AOAC ERP Gluten Assays - Dec 2018




The Official Methods of Analysis SM (OMA) program is AOAC INTERNATIONAL's premier methods program. The program evaluates chemistry, microbiology, and molecular biology methods. It also evaluates traditional benchtop methods, instrumental methods, and proprietary, commercial, and/or alternative methods. In 2011, AOAC augmented the Official Methods SM program by including an approach to First Action Official Methods SM status that relies on gathering the experts to develop voluntary consensus standards, followed by collective expert judgment of methods using the adopted standards. All methods in the AOAC Official Methods SM program are now reviewed by Expert Review Panels for First Action AOAC Official Methods of Analysis SM status, continuance, repeal, and/or to recommend for AOAC Final Action Official Methods status. The OMA program has undergone a series of transitions in support of AOAC's collaborations, evolving technology, and evolving technical requirements. Methods approved in this program have undergone rigorous scientific and systematic scrutiny such that analytical results by methods in the Official Methods of Analysis of AOAC INTERNATIONAL are deemed to be highly credible and defensible. The methods are published in the Official Methods of Analysis of AOAC INTERNATIONAL and supporting manuscripts are published in the Journal of AOAC INTERNATIONAL . AOAC Official Methods SM program allows for submissions for all proprietary, single and sole source methods. Methods submitted through the PTM-OMA harmonized process also will be reviewed through the O fficial Methods of Analysis SM (OMA) program. Other complementary products and services include expanded consulting services for validation protocol development and AOAC INTERNATIONAL Organizational Affiliate Membership.

AOAC INTERNATIONAL 2275 Research Blvd, Suite 300 Rockville, Maryland 20850 Phone: (301) 924-7077








REVIEW OF METHODS FOR AOAC FIRST ACTION OFFICAL METHODS STATUS For each method, the ERP members will present a review of the proposed collaborative study manuscript, after which the ERP will discuss the method and render a decision on the status for each method. 1) OMAMAN-47: Quantification of Wheat, Rye, and Barley Gluten in Oat and Oats Products by ELISA RIDASCREEN® Total Gluten Study Director: Patricia Meinhardt, R-Biopharm Inc, 870 Vossbrink Dr., Washington, MO, 63090 USA


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.





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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.

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Version 1 – OMA ERP Meeting Conduct

Official Methods of Analysis SM (OMA) Expert Review Panel MEETING AND METHOD REVIEW GUIDANCE

Reviewing Methods (Cont’d)

Reviewers shall conduct in-depth review of method and any supporting information. 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. All reviews will be discussed during the Expert Review Panel meeting. Any ERP member can make the motion to adopt or not to adopt the method. 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 . 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.

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Version 1 – OMA ERP Meeting Conduct


AOAC Expert Review Panels An Orientation

Deborah McKenzie רב Sr. Dir., Standards Development AOAC INTERNATIONAL Staff Liaison ‐ Official Methods Board



As a



members, organizations, and experts dedicated to developing and validating and of


Analytical  Excellence

AOAC  Strategic  Goals

Core  Programs




Accessible at AOAC homepage

Analytical Excellence addresses emerging issues and influence standards development as a global leader in analytical excellence

Standards Development

Official Methods of Analysis SM (OMA) & Performance Tested Methods SM (PTM)

Laboratory Proficiency Testing & Quality Management

Analytical Excellence




AOAC Method Approval Programs

Official Methods of Analysis SM (OMA)   • AOAC’s premiere methods  program • Approved methods  – published in the Official Methods  of Analysis of AOAC  INTERNATIONAL  (print and  online) – Manuscripts published in the  Journal of AOAC INTERNATIONAL – First Action and Final Action  status

Performance Tested Methods SM (PTM)  • AOAC’s method certification  program • Certified methods – Commercial/proprietary rapid  methods (test kits) – Certifications published on AOAC  website – Manuscripts published in the Journal  of AOAC INTERNATIONAL – Method developers licensed to use  certification mark – Annual review & recertification

AOAC Official Methods SM Program

Submit Methods Responding to issued Call for Methods • Adoption of methods as Official Methods  is contingent upon  standards development activities • No application fee required to submit methods in response to Call  for Methods Submit Individual & Sole Source Methods • Adoption of methods as Official Methods  is contingent upon data  supporting applicability and community based validation guidance  information • Including proprietary/commercial methods and harmonized PTM – OMA methods • Application fee required



Status of Official Methods of Analysis First Action, Final Action, Repeal

AOAC Policies & Procedures

Policy on Use of  Association Name,  Identifying Insignia,  Letterhead, Business  Cards

Policy on Volunteer  Conflict of Interest

Policy on Antitrust

OMA Appendix G ‐ Use  of AOAC Voluntary  Consensus Standards to  Evaluate Characteristics  of a Method of Analysis

Expert Review Panel  Policies and Procedures



Road to First Action OMA Status

1. PTM – OMA Methods 2. Other Sole Source Methods 3. Response to Call for Methods

Method submitted 

Expert Review Panels  review all methods  submitted methods

Notify  Method  author 




Published First  Action OMA

Road to Final Action OMA  Status

Method reproducibility must be  demonstrated before Final Action  consideration. 

