AOAC-RI ERP Book - Gluten.pdf
AOAC Official Methods of Analysis SM (OMA)
AOAC EXPERT REVIEW PANEL FOR GLUTEN
WEDNESDAY, MARCH 15, 2017 4:30PM - 6:30PM
Gaithersburg Marriott Washingtonian Center 9751 Washingtonian Boulevard Gaithersburg, MD 20878 USA
AOAC Official Methods of Analysis SM (OMA) Expert Review Panel for Gluten Assays
TABLE OF CONTENTS A. ABOUT AOAC OFFICIAL METHODS OF ANALYSIS SM .......................................................................3 B. AGENDA ......................................................................................................................................7 C. EXPERT REVIEW PANEL ROSTER ...................................................................................................9 D. AOAC INTERNATIONAL VOLUNTEER CONFLICT OF INTEREST, STATEMENT OF POLICY...................11 E. AOAC INTERNATIONAL ANTITRUST POLICY STATEMENT AND GUIDELINES ...................................13 F. AOAC INTERNATIONAL POLICY ON THE USE OF THE ASSOCIATION NAME, INITIALS, IDENTIFYING INSIGNIA, LETTERHEAD, AND BUSINESS CARDS...........................................................................17 G. MEETING AND METHOD REVIEW INFORMATION ........................................................................21 H. DISCUSS FINAL ACTION REQUIREMENTS FOR FIRST ACTION OFFICIAL METHODS [PRESENTATION]... I. AOAC OFFICIAL METHOD 2014.03:GLUTEN IN RICE FLOUR AND RICE-BASED FOOD PRODUCTS G12 SANDWICH ELISA, FIRST ACTION 2014 ........................................................................................23 I. METHOD FEEDBACK..........................................................................................................27 II. ARTICLE: GLUTEN IN RICE FLOUR AND BAKED RICE PRODUCTS BY G12 SANDWICH ELISA: FIRST ACTION 2014.03 ......................................................................................................37 iii. EXPERT REVIEW PANEL REPORT (MARCH, 2014) ...............................................................47 J. OMAMAN-34: AOAC PERFORMANCE TESTED METHODS SM VALIDATION REPORT #061201, SINGLE LABORATORY VALIDATION I. OMAMAN-34 A: GENERAL METHOD INFORMATION...........................................................51 II. OMAMAN-34 B: VALIDATION STUDY OF THE VERATOX R5 RAPID ELISA FOR DETECTION OF GLIADIN (PTM #061201)....................................................................................................59 III. OMAMAN-34 C: METHOD USER GUIDE/INSTRUCTIONS FOR USE........................................71 K. MODIFICATION OF OMA 2012.01: GLIADIN AS A MEASURE OF GLUTEN IN RICE AND CORN BASED FOODS I. METHOD MODIFICATION APPLICATION ............................................................................83 II. OMA 2012.01 REVISED .....................................................................................................89 III. METHOD PERFORMANCE DATA FOR MODIFICATION .........................................................93
EXPERT REVIEW PANEL (ERP) FOR GLUTEN ASSAYS
Gaithersburg Marriott Washingtonian Center 9751 Washingtonian Boulevard, Gaithersburg, MD 20878 USA Wednesday, March 15, 2017 4:30pm – 6:30pm . MEETING AGENDA
Expert Review Panel Chair Dr. Terry Koerner
I.
Welcome and Introductions Expert Review Panel Chair
II. Review of AOAC Volunteer Policies & Expert Review Panel Process Overview and Guidelines Deborah McKenzie, Senior Director, Standards Development and Method Approval Processes, AOAC INTERNATIONAL and AOAC Research Institute III. Discuss Final Action Requirements for First Action Official Methods (if applicable) ERP will discuss, review and track First Action methods for 2 years after adoption, review any additional information (i.e., additional collaborative study data, proficiency testing, and other feedback) and make recommendations to the Official Methods Board regarding Final Action status. 1) 2014.03 Gluten in Rice Flour and Rice-Based Food Products IV. Review of Methods - Discussion of Reviewing Single Laboratory Validations vs Collaborative Study Validations The ERP will discuss which types of methods will be reviewed and validated through the ERP, after which the ERP will render a decision on the subject matter. 1) OMAMAN-34: Validation Study of the Veratox R5 Rapid ELISA for Detection of Gliadin Study Director: Ryan Viator, Neogen Corporation, 620 Lesher Place, Lansing, MI 48912 2) Modification of OMA 2012.01: Gliadin as a Measure of Gluten in Rice and Corn Based Foods Study Director: Markus Lacorn, R-Biopharm AG, An der neuenBergstrasse 17, 64297 Darmstadt Germany
V.
Next Steps and Upcoming Meetings
VI.
