OMB First to Final Action Review (January 11, 2018)

AOAC INTERNATIONAL Official Methods Board (OMB)

First to Final Action Review CHECKLIST

January 11, 2018

AOAC INTERNATIONAL 2275 Research Blvd., Suite 300 Rockville, MD, 20850

UNITED STATES dboyd@aoac.org 301.924.7077 x126

ERP SUMMARY FOR FIRST TO FINAL ACTION METHOD RECOMMENDATION AOAC 2014.03 – Gluten in Rice Flour and Rice-Based Food Products

AOAC No.

GUIDANCE FOR AOAC ERPS - APPENDIX G 1 Considered? Comments/Reference if applicable MethodApplicability YES ERP First Action to Final Action recommendations & improvements implemented/addressed YES Draft Final Action method reviewed by ERP YES No changes to the method, since First Action Safety Concerns YES ReferenceMaterials YES Single Laboratory Validation YES Reviewed separately and SLV referenced in J. AOAC Int. 98, 103 (2015) Reproducibility/Uncertaintyand Probability of Detection YES Comparison to SMPR ( SMPR criteria met?) NO Feedback from Users of Method YES DOCUMENTATION Available? Comments Safety Evaluation YES ReferenceMaterials YES SLV or PTM YES SLV referenced in J. AOAC Int. 98, 103 (2015) Approved Validation Protocols NO Studied completed through AACC Statistics Review YES Method Published in OMA YES Method Performance vs SMPR criteria NO Feedback Information YES No changes to the method, since First Action Additional Recognition(s) ERP Reports YES March 2014 and March 2017 Manuscript(s) Published in JAOAC YES J. AOAC Int. 98, 103 (2015) ERP Method Recommendation (Final Action/Repeal/Continuation) Final Action

1 Official Methods of Analysis of AOAC INTERNATIONAL , Appendix G: Procedures and Guidelines for the Use of AOAC Voluntary Consensus Standards to Evaluate Characteristics of a Method of Analysis, p.3 “ First Action to Final Action Methods: Guidance for AOAC Expert Review Panels.”

AOAC Official Methods of Analysis SM (OMA) EXPERT REVIEW PANEL FOR GLUTEN ASSAYS

TABLE OF CONTENTS A. AOAC FINAL ACTION REQUIREMENTS FOR FIRST ACTION OFFICIAL METHODS – COVER SHEET I. AOAC OFFICIAL METHOD 2014.03:GLUTEN IN RICE FLOUR AND RICE-BASED FOOD PRODUCTS G12 SANDWICH ELISA, FIRST ACTION 2014......................................................2 1. METHOD FEEDBACK .......................................................................................5 2. ARTICLE: GLUTEN IN RICE FLOUR AND BAKED RICE PRODUCTS BY G12 SANDWICH ELISA: FIRST ACTION 2014.03 .....................................................14 3. EXPERT REVIEW PANEL REPORT (MARCH, 2014) ...........................................23 4. EXPERT REVIEW PANEL REPORT (MARCH, 2017) ...........................................26 5. PTM CERTIFICATION REPORT #061403 ( http://www.aoac.org/aoac_prod_imis/AOAC_Docs/RI/17PTM/17C_061403_RG 12.pdf ) II. REFERENCE AND GUIDANCE DOCUMENTATION ( Access via the link below ) In addition to all AOAC Official Methods Board Guidance regarding AOAC First Action and AOAC Final Action Official Methods status.

1. APPENDIX D : GUIDELINES FOR COLLABORATIVE STUDY PROCEDURES TO VALIDATE CHARACTERISTICS OF A METHOD OF ANALYSIS 2. APPENDIX M: VALIDATION PROCEDURES FOR QUANTITATIVE FOOD ALLERGEN ELISA METHODS: COMMUNITY GUIDANCE AND BEST PRACTICES 3. APPENDIX N: ISPAM GUIDELINES FOR VALIDATION OF QUALITATIVE BINARY CHEMISTRY METHODS

is then added which changes the color from blue to yellow. The microwells are measured optically using a microwell reader with a SULPDU\ DEVRUEDQFH ¿OWHU RI QP 2' 7KH RSWLFDO GHQVLWLHV of the samples are compared to the standards and an interpolated result is determined. B. Apparatus 7KH DSSDUDWXV VSHFL¿HG KDV EHHQ WHVWHG (TXLYDOHQW DSSDUDWXV may be used. ( a ) Osterizer blender .—Used for homogenization of sample 6XQEHDP 2VWHU )W /DXGHUGDOH )/ 86$ ( b ) Centrifuge tubes ² P/ IRU H[WUDFWLRQ 6WDU /DEV International GmbH, Hamburg, Germany). ( c ) Glassware ²:DVK ERWWOH P/ DQG JUDGXDWHG cylinders. ( d ) Water bath .—Grant Sub Aqua 12 (Grant Instruments, Cambridgeshire, UK). ( e ) Stuart roller mixer ²%LEE\ 6FLHQWL¿F /WG 6WDIIRUGVKLUH UK). ( f ) Bench top centrifuge ²6LJPD 6LJPD /DERU]HQWULIXJHQ 2VWHURGH DP +DU] *HUPDQ\ ( g ) Centrifuge tubes .—2 mL; for sample dilution (Star Labs International GmbH). ( h ) Micropipet.— $FFXUDWHO\ GHOLYHULQJ —/ “ ( i ) 0LFURWLWHU SODWH UHDGHU ZLWK D QP ¿OWHU ²7KHUPR )LVKHU 6FLHQWL¿F 6KDQJKDL &KLQD C. Reagents Items ( a )–( i ) are available as a test kit (AgraQuant Gluten G12 (/,6$ ® , Romer Labs UK Ltd, Runcorn, UK). All reagents are VWDEOH IRU PRQWKV IURP GDWH RI PDQXIDFWXUH DW ± ƒ& ± ƒ) Refer to kit label for current expiration. ( a ) Antibody-coated microwell strips .—Monoclonal antibodies DUH FRDWHG LQ P0 SKRVSKDWH EXIIHUHG VDOLQH 3%6 RQWR D VHW RI 12 eight-microwell strips (NUNC, Roskilde, Denmark). ( b ) Gluten ready-to-use standards (antigen) ²)LYH YLDOV FRQWDLQLQJ P/ RI HDFK JOXWHQ * VWDQGDUG DQG PJ NJ ODEHOHG DV SSP SUHSDUHG E\ YLWDO ZKHDW JOXWHQ GLVVROYHG LQ HWKDQRO DW D FRQFHQWUDWLRQ RI PJ P/ 6ROXWLRQ LV IXUWKHU GLOXWHG LQ P0 3%6±7ZHHQ VRGLXP FKORULGH 7ZHHQ FRQWDLQLQJ ¿VK JHODWLQ 6LJPD WR