ERP determines if sufficient  evidence merits a  recommendation for Final Action  status or repeal. • Only the OMB promotes a  method to “Final Action” status or   repeal the method. • Methods that did not meet the  bar would be repealed. • Same for all method submissions



PTM Overview for PTM‐OMA  Harmonized Process • Administered by the Research  Institute in 2003. • Well established and streamlined • Original approved by consensus  with the OAs, OMB, RI Board of  Directors and AOAC  INTERNATIONAL Board of  Directors. • ERP may be formed during  Consulting Service. • Criterion for OMA:  manufacturer’s method claims.

Recruiting Experts and Methods  • AOAC issues  – Call for Methods  (Stakeholder affiliated methods) – Call for Experts  • Sole Source/Individual Method Submissions  – Individually completed Application not associated  with an open Call for Methods



Qualifications for ERP Membership Candidate must meet one of the following: • Demonstrated knowledge in the appropriate scientific  disciplines. • Demonstrated knowledge regarding data relevant to  adequate method performance.

• Demonstrated knowledge of practical application of  analytical methods to bona fide diagnostic requirements.

Candidate application package includes: • Statement of Expertise • Current Abridged CV or Resume

ERP Member Vetting Process

Approved roster  sent to AOAC  President for  volunteer  appointment

Candidate  submits  application  package

Reviewed by  AOAC staff with  recommendation  to OMB

Reviewed by  OMB and roster  approved

• All members serve at the pleasure of the AOAC President • OMB assigns a representative to serve as a resource for every ERP



Candidate Method Assignments  A minimum of primary and secondary reviewers may be assigned  to every method.  In depth review via review form  Prepare to attend and speak on the method and make a recommendation  for ERP discussion and consideration.  Review forms are completed and returned to AOAC staff in advance  of the  meeting.  An email is sent with information on how to access the  candidate methods and how to submit reviews

 Members of both Committee on Safety and Committee on  Statistics serve as  advisory resources for all ERPs

Candidate Method Reviews

 In your judgment, does the method sufficiently meet the Standard Method   Performance Requirements (SMPR) or community‐based guidance?

 In your judgment, is the method scientifically sound and can be followed?  In your judgment, what are the strengths and weaknesses of the method?  In your judgment, how do the weaknesses weigh in your recommendation for   the method?  In your judgment, will the method serve well the stakeholder community that   will use the method?  In your judgment, what additional information may be needed to further   support the method meeting the SMPR or community‐based guidance?  Members of both Committee on Safety and Committee on Statistics serve  as  advisory resources for all ERPs



ERP Meetings  ERPs can meet in person at a minimum of twice a year and up to four times  per year*:  AOAC Mid‐Year meeting  (DC metro area)  AOAC Annual Meeting.  *2 additional designated times for proprietary method Organziational Affiliates  At the ERP meeting:  Reviews will be presented and the reviewers can make a motion to the ERP  whether to adopt the method as First Action OMA.  ERP discusses the method.  ERP renders a decision on First Action status.  ERP renders decisions on modifications to Official Methods .  If the method is adopted  ERP decides on what additional information is needed to recommend the  method for Final Action status

ERP Teleconferences • Only after the initial in‐person ERP meeting  for First Action consideration of methods • Possible for some method modifications • Possible for First Action to Final Action ERP  recommendations



ERP Meetings


Presence of 7  vetted ERP  members 

Presence of  2/3 vetted  ERP members



Method Review Overview

 Method authors may be invited to make a presentation on their method  REVIEWERS PRESENT THEIR REVIEWS AND MAY  INITIATE A MOTION TO ADOPT THE  METHOD IF THEY CHOOSE  Chair recognizes each reviewer  Reviews are presented.

 If in favor, reviewers may make and second a motion to adopt  adopt the method  Chair can then entertain discussion on themethod  Chair can call for a vote once deliberation is complete



Consensus – First Action Adoption

 First Action Official Methods status is granted:

 Method must be adopted by unanimous decision of ERP on first   ballot, if not unanimous, negative votes must be based on scientific reasons.

 Negative voter(s) can be overridden by 2/3 of voting ERP   members after due consideration.