Adjournment
**Agenda is subject to change. V1
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AOAC Research Institute ▪ 2275 Research Blvd, Suite 300 ▪ Rockville, Maryland 20850 Phone: 301-924-7077
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E\ DQ ,QGLYLGXDO 0HPEHU RU 6XVWDLQLQJ 0HPEHU RI WKH $VVRFLDWLRQ DQG WKH PLVXVH FRQWLQXHV DIWHU QRWLILFDWLRQ WKH %RDUG RI 'LUHFWRUV ZLOO WDNH DSSURSULDWH DFWLRQ ,I FRQWLQXHG PLVXVH LV E\ D QRQPHPEHU RI WKH $VVRFLDWLRQ RU LI D PHPEHU FRQWLQXHV PLVXVH LQ VSLWH RI QRWLILFDWLRQ DQG %RDUG DFWLRQ XOWLPDWHO\ WKH $VVRFLDWLRQ ZLOO WDNH OHJDO DFWLRQ WR SURWHFW LWV SURSHUW\ OHJDO LQWHJULW\ UHSXWDWLRQ DQG LPDJH $GRSWHG E\ WKH $2$& %RDUG RI 'LUHFWRUV 6HSWHPEHU 5HYLVHG -XQH )HEUXDU\ 0DUFK 2FWREHU Official Methods of Analysis SM (OMA) Expert Review Panel MEETING AND METHOD REVIEW GUIDANCE The AOAC Research Institute administers AOAC INTERNATIONAL's premier methods program, the AOAC Official Methods of Analysis SM (OMA). The program evaluates chemistry, microbiology, and molecular biology methods. It also evaluates traditional benchtop methods, instrumental methods, and proprietary, commercial, and/or alternative methods and relies on gathering the experts to develop voluntary consensus standards, followed by collective expert judgment of methods using the adopted standards. The Official Methods of Analysis of AOAC INTERNATIONAL is deemed to be highly credible and defensible. All Expert Review Panel (ERP) members are vetted by the AOAC Official Methods Board (OMB) and serve at the pleasure of the President of AOAC INTERNATIONAL. In accordance to the AOAC Expert Review Panel Member and Chair Volunteer Role Description all Expert Review Panel members are expected to 1) serve with the highest integrity, 2) perform duties and method reviews, and 3) adhere to review timelines and deadlines. To assist the ERP Chair and its members, please note the following in preparation for Expert Review Panel meetings and method reviews. Pre-Meeting Requirements 1. Confirm availability and plan to be present to ensure a quorum of the ERP. (Please refer to page 25, Quorum Guidelines, Expert Review Panel Information Packet ) 2. Ensure that your laptop, CPU or mobile device can access online web documentation. 3. Be prepared for the meeting by reviewing all relevant meeting materials and method documentation. In-Person Meeting and Teleconference Conduct 1. Arrive on time. 2. Advise the Chair and ERP members of any potential Conflicts of Interest at the beginning of the meeting. 3. Participation is required from all members of the ERP. All members have been deemed experts in the specific subject matter areas. 4. The ERP Chair will moderate the meeting to ensure that decisions can be made in a timely manner. 5. Follow Robert’s Rules of Order for Motions. 6. Speak loud, clear, and concise so that all members may hear and understand your point of view. 7. Due to the openness of our meetings, it is imperative that all members communicate in a respectful manner and tone. 8. Refrain from disruptive behavior. Always allow one member to speak at a time. Please do not interrupt. 9. Please note that all methods reviewed and decisions made during the Expert Review Panel process are considered confidential and should not be discussed unless during an Expert Review Panel meeting to ensure transparency. Reviewing Methods Prior to the Expert Review Panel meeting, ERP members are required to conduct method reviews. All methods are reviewed under the following criteria, technical evaluation, general comments, editorial criteria, and recommendation status. These methods are being reviewed against their collaborative study protocols as provided in the supplemental documentation. Note: The method author(s) will be present during the Expert Review Panel session to answer any questions. Page 1 of 2 Version 1 – OMA ERP Meeting Conduct Official Methods of Analysis SM (OMA) Expert Review Panel MEETING AND METHOD REVIEW GUIDANCE Reviewing Methods (Cont’d) x Reviewers shall conduct in-depth review of method and any supporting information. x In-depth reviews are completed electronically via the method review form. The method review form must be completed and submitted by the deadline date as provided. x All reviews will be discussed during the Expert Review Panel meeting. x Any ERP member can make the motion to adopt or not to adopt the method. x If the method is adopted for AOAC First Action status, Expert Review Panel members must track and present feedback on assigned First Action Official Methods . x Recommend additional feedback or information for Final Action consideratio n. Here are some questions to consider during your review based on your scientific judgment: 1. Does the method sufficiently follow the collaborative study protocol? 2. Is the method scientifically sound and can be followed? 3. What are the strengths and weaknesses of the method? 4. How do the weaknesses weigh in your recommendation for the method? 5. Will the method serve the community that will use the method? 6. What additional information may be needed to further support the method? 7. Can this method be considered for AOAC First Action OMA status? Reaching Consensus during Expert Review Panel Meeting 1. Make your Motion. 2. Allow another member to Second the Motion. 3. The Chair will state the motion and offer the ERP an option to discuss the motion. 4. The Chair will call a vote once deliberations are complete. 5. Methods must be adopted by unanimous decision of ERP on first ballot, if not unanimous, negative votes must delineate scientific reasons. Negative voter(s) can be overridden by 2/3 of voting ERP members after due consideration. 6. All other motions will require 2/3 majority for vote to carry. Page 2 of 2 Version 1 – OMA ERP Meeting Conduct 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 ± & ± ) 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 $2$& 2I¿FLDO 0HWKRG *OXWHQ LQ 5LFH )ORXU DQG 5LFH %DVHG )RRG 3URGXFWV * 6DQGZLFK (/,6$ )LUVW $FWLRQ $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 7DEOH $ 3HUIRUPDQFH VWDWLVWLFV IRU RYHUDOO * VDQGZLFK (/,6$ UHVXOWV 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 a © 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 ± & ± ) DQG OHW DOO FRPSRQHQWV HTXLOLEUDWH WR ± & ± ) 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 & ( 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 & ( 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 %UDFKDQ <4 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 $PDUDQWK © 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 ± & ± ) 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 & ) 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 ± & ± ) ZLWK D URWDU\ VKDNHU $IWHU DERXW PLQ LQ WKH rotator, check the vials visually if all sample material has suspended in the liquid. 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" ! / )" ! * + ,- . / ) 4 * 1 5 6 ) 0 ! ) 1 2 # ) # , # !7 8 ! % + 7 9 0! 34 . !7 1 !: 7 0! 7 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
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