OMB Meeting Book - Jan. 11, 2018

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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