 Method becomes First Action on the date when ERP decision is   made.

Consensus – First Action to Final Action

 The ERP may then reach consensus on any additional   information that it needs to review to be able to make a   recommendation for Final Action Official Methods   status.

 This is a separate motion.



ERP Meetings – Review for First Action  METHOD AUTHOR:    present any method and any resulting changes to  the method since submission for review, summary of SLV and/or  reproducibility evaluation, any recognitions (from AOAC or external)  and, final draft of method proposed for decision

ERP CHAIR & MEMBERS:    present reviews and discuss any resulting  issues or questions on the method, review and agree upon final draft of  method proposed for decision, and chair calls for ERP decision in  accordance to procedures.

CONSENSUS:   Method 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 non‐ negative voting ERP members after due consideration.    Abstentions do not count towards vote; in case of multiple abstentions the results  will need to be evaluated.  Staff will monitor  and record consensus voting.

STAFF:   Will organize and coordinate meeting,  record  ERP  actions and decisions, draft ERP report and distribute after  chair approval,  work with chair and OMB liaison to complete  checklist and assemble recommendation package  for OMB.

ERP Methods Review & Approval

Methods should be scientifically sound with demonstrating  that it will meet the needs of those using the method  (evidenced by meeting the standard, or other acceptance  criteria) 

ERPs have approved methods with evidence of high potential  to First Action and request additional work or support be  submitted for review prior to ERP convening to recommend an  action to OMB

OMB requires a justification or rationale for methods that are  deemed acceptable and adopted but may not fully meet the  standard set or acceptance criteria.



OMB Expectations for First Action

Safety review needed  prior to First Action  status

SLV type of supporting  information available  per the SMPR

• Applicability, Method Performance Requirements  Table, System Suitability, Reference Materials, and  Validation Guidance

• Documented method performance versus a SMPR • Document reasons for acceptability if method  does not meet the SMPR

Comparison to SMPR

Any approved  method(s) along with  supporting  manuscript(s) and   documentation sent to  AOAC Publications  after the meeting.

Method incorporating ERP revisions (preferably  in AOAC Format) Method Manuscript incorporating specified ERP  revisions (in AOAC  Format) Signed AOAC Copyright Authorization form


Publication  of First  Action Methods

Method and method manuscript prepared for  publication  in the Official Methods of  Analysis of AOAC  INTERNATIONAL and in  Journal of AOAC INTERNATIONAL Updates on methods approved or status  changes are  published in the Inside  Laboratory Management magazine  and on  the AOAC website



ERP Meetings – Method Tracking METHOD AUTHOR:    present any method feedback obtained  and any resulting changes to the method, any reproducibility  information, any implemented ERP recommendations, final  draft of method proposed for decision ERP MEMBERS:    present any method feedback obtained and 

discuss any resulting changes to the method, any  reproducibility information, any implemented ERP  recommendations, review and agree upon final draft of  method proposed for decision, and make a recommendation  to OMB. CONSENSUS:    2/3 vote in favor of a motion.    Abstentions do not count towards vote; in case of  multiple abstentions.  Staff will monitor  and record  consensus voting.

STAFF:   Will organize and coordinate meeting,  record   ERP actions and decisions, draft ERP report and  distribute after chair approval,  work with chair and  OMB liaison to complete checklist and assemble  recommendation package  for OMB.

Documentation Needed

Method Safety Evaluation

Reference Materials

Evidence of Single Laboratory Validation or equivalent 

Evidence of Reproducibility Assessment 

Published First Action OMA

Method Performance versus SMPR or acceptance criteria

Final draft of First Action OMA to be considered for status update

Rationale or Justification for Repeal or Continuance of First Action OMA



OMB Meeting for Review of ERP  Recommendations

OMB Review (renders decision on  recommendation) 

ERP Chair/or  designee  (addresses  questions/comment)

OMB Liaison (presents  recommendation)

Modifications to Official Methods • Types of Modifications – Editorial

– Major – Minor

• Applicable to First Action and Final Action  OMA

• Relevant to all ERPs



Editorial Modifications • The applicant must submit a written explanation of  the change(s) including a statement that the  modification does not alter the validated  performance of the method.

• Examples include: Typos or editorial corrections or  clarifications that strengthen instruction.

• Methods that have undergone an editorial  modification will retain the same number. 

Editorial Changes

• Editorial changes to methods only require AOAC staff review and  the change is made to the OMA with changes noted in next printed  edition of OMA. • A list of the methods with editorial modifications will be published  in  Inside Laboratory Management and on  the Website.