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$SSOLFDEOH IRU GHWHUPLQDWLRQ RI JOXWHQ LQ ULFH ÀRXU DQG rice-based unprocessed and processed foods as evaluated in a multilaboratory study.) Caution : Wear protective gloves and safety glasses. The stop solution contains acid. Avoid contact with skin or eyes. ,I H[SRVHG ÀXVK ZLWK ZDWHU see Material Safety Data Sheet). The extraction solution contains chemicals which are harmful to health. Perform sample extraction under a chemical hood and avoid contact with skin. Dispose of all materials, containers, and devices appropriately after use. See Table 2014.03A for results of the interlaboratory study supporting acceptance of the method. A. Principle The method is based on an enzyme immunoassay format using a monoclonal G12 antibody that can determine gluten derived from wheat, rye, barley, and cross-bred varieties. The G12 antibody binds to the celiac toxic amino acid sequence QPQLPY and related sequences in rye and barley. The antibody detects prolamins in QRQKHDWHG DQG KHDWHG IRRG E\ XVLQJ D VSHFL¿F SURSULHWDU\ H[WUDFWLRQ solution. No cross-reactivity has been determined to maize, rice, teff, millet, buckwheat, quinoa, amaranth, and soy ( see Table 2014.03B ). Gluten is extracted from samples using proprietary extraction solution containing reducing agents followed by ethanol extraction. After centrifugation the supernatant is used in a sandwich enzyme-linked immunoassay. When incubated on monoclonal antibody-coated microwells, the analyte is forming an antibody- antigen complex. After a washing step, an enzyme-conjugated monoclonal antibody is applied to the well and incubated. After a second washing step, an enzyme substrate is added and blue color develops. The intensity of the color is directly proportional to the concentration of gluten in the sample or standard. A stop solution

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Sample ID a

Parameter

Symbol

1

2

3

4

5

6

7

8

9

10 18 36

11

12 18 36

Total No. laboratories

17 34

18 36

18 36

18 36

16 32

18 36

18 36

16 32

17 34

18 36

P

Total No. replicates

Sum [n(L)]

Overall mean of all data (grand mean; mg/kg) Repeatability SD, mg/kg Reproducibility SD, mg/kg

xbarbar

1.6 13.5 26.2 101.2 0.1 6.2 13.1 63.5 4.1 14.9 26.6 112.7

s r 0.8 2.5 8.1 14.8 1.2 1.2 1.3 5.1 1.9 1.5 4.3 20.4 R 1.9 4.0 11.6 31.8 1.2 1.8 2.5 13.5 2.8 4.5 8.9 33.2 48.2 18.5 30.7 14.7 2348 19.2 10.2 8.0 46.2 10.4 16.2 18.1 R 115.8 29.6 44.2 31.4 2348 28.3 19.1 21.2 69.0 30.3 33.6 29.4 1.6 3.5 6.2 1.2 0.1 –3.8 –6.9 –36.5 –0.4 –0.1 2.6 10.7

s

Repeatability RSD, %

RSD r

Reproducibility RSD, %

RSD

Bias (mg/kg) observed-nominal

Recovery, % = observed/nominal × 100

135.0 131.0 101.2

62.0 65.5 63.5 91.1 99.3 110.8 110.5

*OXWHQ IUHH ULFH ÀRXU ULFH ÀRXU PJ JOXWHQ NJ JOXWHQ IUHH FKRFRODWH FDNH FKRFRODWH FDNH PJ JOXWHQ NJ FKRFRODWH FDNH PJ JOXWHQ NJ FKRFRODWH FDNH PJ JOXWHQ NJ FULVS EUHDG PJ JOXWHQ NJ 10 = crisp bread 15 mg gluten/kg; 11 = crisp bread 24 mg gluten/kg; and 12 = crisp bread 102 mg gluten/kg. PJ JOXWHQ NJ ULFH ÀRXU PJ JOXWHQ NJ ULFH ÀRXU

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© 2015 AOAC INTERNATIONAL

DQG QJ P/ JOXWHQ FDOLEUDWHG WR WKH :*3$7 JOLDGLQ KLJKO\ SXUL¿HG JOLDGLQ IURP GLIIHUHQW (XURSHDQ ZKHDW varieties). ( c ) Conjugate solution (peroxidase-labeled antibody, ready-to- use) ²2QH ERWWOH FRQWDLQLQJ P/ ( d ) Substrate solution (stabilized peroxide substrate and 3,3 ƍ ,5,5 ƍ -tetramethyl-benzidine in a dilute buffer solution) ²2QH ERWWOH FRQWDLQLQJ P/ ( e ) Stop solution (1 N H 2 SO 4 ) ²2QH ERWWOH FRQWDLQLQJ P/ ( f ) Diluent buffer ²2QH ERWWOH FRQWDLQLQJ P/ RI î FRQFHQWUDWHG GLOXHQW EXIIHU &RQWDLQV D ¿QDO FRQFHQWUDWLRQ RI P0 3%6 7ZHHQ VRGLXP FKORULGH 7ZHHQ ZLWK ¿VK JHODWLQ 6LJPD DQG 3URFOLQ DV D SUHVHUYDWLYH ( g ) Wash buffer ²2QH ERWWOH FRQWDLQLQJ P/ RI î FRQFHQWUDWHG ZDVK EXIIHU &RQWDLQV D ¿QDO FRQFHQWUDWLRQ RI P0 3%6 7ZHHQ VRGLXP FKORULGH 7ZHHQ ZLWK Proclin as preservative. ( h ) Extraction solution ²2QH ERWWOH FRQWDLQLQJ P/ RI ready-to-use proprietary extraction solution containing reducing agents. ( i ) Fish gelatin ²2QH VDFKHW FRQWDLQLQJ J Additional reagents needed, but not provided with the test kit: ( j ) Distilled or deionized water . 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 VXFK DV VSDWXODV DUH FOHDQ &OHDQLQJ FDQ EH GRQH E\ XVLQJ D alcoholic solution. Spatula should be cleaned after each sample ZHLJKLQJ E\ D DOFRKROLF VROXWLRQ 6WRUH NLW DW ± ƒ& ± ƒ) DQG OHW DOO FRPSRQHQWV HTXLOLEUDWH WR ± ƒ& ± ƒ) EHIRUH XVH 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 SHUIRUP WKH DVVD\ 1R PRUH WKDQ VDPSOHV DQG VWDQGDUGV WRWDO should be run in one experiment when using an eight-channel SLSHWWRU ZKHQ VDPSOHV DQG VWDQGDUGV DUH DGGHG LQ GXSOLFDWH 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 ZLWK GLVWLOOHG ZDWHU H J DGG P/ RI FRQFHQWUDWHG GLOXHQW EXIIHU WR P/ GLVWLOOHG ZDWHU 'LOXWLRQ EXIIHU PD\ EH XVHG ZLWKLQ K LI VWRUHG DW ƒ& ( b ) Wash buffer .—If a precipitate is formed during storage of the wash buffer concentrate, the concentrate should be warmed XS XQWLO LW LV GLVVROYHG 'LOXWH ZDVK EXIIHU FRQFHQWUDWH ZLWK GLVWLOOHG ZDWHU H J DGG P/ RI FRQFHQWUDWHG ZDVK EXIIHU WR P/ GLVWLOOHG ZDWHU :DVK EXIIHU PD\ EH XVHG ZLWKLQ ZHHN LI VWRUHG DW ƒ& ( k ) Ethanol ² Y Y D. General Instructions

7DEOH % &URVV UHDFWLYLW\ RI WKH * DQWLERG\ * DQWLERG\ VKRZV QR FURVV UHDFWLYLW\ WR YDULRXV QXWV RLOV VHHGV VWDUFKHV RU JOXWHQ IUHH JUDLQV

Romer extraction solution, mg/kg gluten Gluten, %

Food category

Food sample

Gluten-containing grains

:KHDW ÀRXU

72222

7.2

Barley (Cumion) 'XUXP ZKHDW Spelt (Ostro) Rye (Capitan)

