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108 

H

albmayr

-J

ech

et al

.

:

J

ournal of

AOAC I

nternational

V

ol

. 98, N

o

. 1, 2015

10, 50, 200 and 500 ng/mL gluten, calibrated to the WGPAT

gliadin (86% highly purified gliadin from 40 different European

wheat varieties).

(

c

) 

Conjugate solution (peroxidase-labeled antibody, ready-

to-use)

.—One bottle containing 13 mL.

(

d

) 

Substrate solution (stabilized peroxide substrate and

3,3

,5,5

-tetramethyl-benzidine in a dilute buffer solution)

.—

One bottle containing 15 mL.

(

e

) 

Stop solution (1 N H

2

SO

4

)

.—One bottle containing

15 mL.

(

f

) 

Diluent buffer

.—One bottle containing 20 mL of 5×

concentrated diluent buffer. Contains a final concentration of

20 mM PBS-Tween (0.9% sodium chloride, 0.07% Tween 80)

with 0.25% fish gelatin (Sigma) and 0.01% Proclin as a

preservative.

(

g

) 

Wash buffer

.—One bottle containing 60 mL of 10×

concentrated wash buffer. Contains a final concentration of

20 mM PBS-Tween (0.9% sodium chloride, 0.05% Tween 20)

with 0.01% Proclin as preservative.

(

h

) 

Extraction solution

.—One bottle containing 105 mL

of ready-to-use proprietary extraction solution containing

reducing agents.

(

i

) 

Fish gelatin

.—One sachet containing 10 g.

Additional reagents needed, but not provided with the test kit:

(

a

) 

Distilled or deionized water

.

(

b

) 

Ethanol

.—80% (v/v).

D. General Instructions

Due to the sensitivity of the assay, a gluten-free environment

must be maintained. It is preferable to perform the assay in

a separate room from that used for sample preparation and

extraction. Make sure balance and the surrounding space, as well

as equipment such as spatulas, are clean. Cleaning can be done

by using a 70% alcoholic solution. Spatula should be cleaned

after each sample weighing by a 70% alcoholic solution.

Store kit at 2–8°C (35–46°F) and let all components

equilibrate to 20–25°C (68–77°F) before use.

Include ready-to-use standards in duplicates to each run of

samples. Use separate pipet tips for each standard and each

sample extract to avoid cross-contamination.

It is recommended that an eight-channel pipettor is used to

perform the assay. No more than 48 samples and standards total

should be run in one experiment when using an eight-channel

pipettor (24 when samples and standards are added in duplicate,

e.g., six test strips). If using only single-channel pipets, it

is recommended that no more than a total of 16 samples and

standards are analyzed in one experiment (eight when standards

and samples are added in duplicate, e.g., two test strips).

E. Preparation of Components Delivered with the Kit

(

a

) 

Sample dilution buffer

.—Dilute diluent buffer concentrate

1:5 with distilled water (e.g., add 20 mL of concentrated diluent

buffer to 80 mL distilled water). Dilution buffer may be used

within 24 h, if stored at 4°C.

(

b

) 

Wash buffer

.—If a precipitate is formed during storage of

the wash buffer concentrate, the concentrate should be warmed

up until it is dissolved. Dilute wash buffer concentrate 1:10 with

distilled water (e.g., add 10 mL of concentrated wash buffer to

90 mL distilled water). Wash buffer may be used within 1 week,

if stored at 4°C.

F. Sample and Test Portion Preparation

Obtain a representative sample and homogenize a minimum

of 5 g in a mortar or blender as fine as possible. Weigh out 0.25 g

of homogenized sample into a vial with a minimum 10 mL

capacity, which can be tightly sealed. For chocolate-containing

samples, additionally add 0.25 g of powdered fish gelatin. Add

2.5 mL extraction solution (under a fume/chemical hood),

close vials, and mix vigorously on a vortex. Visually check for

clumps, and continue mixing until samples are well dispersed in

the extraction solution.

Incubate at 50°C (122°F) for 40 min in a water bath. Allow

the extracts to cool to room temperature and add 7.5 mL of

80% ethanol; mix well. Shake for a total of 60 min at room

temperature (20–25°C/68–77°F) with a rotary shaker. (After

about 30 min in the rotator, check the vials visually if all sample

material has suspended in the liquid. If clumps have formed,

vortex and let the vials rotate for the second 30 min to complete

the extraction procedure).

Centrifuge samples for 10 min at 2000 ×

g

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 into a new vial. Dilute supernatant at

least 1:10 (0.1 + 0.9 mL) with prediluted sample dilution buffer

(depending on the expected prolamin content of the sample). If

prediluted samples are not immediately used for determination

by ELISA, close vials and keep in the dark at room temperature

(20–25°C/68–77°F) for a maximum of 7 days until ELISA

experiments.

G. Determination (Assay)

Bring all reagents to room temperature (20–25°C, 68–77°F)

before use.

Use dilution of the sample extract to carry out ELISA

experiments. 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.

Using a single-channel pipettor, add 100 µL of each ready-to-

use 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.

Incubate at room temperature (20–25°C, 68–77°F) for

20 min. Empty the contents of the microwell strips into a

waste container. Wash by filling each microwell with diluted

wash buffer, and then emptying the buffer from the microwell

strips. Repeat this step four times for a total of five washes.

Take care not to dislodge the strips from the holder during the

wash procedure. Lay several layers of absorbent paper towels

on a flat surface and tap microwell strips on towels to expel all

of the residual buffer after the fifth wash. Dry the bottom of the

microwells with a dry cloth or towel.

Measure the required amount of conjugate from the

green-capped bottle (about 120 µL/well or 1 mL/strip) and

place in a separate container (e.g., reagent boat when using the