Minor Modifications • Results in no changes to the current validated  performance. There is no significant effect to the  results. The method will retain the original number. • Supporting data to justify the proposed modification  must be submitted. Equivalency data is required unless  adequate Justification to exclude this data is provided. • Examples include: Reagent change, a change in a  column or consumables that do not impact the  validated method performance.

Major Modifications • Results in a change to the current validated  performance of the method.  • This level of modification will result in a new method  as part of AOAC standards development and will  receive a new method number. • Examples include: significant change to the  technology, sample preparation, or chemistry.



Minor & Major Modifications

Based on AOAC staff review, a public comment  period for the proposed modification is required.

Applicant Options

• Following the comment period, any comments are reconciled and  recommends a response to the applicant.  • The applicant can decide to proceed based on the reconciled comments



Pathways for Minor & Major  Modification • If applicant  decides to 

proceed, an ERP is  formed – Level of  modification  determined by ERP

– Applies to 

modifications of  First Action and  Final Action  methods

Documentation and Communication • AOAC carefully documents the actions of Stakeholder Panel, Working  Groups, and Expert Review Panels • AOAC will prepare summaries of the meetings  – Communicate summaries to the stakeholders – Publish summaries in the Referee section of AOAC’s  Inside  Laboratory Management • AOAC publishes its voluntary consensus standards and Official  Methods – Official Methods of Analysis of AOAC INTERNATIONAL – Journal of AOAC INTERNATIONAL • AOAC publishes the status of standards and methods in the Referee  section of AOAC’s  Inside Laboratory Management



Requirements for ERP Service

 Must have demonstrated expertise in the method, technology,   analyte/matrix, etc… Be a subject matter expert.  Must be able to attend ERP meetings  Must be able to complete assigned reviews on time  Must be prepared to speak on the method and share reviews   during the meeting  Must be proactive in tracking assigned First Action Official   Methods  Must be able to assist in peer reviewing paper for publication  Must sign and submit AOAC Volunteer Acceptance Form

General Expectations for ERPs • You can expect to have a minimum of three weeks to review  methods prior to ERP meeting.  – You are requested to submit written reviews by specified deadline.  Please  alert staff if you are not able to complete on time. – You may have individually assigned methods to review or all of the methods  to review.  Please be prepared to discuss these methods during meeting. – You may use the OMA appendices as guidance for types of validation work  that can be expected.  If additional information is needed, please ask staff. • ERP Meeting Quorum – If there is no quorum, there is no official meeting.  Please alert staff as early  as possible if you are not able to attend a meeting. • ERP Consensus – ERP consensus may not reflect your own personal view – There may be times when a method may not meet all of the criteria exactly;  however, the ERP can adopt the method.



Ethical Expectations of AOAC Expert  Review Panel Members • Respect for your peer ERP members and chair – Each member has been vetted for expertise relevant to the  review of the method(s) in the ERP  • Be considerate of each others perspectives and points of view • Be considerate of the ERP’s consensus even if you disagree – Inform staff as early as possible if you cannot attend the  scheduled ERP meeting • Be considerate in that your absence can impact the quorum of the  ERP and its ability to have an official meeting to make decisions – Notify staff and/or disclose in the ERP meeting if you have a  direct or perceived conflict of interest for a specific method • Please review AOAC’s policy on Volunteer Conflict of Interest Ethical Expectations of Expert Review Panel  Members  (con’t) • Respect for Method Authors and Intellectual Property – Each Method Author is encouraged to attend the ERP meeting – Each candidate methods (not yet adopted or published as Official  Methods of Analysis of AOAC INTERNATIONAL ) are still the intellectual  property of the method author.  Therefore, the information is shared only  with the vetted ERP members and is available during the meetings.  Please  do not distribute the information without expressed written permission  from an appropriate AOAC staff liaison.  – Be clear about and justify how additional recommended work is a  requirement for First Action, a requirement for Final Action consideration,  or something recommended, but not necessary. – Keep your focus on the science