292390 15733 81926 41577

29.2

1.6 8.2 4.2

Naturally gluten-free grains

Soya bean

<4

Soya mince %XFNZKHDW

<4 <4 <4 <4 <4 <4 <4

5LFH ÀRXU

Quinoa

Corn kernels

7HII ÀRXU

Millet

Oats

Bastion

4.3 7.4

00-61 Cn

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Husky

6.3 6.6

Fusion Pecan Walnut Almond &DVKHZ

Nuts

<4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4

Macadamia

Peanut

Hazelnut Pine nut 3LVWDFKLR

Seeds

Golden linseed Brown linseed

Poppy

Sesame Mustard

Oils

Hazelnut oil

Walnut oil

Vegetable oil 6XQÀRZHU RLO 7DSLRFD VWDUFK :KHDW VWDUFK 3RWDWR VWDUFK

6WDUFKHV

Miscellaneous

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© 2015 AOAC INTERNATIONAL

WRZHOV WR H[SHO DOO RI WKH UHVLGXDO EXIIHU DIWHU WKH ¿IWK ZDVK 'U\ WKH bottom of the microwells with a dry cloth or towel. Measure the required amount of substrate from the blue-capped ERWWOH DERXW —/ ZHOO RU P/ VWULS DQG GLVSHQVH LQWR D separate container (e.g., reagent boat for an eight-channel pipettor). 3LSHW —/ RI WKH VXEVWUDWH LQWR HDFK PLFURZHOO XVLQJ DQ HLJKW FKDQQHO SLSHWWRU ,QFXEDWH DW URRP WHPSHUDWXUH ± ƒ& ± ƒ) IRU PLQ LQ WKH GDUN Measure the required amount of stop solution from the UHG FDSSHG ERWWOH DERXW —/ ZHOO RU P/ VWULS DQG GLVSHQVH into a separate container (e.g., reagent boat for an eight-channel pipet). 3LSHW —/ RI VWRS VROXWLRQ LQWR HDFK PLFURZHOO XVLQJ DQ HLJKW channel pipettor. The color should change from blue to yellow. H. Reading (OLPLQDWH DLU EXEEOHV SULRU WR UHDGLQJ ZHOOV DV WKH\ DUH OLNHO\ WR affect analytical results. Read the absorbance of wells with a microwell reader using a QP ¿OWHU 5HFRUG 2' UHDGLQJV IRU HDFK PLFURZHOO I. Calculations 8VH XQPRGL¿HG 2' YDOXHV RU 2' YDOXHV H[SUHVVHG DV D SHUFHQWDJH RI WKH 2' RI WKH SSP VWDQGDUG WR FRQVWUXFW D GRVH UHVSRQVH FXUYH XVLQJ WKH ¿YH VWDQGDUGV DQG SSP gluten). Gluten concentration given for the standards already FRQVLGHU VDPSOH SUHSDUDWLRQ DQG GLOXWLRQ DFFRUGLQJ WR PHWKRG 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 VWDQGDUG ! SSP WKH VDPSOH H[WUDFW VKRXOG EH IXUWKHU GLOXWHG with dilution buffer such that the diluted sample results are in the UDQJH RI WR SSP DQG UHDQDO\]HG WR REWDLQ DFFXUDWH UHVXOWV 7KH GLOXWLRQ IDFWRU PXVW EH LQFOXGHG ZKHQ WKH ¿QDO UHVXOW LV FDOFXODWHG J. Criteria for Acceptance of Standard Curve $Q H[DPSOH IRU WKH FDOLEUDWLRQ FXUYH LV VKRZQ LQ WKH &HUWL¿FDWH RI $QDO\VLV LQFOXGHG LQ HDFK WHVW NLW +LJKHU 2' YDOXHV RI WKH DEVRUEDQFH DW QP FRPSDUHG WR WKH FHUWL¿FDWH PD\ LQGLFDWH LQVXI¿FLHQW ZDVKLQJ RU JOXWHQ FRQWDPLQDWLRQ )RU VDPSOHV VKRZLQJ 2' YDOXHV KLJKHU WKDQ WKH SSP VWDQGDUG D IXUWKHU GLOXWLRQ DQG 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 2' YDOXH RI OHVV WKDQ DEVRUEDQFH XQLWV IRU SSP VWDQGDUG may indicate instability or deterioration of reagents. Reference: J. AOAC Int . 98 '2, MDRDFLQW Posted: March 9, 2015

F. Sample and Test Portion Preparation 2EWDLQ D UHSUHVHQWDWLYH VDPSOH DQG KRPRJHQL]H D PLQLPXP RI J LQ D PRUWDU RU EOHQGHU DV ¿QH DV SRVVLEOH :HLJK RXW J RI KRPRJHQL]HG VDPSOH LQWR D YLDO ZLWK D PLQLPXP P/ FDSDFLW\ ZKLFK FDQ EH WLJKWO\ VHDOHG )RU FKRFRODWH FRQWDLQLQJ VDPSOHV DGGLWLRQDOO\ DGG J RI SRZGHUHG ¿VK JHODWLQ $GG P/ H[WUDFWLRQ VROXWLRQ XQGHU D IXPH FKHPLFDO KRRG FORVH YLDOV and mix vigorously on a vortex. Visually check for clumps, and continue mixing until samples are well dispersed in the extraction solution. ,QFXEDWH DW ƒ& ƒ) IRU PLQ LQ D ZDWHU EDWK $OORZ WKH H[WUDFWV WR FRRO WR URRP WHPSHUDWXUH DQG DGG P/ RI HWKDQRO PL[ ZHOO 6KDNH IRU D WRWDO RI PLQ DW URRP WHPSHUDWXUH ± ƒ& ± ƒ) ZLWK D URWDU\ VKDNHU $IWHU DERXW PLQ LQ WKH rotator, check the vials visually if all sample material has suspended in the liquid. If clumps have formed, vortex and let the vials rotate IRU WKH VHFRQG PLQ WR FRPSOHWH WKH H[WUDFWLRQ SURFHGXUH &HQWULIXJH VDPSOHV IRU PLQ DW î g to obtain a clear aqueous layer between the particulate sediment and supernatant. Note, in some cases, a thin fatty layer creaming on top of the supernatant. Collect the aqueous supernatant (extract) and transfer LQWR D QHZ YLDO 'LOXWH VXSHUQDWDQW DW OHDVW P/ with prediluted sample dilution buffer (depending on the expected prolamin content of the sample). If prediluted samples are not LPPHGLDWHO\ XVHG IRU GHWHUPLQDWLRQ E\ (/,6$ FORVH YLDOV DQG NHHS LQ WKH GDUN DW URRP WHPSHUDWXUH ± ƒ& ± ƒ) IRU D PD[LPXP RI GD\V XQWLO (/,6$ H[SHULPHQWV G. Determination (Assay) %ULQJ DOO UHDJHQWV WR URRP WHPSHUDWXUH ± ƒ& ± ƒ) before use. 8VH GLOXWLRQ RI WKH VDPSOH H[WUDFW WR FDUU\ RXW (/,6$H[SHULPHQWV 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. 8VLQJ D VLQJOH FKDQQHO SLSHWWRU DGG —/ RI HDFK UHDG\ WR XVH 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. ,QFXEDWH DW URRP WHPSHUDWXUH ± ƒ& ± ƒ) IRU PLQ (PSW\ WKH FRQWHQWV RI WKH PLFURZHOO VWULSV LQWR D ZDVWH FRQWDLQHU :DVK E\ ¿OOLQJ HDFK PLFURZHOO ZLWK GLOXWHG ZDVK EXIIHU DQG WKHQ emptying the buffer from the microwell strips. Repeat this step four WLPHV IRU D WRWDO RI ¿YH ZDVKHV 7DNH FDUH QRW WR GLVORGJH WKH VWULSV from the holder during the wash procedure. Lay several layers of DEVRUEHQW SDSHU WRZHOV RQ D ÀDW VXUIDFH DQG WDS PLFURZHOO VWULSV RQ WRZHOV WR H[SHO DOO RI WKH UHVLGXDO EXIIHU DIWHU WKH ¿IWK ZDVK 'U\ WKH bottom of the microwells with a dry cloth or towel. Measure the required amount of conjugate from the green-capped ERWWOH DERXW —/ ZHOO RU P/ VWULS DQG SODFH LQ D VHSDUDWH container (e.g., reagent boat when using the eight-channel pipettor). 8VLQJ DQ HLJKW FKDQQHO SLSHW GLVSHQVH —/ RI FRQMXJDWH LQWR each well. ,QFXEDWH DW URRP WHPSHUDWXUH ± ƒ& ± ƒ) IRU PLQ (PSW\ WKH FRQWHQWV RI WKH PLFURZHOO VWULSV LQWR D ZDVWH FRQWDLQHU :DVK E\ ¿OOLQJ HDFK PLFURZHOO ZLWK GLOXWHG ZDVK EXIIHU DQG WKHQ emptying the buffer from the microwell strips. Repeat this step four WLPHV IRU D WRWDO RI ¿YH ZDVKHV 7DNH FDUH QRW WR GLVORGJH WKH VWULSV from the holder during the wash procedure. Lay several layers of DEVRUEHQW SDSHU WRZHOV RQ D ÀDW VXUIDFH DQG WDS PLFURZHOO VWULSV RQ

