. AOAC Int

'2,

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Posted: March 9, 2015