ERP Chair Responsibilities

Before Meeting

During Meeting

Moderate discussions based  on agenda

Work with staff on meeting  coordination

Engage staff to encourage  members to reach decision  points

Review submitted and/or  assigned methods

Engage staff on procedural  questions

Review method reviews if  applicable

Engage discussion on feedback  mechanism

Review SMPR(s) and/or  relevant guidance and criteria

ERP Chair Responsibilities

Other Efforts and  Recognitions Can nominate methods for  OMB Award

After Meeting Review Meeting Report  and Approve Final Version

Can nominate ERP members  for OMB Award

Assist with any follow up on  methods

Can assist in identifying  methods for review

Assist in Publication  Reviews

Can serve as a guest editor for  the Journal



Roles and Responsibilities

AOAC Official Methods Board Vet and approve stakeholder panel chair & voting members Vet and approve ERP membership and AOAC Experts Render decisions on status of First Action methods (Final Action,  repeal, etc…) Assign a liaison to each stakeholder panel and ERP Coordinate OMB Awards AOAC Expert Review Panels Review methods and meet in person to render decisions on  methods for First Action Official Methods SM status. Track First Action Official Methods SM and modify, if necessary Recommend First Action methods after 2 years or less to OMB  for Final Action, continuance, or Repeal Participate in Consulting Service and PTM reviews for OMA and  harmonized PTM and harmonized OMA method studies AOAC Experts Review and approve PTM validationtesting protocol documentation Peer review of PTM validation manuscript and supporting  documentation AOAC Research Institute ‐ PTM Expert Reviewers Peer Review of PTM validationmanuscripts and supporting  documentation

AOAC Research Institute Independent Laboratories Conduct independent evaluation of candidate method using AOAC  approved testing protocols AOAC Stakeholder Panels Develop  voluntary consensus standards  Assign working groups to  draft standards method performance  requirements Voting members demonstrate  consensus on behalf of  stakeholders AOAC Staff Coordinate method reviews and method approval activities Coordinate OMB meetings Provide trainings and orientations Maintain website and communication Document and publish actions and decisions Coordinate standards development activities Publish standards and methods AOAC Research Institute Technical Consultants Draft validation protocols in Consulting Service for assigned methods

Facilitate PTM evaluation of assigned candidate methods Facilitate comments/responses for assigned OMA reviews


Thank you 


AOAC SMPR® 2017.021 (Revised 8-2018)

Reproducibility (ISO 5725-1). —Variation arising when identical test materials are analyzed in different laboratories by different operators on different instruments. The standard deviation or relative standard deviation calculated from among-laboratory data. Expressed as the reproducibility standard deviation (SD R ); or % reproducibility relative standard deviation (%RSD R ). 5 Method Performance Requirements See Table 1. 6 System Suitability See information on antibodies, cross reactivity, and calibrators in Appendix M. 7 Reference Material(s) Samples of oat flour spiked with wheat, rye, and barley for validation studies are available from USP. Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F : Guidelines for Standard Method Performance Requirements , Official Methods of Analysis of AOAC INTERNATIONAL (2016) 20th Ed., AOAC INTERNATIONAL, Rockville, MD, USA (http:// 8 Validation Guidance For all candidate methods, developers must: ( 1 ) Provide antibody information, cross reactivity data, and information on calibrators according to Appendix M ( 2 ) Wherever possible, identify peptide sequences or target epitopes for all antibodies used ( 3 ) Determine and submit estimates for recovery for each gluten source (wheat, rye, and barley) using the oat flour testing materials ( see Reference Material(s) section for source information) For all claimed matrices, developers must submit: ( 1 ) A sample processing procedure for homogenization, particle size reduction, and test portion size ( 2 ) Data and estimates for LOD and LOQ ( 3 ) Precision estimates (RSD r and/or RSD R ) Method developers may use spiked samples to determine the recovery performance of candidate methods for specific claimed matrices. Guidance on spiking for recovery evaluation are provided at: ( 1 ) Koerner et al. (2013) J. AOAC Int . 96 , 1033–1040. doi. org/10.5740/jaoacint.13-043 ( see section on Spiking Methods ) ( 2 ) Estimating Recovery of a Gluten ELISA Kit Method ( see supporting information following this SMPR) ( 3 ) Spiking Materials for Barley and Rye ( see supporting information following this SMPR) ( 4 ) Appendix D: Guidelines for Collaborative Study Procedures to Validate Characteristics of a Method of Analysis , Official Methods of Analysis of AOAC INTERNATIONAL (2016) 20th Ed., AOAC INTERNATIONAL, Rockville, MD, USA (http://www. ( 5 ) Appendix F: Guidelines for Standard Method Performance Requirements , Official Methods of Analysis of AOAC INTERNATIONAL (2016) 20th Ed., AOAC INTERNATIONAL, Rockville, MD, USA ( Approved by the International Stakeholder Panel on Alternative Methods (ISPAM) on September 24, 2017. Final Version Date: November 1, 2017. Revision Date: August 26, 2018.