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AOAC Official Method 2014.03 Gluten in Rice Flour and Rice-Based Food Products G12 Sandwich ELISA First Action 2014

1. Test principle The method is based on an enzyme immunoassay format using a monoclonal G12 antibody that can determine gluten derived from wheat, rye, barley, and cross-bred varieties. The G12 antibody binds to the celiac toxic amino acid sequence QPQLPY and related sequences in rye and barley. The antibody detects prolamins in non-heated and heated food by using a specific proprietary extraction solution. Gluten is extracted from samples using proprietary extraction solution containing reducing agents followed by ethanol extraction. After centrifugation the supernatant is used in a sandwich enzyme-linked immunoassay. When incubated on monoclonal antibody-coated microwells, the analyte is forming an antibody-antigen complex. After a washing step, an enzyme-conjugated monoclonal antibody is applied to the well and incubated. After a second washing step, an enzyme substrate is added and blue color develops. The intensity of the color is directly proportional to the concentration of gluten in the sample or standard. A stop solution is then added which changes the color from blue to yellow. The microwells are measured optically using a microwell reader with a primary absorbance filter of 450 nm (OD450). The optical densities of the samples are compared to the standards and an interpolated result is determined. 2. Method Applicability The test method is applicable for determination of gluten in rice flour and rice-based unprocessed and processed foods. No limitations of the intended use have been discovered over the past two years. None of the reagents or required apparatuses have changed since the first approval of the method. The same general instructions, precautions and instructions for solution and sample preparation, extraction, assay and reading and calculation of the results still apply as stated in the method publication. The method performs as desired in the above mentioned matrices and furthermore shows satisfying results routine samples composed of other matrices as well.

3. Safety Concerns

No additional safety concerns have been identified. The following general instructions and precautions still apply:

It is recommended to perform the assay in a room separate from that used for sample preparation and extraction in order to maintain a gluten-free environment. The lab and equipment has to be kept clean, ideally by using a 70% alcoholic solution. Good laboratory practice applies when performing the assay. Instructions in the package insert need to be followed accurately to ensure reliable test results.

4. Reference materials No certified reference material for gluten is available at the time of submission.

Page 1 of 6

© Romer Labs, 2016

5. Single Laboratory Validation An internal evaluation of spiked and incurred samples was conducted by Romer Labs ® in Tulln, Austria to confirm the performance of the G12 antibody-based sandwich ELISA test kit. Two different operators investigated heat-treated and non-heat treated samples with incurred gluten levels up to 124 mg/kg. In the validation it was shown that gluten could be accurately quantified by G12 Sandwich ELISA with reproducibility RSD(R) of 12.6% and repeatability RSD(r) of 10.8%. Tested samples showed a recovery between 89 % and 118%. Details are indicated in table 1.

Table 1: Performance statistics for overall G12 sandwich ELISA results

Gluten- free cookie

cookie powder mixture 1

cookie powder mixture 2

Incurred cookie

cookie dough

Nominal gluten content

0 mg/kg 106 mg/kg 102 mg/mg 124 mg/kg 124 mg/kg

No. of operators

2

2

2

2

2

Total No. of replicates Overall mean, mg/kg Repeatability SD, mg/kg Reproducibility SD, mg/kg Repeatability RSD, % Reproducibility RSD, %

18

17

18

18

18

1.63*

94.3

113.7

146.5

116.9

0.85 0.96 51.8 59.1

5.9 9.9 6.3

11.1 14.3

15.8 15.4 10.8 10.5

10.4 10.6

9.7

8.9 9.0

10.5

12.6

Bias (mg/kg) observed - nominal

1.63

-11.7

11.7

22.5

-7.1

Recovery, %=observed/nominal x 100

-

89.0

111.4

118.1

94.3

*result

Gluten-free blend flours were spiked with PWG gliadin delivered from the Working Group on Prolamin Analysis and Toxicity (Germany) at levels in the middle of the quantification range of the ELISA. The theoretical gluten concentration of the cookie materials was calculated according to the certified protein content of the PWG gliadin (van Eckert et al., 2006), considering the weight loss during heat treatment. The incurred materials were produced under the guidance of the Food Allergen Working Group of the Budapest University of Technology and Economics (Bugyi et al., 2012). Samples consisted of PWG gliadin, gluten-free blend flour, salt, sugar, sodium-bicarbonate, margarine and water. For measurements, two different cookie powder mixtures (without margarine), the lyophilized dough (with margarine, non-heat treated) and the baked cookies were used. Sample preparation, extraction, ELISA procedure and interpretation of results were conducted as described in the official method protocol.

Page 2 of 6

© Romer Labs, 2016

6. Batch-to-Batch Variation

Measurements of kit standards of several batches were conducted at 450 nm and OD values were tracked over a 1½ year period. OD values slightly differed from lot to lot, but the shape of the standard curve stayed the same.

Batch-to-Batch Variation of Standards

3,0

2,5

2,0

1,5

1,0 OD [450 nm]

0,5

0,0

0

20

40

60

80

100 120 140 160 180 200

Kit standards [mg/kg gluten]

Figure 1: OD values of standards from various batches measured at 450 nm

Table 2: OD values of standards from various batches measured at 450 nm

Standard OD [450 nm]

Test Date 05.01.2015 07.01.2015 27.01.2015 30.01.2015 25.02.2015 28.04.2015 30.04.2015 26.06.2015 19.08.2015 12.09.2015 03.01.2016 04.02.2015 19.02.2015 04.03.2016 31.03.2016 29.04.2016 04.05.2016 06.05.2016 09.05.2016

0

4

20

80

200

0,032 0,039 0,035 0,038 0,064 0,053 0,052 0,050 0,120 0,069 0,026 0,059 0,039 0,043 0,027 0,069 0,038 0,043 0,038 0,049

0,252 0,259 0,242 0,234 0,242 0,178 0,182 0,167 0,390 0,368 0,282 0,197 0,309 0,170 0,279 0,301 0,285 0,290 0,283 0,258

0,745 0,738 0,708 0,678 0,825 0,606 0,628 0,507 0,999 0,875 0,711 0,508 0,948 0,530 0,889 0,921 0,770 0,853 0,854 0,752

1,715 1,829 1,669 1,730 1,643 1,437 1,450 1,279 1,617 1,607 1,415 1,248 1,708 1,022 1,682 1,579 1,404 1,577 1,595 1,537

2,363 2,318 2,171 2,399 2,425 2,035 2,118 1,950 2,445 2,055 1,953 1,959 2,270 1,825 2,113 2,272 1,957 2,195 2,245 2,161

Overall mean, OD

Reproducibility SD, OD Reproducibility RSD, %

0,02

0,06

0,15

0,20

0,19

44

24

20

13

9

Page 3 of 6

© Romer Labs, 2016

7. Repeatability

A gluten-free beverage sample was measured in 9-fold replicates to assess the variation of repeated measurements in a blank sample. The same sample was then spiked with gluten to obtain final theoretical contamination levels of 4, 50 and 200 mg/kg gluten to cover the whole quantitation range of the method. The samples were measured 9 times, only the spike at the lower limit of quantification was determined 20 times. The recovery was calculated for each gluten level and ranged from 95 to 111%. The repeatability RSD of the spiked samples ranged from 2 to 13%, depending on the gluten concentration. Data of all repeatability measurements are shown in tables 3 to 6.

Table 3: Repeated measurements of a gluten-free beverage sample

Sample

OD [450 nm]

1 2 3 4 5 6 7 8 9

0,08

0,082 0,074 0,051 0,054 0,063 0,045 0,044 0,043 0,060

Overall mean

Repeatability SD

0,02

Repeatability RSD, %

26

Table 4: Repeated measurements of a beverage sample spiked with 200 mg/kg gluten

Spike level [mg/kg]

OD [450 nm]

Gluten conc. [mg/kg]

Recovery, %=observed/nominal x 100

Replicate

1 2 3 4 5 6 7 8 9

1,797 1,795 1,812 1,821 1,799 1,771 1,815 1,795 1,800

189 189 193 195 190 183 193 189 190 190 3,57

95 94 97 97 95 91 97 95 95 95

200

Overall mean

Repeatability SD

Repeatability RSD, %

2

Page 4 of 6

© Romer Labs, 2016

Table 5: Repeated measurements of a beverage sample spiked with 50 mg/kg gluten

Spike level [mg/kg]

OD [450 nm]

Gluten conc. [mg/kg]

Recovery, %=observed/nominal x 100

Replicate

1 2 3 4 5 6 7 8 9

1,040 1,030 1,018 0,999 1,018 1,001 1,027 1,010 1,048

50,2 49,3 48,2 46,4 48,2 46,6 49,0 47,4 51,0 48,5 1,57

100

99 96 93 96 93 98 95

50

102

Overall mean

97

Repeatability SD

Repeatability RSD, %

3

Table 6: Repeated measurements of a beverage sample spiked with 4 mg/kg gluten

Spike level [mg/kg]

OD [450 nm]

Gluten conc. [mg/kg]

Recovery, %=observed/nominal x 100

Replicate

1 2 3 4 5 6 7 8 9

0,250 0,259 0,244 0,259 0,266 0,265 0,266 0,262 0,258 0,256 0,268 0,269 0,300 0,269 0,260 0,262 0,259 0,323 0,268 0,273

3,91 4,15 3,80 4,15 4,40 4,36 4,40 4,26 4,12 4,05 4,46 4,50 5,57 4,50 4,19 4,26 4,15 6,36 4,46 4,64 4,43 0,58

98

104

95

104 110 109 110 107 103 101 112 113 139 113 105 107 104 159 112 116 111

10 11 12 13 14 15 16 17 18 19 20

4

Overall mean

Repeatability SD

Repeatability RSD, %

13

Page 5 of 6

© Romer Labs, 2016

H ALBMAYR -J ECH ET AL .: J OURNAL OF AOAC I NTERNATIONAL V OL . 98, N O . 1, 2015 103

FOOD COMPOSITION AND ADDITIVES

Gluten in Rice Flour and Baked Rice Products by G12 Sandwich ELISA: First Action 2014.03 E LISABETH H ALBMAYR -J ECH Romer Labs Division Holding GmbH, Technopark 1, 3430 Tulln, Austria A DRIAN R OGERS Romer Labs UK Ltd, Block 5, The Heath Technical and Business Park, Runcorn, Cheshire WA7 4QX, United Kingdom C LYDE D ON Foodphysica, Vogelwikke 12, 6665 HP Driel, The Netherlands M ICHAEL P RINSTER Romer Labs Inc, 1301 Stylemaster Dr, Union, MO 63084-1156 Collaborators: G. Augustin; C. Brewe; Z. Bugyi; D. Clarke; P. Cressey; A. Firzinger; J. Gelroth; M. Hemingway; R. Hochegger; J. Jolly; P. Kasturi; P. Koehler; T. Koerner; M. Marquard; C. Poirier; A. Rogers; G. Sharma; R. Sherlock; C. Sousa; S. Taylor; S. Tomoszi, J. Topping; P. Wehling

done in 60 min. Ready-to-use standards of the ELISA test kit are calibrated against the Working Group on Prolamin Analysis and Toxicity (WGPAT) gliadin standard material and cover a range from 4 to 200 mg gluten/kg sample ( see Figure 1). The preparation of ready-to-use standards was described at Halbmayr- Jech et al. (3). Single-laboratory validation (SLV), performed by Romer Labs UK Ltd in May 2011, determined an LOD of 2 mg gluten/kg sample and an LOQ of 4 mg gluten/kg sample ( see Table 1) as well as a recovery rate ranging from 90 to 145% ( see Table 2) for WKH *OXWHQ * 6DQGZLFK (/,6$ DVVD\ &RHI¿FLHQW RI YDULDWLRQ for repeatability and lot-to-lot variation (reproducibility) was 15% or less determined within the SLV ( see Tables 3–5). The AgraQuant Gluten G12 kit furthermore produced results similar to those assigned values for the current Codex type I approved R5 Mendez method in three Food Analysis Performance Assessment Scheme (FAPAS) rounds in 2011 ( see Table 6). The Gluten G12 Sandwich ELISA assay has been evaluated in a collaborative study with 20 participants. The main target for an allowable immunogenic gluten method according to the Codex Alimentarius is that it should have a detection limit of 10 mg/kg RU EHORZ 7KLV SDSHU UHSRUWV WKH ¿QGLQJV RI WKH FROODERUDWLYH study and discusses the results in relation to current thresholds (20 mg/kg) for gluten-free products. Study Design The study was conducted on 12 different food samples prepared in the laboratory of the Deutsche Forschungsanstalt für Lebensmittelchemie, Freising, Germany. Blind-coded samples in duplicate, ELISA test kits including extraction solution, method instructions, and result reporting sheets were sent to all participating laboratories. Collaborative Study