Standard Method Performance Requirements (SMPRs®) for Quantitation of Wheat, Rye, and Barley Gluten in Oats

Intended Use: Quantitation of Gluten in the Context of Food Manufacturing 1 Purpose AOAC Standard Method Performance Requirements (SMPRs) describe the minimum recommended performance characteristics to be used during the evaluation of a method. The evaluation may be an on-site verification, a single-laboratory validation, or a multi-site laboratory collaborative study. SMPRs are written and adopted by AOAC stakeholder panels composed of representatives from industry, regulatory organizations, contract laboratories, test kit manufacturers, and academic institutions. AOAC SMPRs are used by AOAC expert review panels (ERPs) in their evaluation of validation study data for a method(s) being considered to determine if it meets the requirements for Performance Tested Methods SM or AOAC Official Methods of Analysis SM , and can be used as acceptance criteria for verification at user laboratories. 2 Applicability Quantitation of total wheat, rye, and barley gluten in groats, rolled oats, steel cut oats, oat flour, oat bran, and extruded/cooked/ finished oat products. 3 Analytical Technique Enzyme-linked immunosorbent assay (ELISA) or related binding-based technologies. 4  Definitions Enzyme-linked immunosorbent assay (ELISA) .—For the purposes of this document, ELISA is defined as “an analytical procedure characterized by the recognition and binding of specific antigens by antibodies” [Appendix M: Validation Procedures for Quantitative Food Allergen ELISA Methods: Community Guidance and Best Practices , Official Methods of Analysis of AOAC INTERNATIONAL (2016) 20th Ed., AOAC INTERNATIONAL, Rockville, MD, USA,; J. AOAC Int . 93 , 442–450(2010)]. This definition is not meant to be restrictive and encompasses other related binding-based technologies. Gluten .—Protein fraction from wheat, rye, barley, or their crossbred varieties and derivatives thereof, to which some persons are intolerant and that is insoluble in water and 0.5 M NaCl. Limit of detection (LOD; Appendix M) .—Lowest concentration or mass of analyte in a test sample that can be distinguished from a true blank sample at a specified probability level. Limit of quantitation (LOQ; AppendixM). —Lowest level of analyte in a test sample that can be quantified at a specified level of precision. Recovery .—The fraction or percentage of analyte that is recovered when the test sample is analyzed using the entire method. Repeatability (ISO 5725-1) .—Variation arising when all efforts are made to keep conditions constant by using the same instrument and operator (in the same laboratory) and repeating during a short time period. Expressed as the repeatability standard deviation (SD r ); or % repeatability relative standard deviation (%RSD r ).


Table 1. Method performance requirements Parameter

Acceptance criteria

Analytical range, ppm

≤5 to ≥15

LOQ, ppm LOD, ppm

≤5 ≤5

Recovery, % a 50 to 200 b a  For validation purposes, individually measured as gluten from wheat, rye, and barley spiked individually in the prepared oat flour test samples, calculated from the slope of the dose response curve.  A sample series shall consist of one sample of unspiked oat flour; two samples spiked with wheat; two samples spiked with rye; and two samples spiked with barley.  Gluten content for wheat, rye, and barley used for spiking will be estimated as follows: See Estimating Recovery of a Gluten ELISA Kit Method for details on procedures for spiking the flour samples ( see supporting information following this SMPR). Sample providers shall develop an SOP for producing, storing, and shipping the materials. b  Criteria for recovery are larger than traditionally used for SMPRs. First, method results for gluten in oats are highly variable due to sample inhomogeneity. This lack of homogeneity may result in a wider range of recovery estimates than would normally be expected at ppm levels. Second, the different relative specificities of the antibodies against wheat, rye, and barley make balancing the response difficult. [ Note : The AOAC Working Group on Gluten determined not to set standards for repeatability (RSD r ) or reproducibility (RSD R ) because of the inhomogeneity issue discussed above about the recovery range. Method developers are directed to determine RSD r using standard oat flour samples.]  Wheat: Gluten = (Dumas nitrogen content) x 5.68 x 0.80  Rye: Gluten = (Dumas nitrogen content) x 5.68 x 0.60  Barley: Gluten = (Dumas nitrogen content) x 5.68 x 0.60


AOAC Official Method ####.##

Quantification of Wheat, Rye, and Barley Gluten in Oat and Oats Products

by ELISA RIDASCREEN® Total Gluten: Collaborative Study, First Action ####.##

Authors: Markus Lacorn a , Thomas Weiss a , Paul Wehling b , Mark Arlinghaus b and Katharina Scherf c a R-Biopharm AG, An der neuen Bergstraße 17, 64297 Darmstadt, Germany Phone: +49 6151 8102 464; Fax: ext-40; Email: b Medallion Labs, 9000 Plymouth Avenue North, Minneapolis, MN 55427; USA c Leibniz-Institute for Food Systems Biology at the Technical University of Munich, Lise-Meitner Str. 34,