Received August 5, 2014. The method was approved by the Expert Review Panel for Food Allergens-Gluten as First Action. The Expert Review Panel for Food Allergens-Gluten invites method users to provide feedback on the First Action methods. Feedback from PHWKRG XVHUV ZLOO KHOS YHULI\ WKDW WKH PHWKRGV DUH ¿W IRU SXUSRVH and are critical to gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@aoac.org. Corresponding author’s e-mail: elisabeth.halbmayr@romerlabs.com Copyright permission has been received by Cereal Foods World to reprint the tables. DOI:10.5740/jaoacint.14-197 antibody utilized in the test kit binds to the celiac toxic amino acid sequence QPQLPY and related sequences in rye and barley (1,2). A homogenized sample is extracted with ethanol and a proprietary extraction solution containing reducing agents. The gluten determination is based on a microtiter plate coated ZLWK VSHFL¿F PRQRFORQDO * DQWLERG\ *OXWHQ LV GHWHFWHG ZLWK a peroxidase-labeled G12 antibody. The determination can be The Protein and Enzymes Technical Committee of American Association of Cereal Chemists initiated D FROODERUDWLYH VWXG\ WR FRQ¿UP ZKHWKHU WKH * DQWLERG\ EDVHG VDQGZLFK (/,6$ WHVW NLW LV DEOH WR GHWHFW JOXWHQ LQ WKH ORZHU PJ NJ SSP OHYHO 7ZHQW\ ODERUDWRULHV LQYHVWLJDWHG KHDW WUHDWHG DQG QRQ KHDW WUHDWHG EOLQG FRGHG VDPSOHV ZLWK LQFXUUHG JOXWHQ OHYHOV XS WR PJ NJ 7KH PHWKRG has been validated for testing foods to conform WR WKH GH¿QHG &RGH[ WKUHVKROGV IRU JOXWHQ LQ JOXWHQ IUHH SURGXFWV DW OHVV WKDQ PJ JOXWHQ NJ 7KH FROODERUDWLYH VWXG\ VKRZHG WKDW ORZ OHYHOV RI JOXWHQ FRXOG EH GHWHFWHG E\ * 6DQGZLFK (/,6$ ZLWK UHSURGXFLELOLW\ 56' 5 RI DQG UHSHDWDELOLW\ 56' r of ,QFXUUHG VDPSOHV VKRZHG D UHFRYHU\ EHWZHHQ DQG ,W LV UHFRPPHQGHG WKDW WKH PHWKRG EH DFFHSWHG E\ $2$& DV 2I¿FLDO )LUVW $FWLRQ A graQuant ® Gluten G12 is a sandwich ELISA for TXDQWL¿FDWLRQ RI JOXWHQ IURP ZKHDW U\H EDUOH\ DQG cross-bred varieties in various foodstuffs. The G12

Collaborators

The collaborative study was coordinated by Clyde Don, Foodphysica, Driel, The Netherlands. Twenty laboratories from the food producing industry, universities, governments, contract

104 H ALBMAYR -J ECH ET AL . : J OURNAL OF AOAC I NTERNATIONAL V OL . 98, N O . 1, 2015

7DEOH 6SLNH UHFRYHU\ GDWD IURP VLQJOH ODERUDWRU\ YDOLGDWLRQ GDWD VDPSOHV ZHUH WHVWHG ERWK LQ WKHLU RULJLQDO VWDWH DQG VSLNHG ZLWK PJ NJ RI 9LWDO ZKHDW JOXWHQ H[WUDFW 3HUFHQWDJH UHFRYHU\ ZDV FDOFXODWHG DJDLQVW D SRVLWLYH FRQWURO VSLNHG LQWR H[WUDFWLRQ EXIIHU 5HFRYHU\ RI PJ NJ VSLNH ZDV DFKLHYHG IURP D UDQJH RI SURFHVVHG IRRG VDPSOHV ZLWKLQ DQ DFFHSWDEOH UDQJH ± 7KH DGGLWLRQ RI JHODWLQ WR WKH H[WUDFWLRQ VROXWLRQ VLJQL¿FDQWO\ LQFUHDVHG WKH H[WUDFWLRQ HI¿FLHQF\ IURP FKRFRODWH Romer extraction solution

Spike (10 ppm gluten)

Spike CV, % Recovery, %

Sample

No spike

laboratories, and kit suppliers fromEurope, United States, Canada, Australia, and New Zealand participated in the collaborative study.All collaborators are listed in the Acknowledgments section. &DOLEUDWLRQ FXUYH RI PRQRFORQDO * (/,6$ 6L[ UHSOLFDWHV HDFK RI WKH 9LWDO ZKHDW JOXWHQ DQG 3:* JOLDGLQ VWDQGDUGV ZHUH UXQ RQ WKH $JUD4XDQW *OXWHQ * WHVW NLW (UURU EDUV LQGLFDWH î 6' RI VWDQGDUG The following 12 samples were prepared for the collaborative VWXG\ JOXWHQ IUHH ULFH ÀRXU ULFH ÀRXU FRQWDLQLQJ PJ JOXWHQ NJ ULFH ÀRXU FRQWDLQLQJ PJ JOXWHQ NJ ULFH ÀRXU containing 100 mg gluten/kg, gluten-free chocolate cake, chocolate cake containing 10 mg gluten/kg, chocolate cake containing 20 mg gluten/kg, chocolate cake containing 100 mg gluten/kg, crisp bread containing 4.5 mg gluten/kg, crisp bread containing 15 mg gluten/kg, crisp bread containing 24 mg gluten/kg, and crisp bread containing 102 mg gluten/kg. Initial target concentrations of the crisp bread samples had been 0, 10, 20, and 100 mg/kg, but a gluten contamination occurred during the preparation of these samples. The contamination was LQGHSHQGHQWO\ FRQ¿UPHG ZLWK DQRWKHU DQWLERG\ EDVHG (/,6$ giving further reason to allow a re-estimation of gluten content of respective samples. $OO LQJUHGLHQWV H[FHSW ZKHDW ÀRXU ZHUH FRQ¿UPHG WR EH IUHH RI gluten contamination before use by means of the G12 Sandwich ELISA, which was also used in this collaborative study. 7KH JOLDGLQ FRQWHQW RI ZKHDW ÀRXU RI WKH *HUPDQ FXOWLYDU ‘Genius’ was determined by an extraction/RP-HPLC method as described byWieser et al. (5). HPLC absorbance values measured at 210 nm were converted to protein concentration using a standard solution of reference gliadin from the ProlaminWorking *URXS 7KH JOLDGLQ FRQWHQW RI WKH ZKHDW ÀRXU VDPSOH ZDV 67.8±0.16 g/kg ( n = 3) on an “as is” basis. The gluten content of 7DEOH &DOFXODWLRQ RI /2' IURP VLQJOH ODERUDWRU\ YDOLGDWLRQ GDWD UHSOLFDWHV RI EXIIHU EODQNV ZHUH UXQ RYHU LQGLYLGXDO $JUD4XDQW *OXWHQ * DVVD\V 7KH /2' ZDV GHWHUPLQHG E\ FDOFXODWLQJ WKH PHDQ 2' RI WKH PJ NJ VWDQGDUG 6' DQG WKHQ UHDGLQJ WKLV YDOXH EDFN RII WKH VWDQGDUG FXUYH 7KH ORZHU /24 ZDV GHWHUPLQHG E\ WKH ORZHVW VWDQGDUG RI FRQFHQWUDWLRQ Standard, mg/kg Mean (OD) SD (OD) CV, % (OD) Mean + 3 SD (OD) LOD, mg/kg 0 0.14 0.03 21.15 0.23 2.00 )LJXUH Description and Preparation of Samples

Crisps

<4 <4

12.6

1.35 NA a 1.84 5.33 0.16 2.44 0.88 0.31 7.00 1.31

134.0

NA

Chocolate

<4

Chocolate + gelatin <4

10.3

109.6

Cheesy corn snack

<4 <4 <4 <4 <4 <4

8.5

90.4

Paprika Chicken

10.8

114.9 103.2 100.0 131.9 144.7 100.0

9.7 9.4

Yogurt

Curry sauce Margarine

12.4 13.6

Positive control

NA

9.4

a

NA = Not applicable.

WKH ZKHDW ÀRXU ZDV FDOFXODWHG DFFRUGLQJ WR &RGH[ JOXWHQ î prolamin) and was 135.6 g/kg. Samples were heat-treated to a different extent during SURFHVVLQJ DV IRXQG LQ FRQVXPHU SURGXFWV 5LFH ÀRXU ZDV XVHG “as is” (not heat-treated) and represented a base material for the production of gluten-free rice based products. Gluten-free ULFH ÀRXU ZDV SURYLGHG E\ *HQHUDO 0LOOV 0LQQHDSROLV 01 *OXWHQ FRQWDLQLQJ VWRFN ULFH ÀRXU ZLWK D JOXWHQ FRQFHQWUDWLRQ RI PJ NJ ZDV SUHSDUHG E\ PL[LQJ ZKHDW ÀRXU LQWR ULFH ÀRXU DQG VXEVHTXHQWO\ GLOXWLQJ WKH PL[WXUH ZLWK ULFH ÀRXU *OXWHQ FRQWDLQLQJ ULFH ÀRXU VDPSOHV ZHUH SUHSDUHG DV IROORZV PJ NJ J VWRFN ULFH ÀRXU ZDV PL[HG ZLWK J JOXWHQ IUHH ULFH ÀRXU PJ NJ J VWRFN ULFH ÀRXU ZDV PL[HG ZLWK J JOXWHQ IUHH ULFH ÀRXU DQG PJ NJ J VWRFN ULFH ÀRXU ZDV PL[HG ZLWK J JOXWHQ IUHH ULFH ÀRXU 0L[WXUHV ZHUH shaken in an overhead shaker for at least 1 h. Chocolate cake represented a product that had been moderately heat-treated, but with typical chocolate components that are known to be challenging for ELISA tests. Gluten-free chocolate 7DEOH 6LQJOH ODERUDWRU\ YDOLGDWLRQ GDWD RQ UHSHDWDELOLW\ XVLQJ D VLQJOH NLW UHSOLFDWHV RI WKH VWDQGDUG FXUYH ZHUH UXQ XVLQJ D VLQJOH $JUD4XDQW *OXWHQ * WHVW NLW 0HDQ 2' YDOXHV 6' DQG &9 DUH VKRZQ EHORZ $OO &9 YDOXHV IRU LQWUD DVVD\ DQDO\VLV ZHUH OHVV WKDQ PHHWLQJ WKH

PDQXIDFWXUHU¶V 4& FULWHULD Standard, mg/kg Mean (OD)

SD (OD)

CV, % (OD)

0 4

0.138 0.359 0.698 1.340 1.877

0.018 0.035 0.058 0.073 0.109

12.80

9.88 8.34 5.43 5.82

20 80

200

H ALBMAYR -J ECH ET AL .: J OURNAL OF AOAC I NTERNATIONAL V OL . 98, N O . 1, 2015 105

bread) was produced as described, except that gluten-containing VWRFN ULFH ÀRXU PJ JOXWHQ NJ see above) was used. Gluten-containing crisp bread samples for the study were prepared as follows: 10 mg/kg: 17.5 g stock crisp bread was mixed with 332.5 g gluten-free crisp bread; 20 mg/kg, 35 g stock crisp bread was mixed with 315 g gluten-free crisp bread; and 100 mg/kg, 175 g stock crisp bread was mixed with 175 g gluten- free crisp bread. Mixtures were shaken in an overhead shaker for at least 1 h. The analyses of homogeneity ( see below) revealed that the gluten-free crisp bread was contaminated with gluten at a very low concentration of about 4.5 mg gluten/kg. This may have happened during production of the crisp breads, in particular during the grinding and sifting steps. Therefore, the target gluten concentrations of the crisp bread samples (0, 10, 20, and 100 mg/kg) were corrected to the gluten concentrations that were in fact present (4.5, 15, 24, and 102 mg/kg). All samples were checked for homogeneity before they were packaged in airtight bottles and accepted for the collaborative study. This was done by taking 10 representative 1 g aliquots from each bulk sample and then analyzing by the G12 Sandwich ELISA. Ideally, the CV of the 10 determinations should be 15% or less. Most samples with gluten concentration above 4 mg/kg complied with this, except the chocolate cake samples containing D ORZ FRQFHQWUDWLRQ ” PJ NJ RI LQFXUUHG JOXWHQ VKRZHG D CV of 21%. This was considered allowable for a sample like chocolate cake containing below 20 mg/kg gluten, because in an earlier study a beer (>20 mg/kg) and a starch syrup (<20 mg /kg) sample were accepted with a CV of 18–22% (7, 8). Homogeneity of Samples

cake was prepared by mixing one bag (425 g) of gluten-free cake mix (Betty Crocker Gluten-Free Cake Mix, General Mills) with 237 mL water, 112 g baking fat (Sanella, Unilever, Hamburg, Germany), and three eggs with a hand mixer at high speed for 5 min. The mass was poured into a round baking tin [diameter (Ø) = 25 cm] and baked in an oven at 170°C for 45 min. The cake was subjected to cooling for 1 h, sliced with a knife, and air-dried at room temperature (22°C) overnight (16 h). The air- dried cake was then lyophilized and ground with a household grinder (Model 836.820 1, Privileg, Fürth, Germany). Chocolate cake with a gluten concentration of 200 mg/kg (stock chocolate cake) was produced as described, except that cake mix containing ZKHDW ÀRXU FY *HQLXV ZDV XVHG 7KH DPRXQW RI ZKHDW ÀRXU LQ WKH FDNH PL[ PJ JOXWHQ NJ ZDV DGMXVWHG WR SURYLGH D ¿QDO gluten concentration of 200 mg/kg in the chocolate cake. Gluten-containing chocolate cake samples for the study were prepared as follows: 10 mg/kg, 25 g stock chocolate cake was mixed with 475 g gluten-free chocolate cake; 20 mg/kg, 50 g stock chocolate cake was mixed with 450 g gluten-free chocolate cake; and 100 mg/kg, 250 g stock chocolate cake was mixed with 250 g gluten-free chocolate cake. Mixtures were shaken in an overhead shaker for at least 1 h. The rice-based crisp bread represented a more heavily heat-treated sample. Gluten-free crisp bread was prepared by PL[LQJ J RI JOXWHQ IUHH ULFH ÀRXU see above) and 2.7 g NaCl with 270 mL of ice-cold water using a hand mixer at high speed (air incorporation). The mass was distributed in two round baking tins (25 cm diameter) to yield a dough layer of approximately 1 cm. The dough surface was perforated with a needle, and the dough was baked at 230°C for 30 min, then turned upside down and baked for another 30 min. After cooling overnight, the bread ZDV O\RSKLOL]HG DQG JURXQG WR D ¿QH SRZGHU XVLQJ D PRUWDU DQG pestle. Crisp bread containing 200 mg gluten/kg (stock crisp $JUD4XDQW *OXWHQ * DVVD\V FRQWDLQLQJ DOO WKH VWDQGDUGV ZHUH UXQ 0HDQ 2' YDOXHV 6' DQG &9 DUH VKRZQ EHORZ $OO &9 YDOXHV IRU LQWHUDVVD\ DQDO\VLV ZHUH OHVV WKDQ PHHWLQJ WKH PDQXIDFWXUHU¶V 4& FULWHULD Standard, mg/kg Mean (OD) SD (OD) CV, % 0 0.12 0.01 10.79 4 0.28 0.04 14.26 20 0.67 0.05 7.89 80 1.29 0.13 10.33 200 2.00 0.17 8.70 7DEOH 6LQJOH ODERUDWRU\ YDOLGDWLRQ GDWD RQ UHSHDWDELOLW\ XVLQJ GLIIHUHQW NLWV RI WKH VDPH EDWFK LQGLYLGXDO

Shipment

Two independent blinded replicates for each sample were provided to the participating laboratories. The coded sample vials contained 1 g of sample. Samples were shipped together with ELISA kits, instructions, and result sheet to participating laboratories.