85354 Freising, Germany


Since its introduction to the analytical community, the R5 method to quantify gluten led to a strong

improvement of the situation for the food industry and celiac patients. During the last years some questions

arose on the use of the Codex Alimentarius factor of 2 to convert from prolamins to gluten, an

overestimation of rye and barley, inadequate detection of glutelins, and the inhomogeneous distribution of

gluten in oats. These limitations of the R5 method, especially when measuring oat samples led to AOAC

Standard Method Performance Requirement (SMPR®) 2017.021, which was approved by stakeholders in

2017. Here we present a collaborative study of a method for the quantitative analysis of wheat, rye, and

barley gluten in oat and oat products, using a sandwich ELISA that is based on four different monoclonal

antibodies including the R5 mAb. The sandwich ELISA detects intact gliadins and related prolamins from rye

and barley, high-molecular weight (HWM) glutenin-subunits (GS) from wheat, HMW-secalins from rye and

low-molecular-weight (LMW)–GS from wheat. It does not detect D-hordeins from barley. Samples are

extracted by Cocktail solution/80% ethanol and analyzed within 50 minutes. The measurement range is

between 5 mg/kg gluten and 80 mg/kg gluten using a calibrator made out of a gluten extract from four

different wheat cultivars. The results of the collaborative test with 19 participating laboratories showed

recoveries ranging from 99 to 137 % for all three grain sources. Relative reproducibility standard deviations

for samples >10 mg/kg ranged from 10 to 53 %. The collaborative study results confirmed that the method is

accurate and suitable to measure gluten from all three grain sources, and has demonstrated performance on

oat matrices which meets the criteria as specified in SMPR® 2017.021.

Page 1 of 24


With a prevalence of 0.4 to 1.2% of the population in Europe, North America, Australia, and the Middle

East (1), celiac disease (CD) is considered to be one of the most common food hypersensitivities. CD is

an immune-mediated inflammatory disease of the upper small intestine in genetically predisposed

individuals triggered by the ingestion of dietary gluten (2). In the context of CD, gluten is defined as a

protein fraction from wheat, rye, barley, or their crossbred varieties and derivatives thereof, to which

some persons are intolerant and that is insoluble in water and 0.5 mol/L NaCl (3). Gluten is composed of

prolamins that can be extracted by 40-70% of ethanol, and alcohol-insoluble glutelins that can only be

extracted under reducing and disaggregating conditions at elevated temperatures. The prolamins from

wheat, rye, and barley are called gliadins, secalins, and hordeins, respectively, and the prolamin content

of gluten is generally taken as 50% (3). The only known effective treatment for CD is a lifelong gluten-

free diet, which is based on the avoidance of gluten-containing cereals and should contain less than 20

mg gluten per day to prevent a relapse of intestinal damage (4). To guarantee the safety of gluten-free

products for CD patients, a threshold of 20 mg/kg gluten for gluten-free foods is recommended by the

Codex Alimentarius and legislation e.g., in the United States by the Department of Health and Human

Services - Food and Drug Administration (5) and in Europe by the European Commission (6). Specific and

sensitive analytical methods are therefore needed for food quality control. Immunochemical methods

are currently recommended for the quantitative and qualitative determination of gluten in foods (3).

Sandwich and competitive ELISA formats (RIDASCREEN® Gliadin R-Biopharm R7001 and RIDASCREEN®

Gliadin competitive R-Biopharm R7021) based on the R5 monoclonal antibody (7) were successfully

validated as AACCI Approved Method 38-50.01 for intact gluten (8) and 38-55.01 for partially hydrolyzed

gluten (9), respectively. Additionally, the R5 Sandwich ELISA RIDASCREEN® Gliadin was endorsed as a

Codex Alimentarius Type I method for the analysis of gluten (10) and has been adopted by AOAC

International as Official Method™ of Analysis 2012.01 Final Action in 2016 (11). The competitive ELISA

RIDASCREEN® Gliadin competitive (R-Biopharm, R7021) has been adopted by the AOAC International as

Official Method™ of Analysis 2015.05 (12). The R5 based Lateral Flow Device RIDA®QUICK Gliadin (R-

Biopharm, R7003) has been adopted as AOAC International Official Method™ of Analysis Final Action

2015.16 for analysis of foods (13) and as AOAC International Performance Tested Method™ 101702 for

surfaces and cleansing waters (14).

Page 2 of 24

The R5 antibody raised against ω-secalins primarily recognizes the epitope QQPFP, which is present in

gliadins, secalins, and hordeins and occurs in many peptides that are toxic or immunogenic for CD

patients (15-17).

Since its introduction, the R5 method has led to a strong improvement of the situation for CD patients.