Analysis and Data Reporting

Participants were requested to follow the instructions and to extract each sample using the test kit’s standard procedure and to analyze in duplicate in one analytical run. If changes had been made to the analytical protocol, they had to be reported in the “comments” box of the result sheet. The samples were analyzed

7DEOH /RW WR ORW YDULDWLRQ UHSURGXFLELOLW\ WKUHH GLIIHUHQW NLW EDWFKHV RI WKH $JUD4XDQW *OXWHQ * WHVW NLW ZHUH UXQ *8 *8 DQG *8 0HDQ 2' YDOXHV 6' DQG &9 DUH VKRZQ EHORZ $OO &9 YDOXHV IRU LQWHUEDWFK DQDO\VLV ZHUH RU OHVV PHHWLQJ WKH PDQXIDFWXUHU¶V 4& FULWHULD Standard, mg/kg GU1001-1106 GU1002-1108 GU1003-1111 Mean (OD) SD (OD) CV, % 0 0.10 0.10 0.09 0.10 0.01 6.97 4 0.26 0.21 0.21 0.23 0.03 11.55 20 0.72 0.57 0.58 0.63 0.08 13.45 80 1.39 1.13 1.09 1.21 0.16 13.65 200 2.11 1.65 1.60 1.79 0.28 15.92

106 H ALBMAYR -J ECH ET AL . : J OURNAL OF AOAC I NTERNATIONAL V OL . 98, N O . 1, 2015

7DEOH 6DPSOHV IURP )$3$6 3UR¿FLHQF\ 7HVW ZHUH DQDO\]HG GXULQJ WKH VLQJOH ODERUDWRU\ YDOLGDWLRQ E\ 5RPHU /DEV XVLQJ WKH $JUD4XDQW *OXWHQ * WHVW NLW 7HVW PDWHULDOV IURP 5RXQG ZHUH FDNH PL[ WR EH DQDO\]HG IRU JOXWHQ 7HVW PDWHULDOV ZHUH SUHSDUHG XVLQJ D JOXWHQ DQG ZKHDW IUHH FKRFRODWH FDNH PL[ WR ZKLFK D JOXWHQ DQG ZKHDW FRQWDLQLQJ FDNH PL[ ZDV DGGHG 7HVW PDWHULDOV IURP 5RXQG ZHUH SUHSDUHG E\ PL[LQJ LQIDQW VR\D IRUPXOD ZLWK ZKHDW ÀRXU 7HVW PDWHULDOV IURP 5RXQG ZHUH SUHSDUHG E\ FRPELQLQJ FDNH PL[ ZLWK ZKHDW ÀRXU $QDO\VLV RI WKH )$3$6 DQG SUR¿FLHQF\ VDPSOHV XVLQJ WKH $JUD4XDQW *OXWHQ * WHVW NLW SURGXFHG YHU\ VLPLODU UHVXOWV WR WKRVH DVVLJQHG YDOXHV IRU WKH 5 0HQGH] PHWKRG GDWD IURP WKH 5 %LRSKDUP NLW 7KH 5 0HQGH] PHWKRG LV FXUUHQWO\ WKH &RGH[ 7\SH , DSSURYHG PHWKRG IRU JOXWHQ DQDO\VLV Assigned value AgraQuant Gluten G12 test kit, mg/kg gluten R5 ELISA–R-Biopharm R7001, mg/kg gluten Veratox ELISA–Neogen, mg/kg gluten FAPAS 2781 A <4 Negative Negative FAPAS 2781 B 22.6 27.4 42.6 FAPAS 2781 C 95.6 91.6 120.7 R5 ELISA–R-Biopharm R7001, mg/kg gluten AR5 ELISA–R-Biopharm R7002, mg/kg gluten FAPAS 2792 A 119.8 134.2 141.0 FAPAS 2792 B <4 Negative Negative R5 ELISA–R-Biopharm R7001, mg/kg gluten R5 ELISA–R-Biopharm R7002, mg/kg gluten Assigned value Ingenasa–R5 ELISA 30.GLU.K2, mg/kg gluten )HEUXDU\ -XQH DQG 2FWREHU

FAPAS 2795 A FAPAS 2795 B

51.3

58.5

43.4

71.4

<4

Negative

Negative

Negative

Caution : Wear protective gloves and safety glasses. The stop solution contains acid. Avoid contact with skin or eyes. ,I H[SRVHG ÀXVK ZLWK ZDWHU see Material Safety Data Sheet). The extraction solution contains chemicals which are harmful to health. Perform sample extraction under a chemical hood and avoid contact with skin. Dispose of all materials, containers, and devices appropriately after use. See Table 2014.03A for results of the interlaboratory study supporting acceptance of the method. A. Principle The method is based on an enzyme immunoassay format using a monoclonal G12 antibody that can determine gluten derived from wheat, rye, barley, and cross-bred varieties. The G12 antibody binds to the celiac toxic amino acid sequence

by each laboratory. All optical density (OD) values had to be recorded in a ready-to-use Excel sheet. The participants used the calculator, which was provided with the Excel sheet. The model ZDV D VLPSOH OLQHDU SRLQW WR SRLQW FDOFXODWLRQ 7KH ¿QDO GDWD IURP the laboratories were sent to the study coordinator. A statistical evaluation was performed according toAOAC guidelines (9, 10).

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$SSOLFDEOH IRU GHWHUPLQDWLRQ RI JOXWHQ LQ ULFH ÀRXU DQG rice-based unprocessed and processed foods as evaluated in the multilaboratory study.)

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Parameter

Symbol

1

2

3

4

5

6

7

8

9

10 11 12

Total No. laboratories

P 17 18 18 18 16 18 18 16 17 18 18 18

Total No. replicates

Sum [n(L)]

34 36 36 36 32 36 36 32 34 36 36 36

Overall mean of all data (grand mean; mg/kg) Repeatability SD, mg/kg

xbarbar

1.6 13.5 26.2 101.2 0.1 6.2 13.1 63.5 4.1 14.9 26.6 112.7

s r 0.8 2.5 8.1 14.8 1.2 1.2 1.3 5.1 1.9 1.5 4.3 20.4 s R 1.9 4.0 11.6 31.8 1.2 1.8 2.5 13.5 2.8 4.5 8.9 33.2 48.2 18.5 30.7 14.7 2348 19.2 10.2 8.0 46.2 10.4 16.2 18.1

Reproducibility SD, mg/kg

Repeatability RSD, %

RSD r RSD R

115.8 29.6 44.2 31.4 2348 28.3 19.1 21.2 69.0 30.3 33.6 29.4

Reproducibility RSD, %

Bias (mg/kg) observed-nominal

1.6 3.5 6.2 1.2 0.1 –3.8 –6.9 –36.5 –0.4 –0.1 2.6 10.7

Recovery, % = observed/nominal × 100

135.0 131.0 101.2

62.0 65.5 63.5 91.1 99.3 110.8 110.5

a

*OXWHQ IUHH ULFH ÀRXU ULFH ÀRXU PJ JOXWHQ NJ JOXWHQ IUHH FKRFRODWH FDNH 6 = chocolate cake 10 mg gluten/kg; 7 = chocolate cake 20 mg gluten/kg; 8 = chocolate cake 100 mg gluten/kg; 9 = crisp bread 4.5 mg gluten/kg; 10 = crisp bread 15 mg gluten/kg; 11 = crisp bread 24 mg gluten/kg; and 12 = crisp bread 102 mg gluten/kg. PJ JOXWHQ NJ ULFH ÀRXU PJ JOXWHQ NJ ULFH ÀRXU