The advantages of the method are that its response is well-characterized and well-understood. There is

a deep understanding of the method performance thanks to the comprehensive initial validation and

therefore, limitations of the R5 system are well known. Additionally, with a limit of quantification of 5

mg/kg gluten, the method is sensitive enough to reliably control and regulate gluten-free products.

However, every analytical method has limitations. For the R5 methods these limitations are the

following: [ 1] factor of 2 to convert from prolamins to gluten, which is not accurate in many cases (18),

[2] overestimation of rye and barley, and [3] inadequate detection of glutelins, which are not detected

by the R5 or any other antibody used in commercial kits, except the Skerritt monoclonal antibody (19).

All these limitations led to increased security for CD patients since analytical results tended to be biased

higher than true contamination levels (limitations 1 and 2). Limitation 3, the non-detectability of

glutelins is only important if a food product shows enriched proportions of glutelins to prolamins as e.g.

in wheat starch (20, 21). One additional limitation for all methods that measure gluten that was raised in

the past years was high observed repeatability standard deviations in some oat samples, which has been

attributed to inhomogeneous distribution of gluten in oats. This may lead to an unsecure situation for

CD patients if a laboratory is not able to manage this inhomogeneity by increasing the test portion and

the number of replicates for extraction. Sample inhomogeneity is a sample-intrinsic problem and not a

shortcoming of analytical systems. Nevertheless, it is an issue that needs to be addressed by all

analytical systems quantifying gluten in oats.

Because of these limitations to the R5 method, a group of oat processors and test kit manufacturers

founded an initiative through AOAC International in 2016. The resulting Standard Method Performance

Requirement (SMPR®) 2017.021 was adopted by stakeholders in 2017 (22). The method acceptance

criteria given in the SMPR are that mean recoveries for gluten from wheat, rye, and barley in oats and

oat products must be between 50% and 200%. Another important requirement in the SMPR is the

availability of “reference materials” with wheat or rye or barley gluten concentrations of 10 or 20 mg/kg

in oats including a blank material. These reference materials can be used to make test kits traceable to

the gluten content of these materials and therefore allow for more precise comparison of methods in

the future.

Page 3 of 24

The new sandwich ELISA RIDASCREEN® Total Gluten (R-Biopharm, R7041) presented here employs a

combination of four monoclonal antibodies including the R5 to detect the majority of gluten fractions

from wheat, rye, and barley including glutelins. It is calibrated to a wheat gluten preparation in the

range of 5 mg/kg up to 80 mg/kg. In consequence, no conversion factor of 2 is needed to convert from

prolamin to gluten. For extraction, the test portion was increased to 1 g compared to 0.25 g for OMA

2012.01 to account for inhomogeneity of gluten in oats. The test portion may be increased even more if

needed. When increasing test portion mass, the amount of Cocktail (patented) (R-Biopharm R7006 and

R7016) and ethanol must be increased proportionately. For this new method, Cocktail (patented) and

80% ethanol are incubated simultaneously and not consecutively as for former methods. The recovery

of the reference materials introduced by SMPR 2017.021 was found to be within the acceptance criteria

prescribed in the SMPR. A collaborative test using oats and oat products was performed in September

2018 with 19 participants worldwide (Australia, Austria, Canada, Finland, Germany, Hungary, Ireland,

Italy, Sweden, USA, and United Kingdom). The study was coordinated by Katharina Scherf (Leibniz-

Institute for Food Systems Biology at the Technical University of Munich).

Scope of the method: RIDASCREEN ® Total Gluten is used for the quantitative analysis of wheat, rye, and barley gluten in oat

flour, groats, oat flakes, and oat cereals which are declared as “gluten-free”. The sandwich ELISA detects

intact gliadins and related prolamins from rye and barley, high-molecular weight (HMW) glutenin-

subunits (GS) from wheat, HMW-secalins from rye and low-molecular-weight (LMW)–GS from wheat. It

does not detect D-hordeins from barley. Samples are extracted by Cocktail (patented) solution/80%

ethanol and analyzed within 50 minutes. The measurement range is between 5 mg/kg gluten and 80

mg/kg gluten using a calibrator made out of a gluten extract from a mixture of four wheat cultivars.

Collaborative Study

Study Design

Following the AOAC guidelines which are published as Appendix D (23) and Appendix M (24), an international collaborative study was set up to validate the RIDASCREEN ® Total Gluten for quantitative

gluten measurement in oat and oat-based foods as an Official Method of Analysis of AOAC International

(OMA). It was coordinated by Katharina Scherf (Leibniz-Institute for Food Systems Biology at the

Technical University of Munich). Before the participants were allowed to analyze the collaborative test

samples, they needed to show analytical competency by analyzing three control samples and the buffer

Page 4 of 24

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