SN T 1959-2007 动物源性食品中醋酸甲羟孕酮残留量的检测方法.酶联免疫法.pdf

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1、SEU 中华人民共和国出入境检验检疫行业标准SN/T 1959-2007 动物源性食品中醋酸甲经孕酣残留量的检测方法酶联免疫法Determination of medroxyprogesterone acetate residue in animal origin food Enzyme Iinked immunosorbent assay method 2007-08-06发布2008-03-01实施精回叫u毗VSNE山叫中华人民共和国发布国家质量监督检验检疲总同前言本标准附录A为规范性附录，附录B为资料性附录。本标f自由f国家认证认可监督管理委员会提出并归口。SNjT 1959-2007

2、本标准起卒单位z中华人民共和国天冲出人挠检验检疫局、北京中检维康技术有限公司。本标准主要起草人z主口Jm、郑文杰、贺他、赵卫东、王雄、王莲、文IJ秀红。本标准系首次发布的出入境检验检疫行业标洁。1 范围动物源性食品中醋酸甲瓷孕酣残留量的检测方法酶联免疫法本标准规定了动物源性食品中附自主qJ起孕附残留足的r#联免疫测定方法。SN/T 1959-2007 本标准适用于1的肉、牛肉、鸡肉、羊肉等肉制品及负和i虾等水产品中陆自主甲经孕自同残留贵的筛选检测。2 规范性引用文件下列文件巾的条款JlR过本标准的引IH而成为本标准的条款。凡是注日期的引用文作，Jt随后所有的修改1，(不包括助误的内容)或修订版

3、均不适用于本标币，然而，鼓励根据本标准达成协议的各方研究是否可使用这峡文件的放新版本。凡是不注明日期的iJ11ll文件，其后新版本适用于本标准。GB/T 6682 分析实室用水J:.IH吕刊i式;L方法3 测定方法3. 1 方法提要JJ乙酸乙陆提取样品中lt酸甲rl:JJ.酬，月i间tl:竞争性酶联免疫11;进行检测。3.2 试剂和材料除刃有要求外，所有试剂均为分忻纯，水fJC;3/T 6682规定的一级水。3.2. 1甲111.3.2.2 乙限乙郎。3.2.3 正己炕。3.2.4 元水硫酸讷，650.C灼烧4h，在1mi器内冷却至室温.贮于密封瓶巾备用。3.2.5 针筒式微孔过滤胶(0.22

4、Im，水性)。3.2.6 陪同空甲运孕阳Nj联免疫试剂企3. 2. 6. 1 96孔板(2条X8孔)包被有陆的甲经孕削和载体蛋白的复合物。3.2.6.2 抗醋酸吁Ii孕酣抗休(浓缩液)，4.C保jf，于使用时用抗体和j释缓冲浪50衍稀秤，抗休稀释8 h后不能继续使用。3.2.6.3 鸥标记物浓缩液)，辣根过氧化物Ij和l羊抗兔二抗的结合物，4QC保存，于使用时月HIj标稀fF缓冲浪50俏丽拌，酶标物衍释8h后不能继续使用。3.2.6.4 洗涤液(浓缩液)，按附录A配制。3. 2. 6. 5 I1点物液A，投附录A自己制。3.2.6.6 J民物液日，按附录A配;1币。3.2.6.7 10 X浓缩

5、抗体稀f缓冲液(pH7.2)，佼用前川菜r水稀fjlO倍。按附录A自己制。3.2. 6. 8 i标稀n缓冲液CpH7.4)，按附录A配制。3.2.6.9 终止液2mol/L硫股水nf议。1) 酣酸甲fi孕阳自吁联免疫吸阵I测定试剂盒是由中检维f;在技术有限公司提供的产品的商品名称。给出这一信息是为了方便本标准的使用者.井不表示对1生产品的认可。如果其他等效产品具1;相同的效果.则可使用这些等效产品。SN/T 1959-2007 3. 2. 6. 10 乙股甲扫孕阳系列标准液(6个倍比衍秤浓度水平，外加l个空白人3. 2. 6. 11 标准品贮备浓z配制成111g/mL可1m贮备液.C保汗。低温

6、保存试剂.放置于室温不要超过J1j己窍。取等1中积的J飞液和lB液混合指匀使用。3. 5. 1. 4 洗涤提供的洗法HLh10111浓缩i伍。使用前1日茶馆水将浓缩洗涤iH奇特10 f8 i1E III 2 SN/T 1959-2007 3.5.2 样品测定在西i机、极巾分别加入100L不同浓度的M!1甲1-2孕回标准溶液和样品待测液g在空白和对照孔中分别加入100L(Jj抗体稀fi缓冲液:除空白孔外.在每个孔中却l人50，稀择的抗体液g空白孔中则加l人50flL抗体稀释缓冲液g轻tM昆匀.1日帖胶纸N1t微孔以防溶液挥发.37C孵f730mIno弃掉tt;标板中的液体.fII i先浓浓洗板三

7、次。加入的n好的阴标记物i夜100，儿，轻轻混匀，37C孵育30mino弃f，li标板中的液体，用洗涤液洗板三次。加入混合好的底物液1001L.轻轻混匀，室温放置30min后，每个孔加入50L的终止Il.轻轻晃动i(j标板，10min内在网标仪上读出每孔450nm吸光值。3.5.3 空白试验除不称取样品外，均按上述步骤进行。4 检测低限、回收率4. 1 检jj!IJ低限本方法中aiJfll限为1,g/kg, 4.2 回收率醋股Ffl瓷孕州的添加浓度和问收率的实验数据参见附录B。实验约果为阳性应用仪器方法进行确证。. ( 1 ) 3 SN/T 1959-2007 附录A(规范性附录)试剂的制备A

8、. 1 10 x浓缩抗体稀释缓冲液100 mmoljL磷酸盐-9%氯化纳(NaCll，pH7.2磷酸氢二纳(Na，HPO, 12日，0)磷酸二氮纳(NaH，PO， H,() 氧化钩(NaCD调pH倪至7.2用水定容至0.5L. 使用时，用水1 10稀释。A.2 黯标稀释缓冲波10 mmoljL磷股盐0.9%氯化纳(只aCD, pH7. 4 磷酸氢二纳(Na，HPO, 12H, O) 磷酸二氢纳(NaH，PO， H,() 氮化纳(NaCD词pH值至7.4用水定容至0.5L。A.3 10 x浓缩洗涤液磷酸氢二纳(Na，HPO, 12H, O) 磷酸二氢何(KH，PO，)氧化何(KCD氮化钧(NaC

9、)Tween-20 加水定容至100mL，室温保存3周4用。使用时，用水1 10稀释。A.4 底物液4 底物液A，磷酸氢二的(Na，HPO, 12H, O) C, H80 , H , O 30%双氧水(H，O，) 调pH值至5.0加蒸馆水定容至100mL。底物液也3.31.5.5四甲基对二氨基联苯TMBlC, H, O, H, O EDTA 12.89 g 2. 18 g 43.9 g 1. 451 g 0.148 g 4. 39 g 2.9 g 0.2 g 0.2 g 8 g 0.5 mL 1. 841 g 0.51 g 335 f1 L 39 mg 115g 19 mg 二吁:基亚枫DMS

10、Ol甘泊(丙三醇)加蒸假水定容至100mL , SN/T 1959-2007 3 mL 20 mL 使用前室温i应光保抒。使用时，按底物液A底物液B为1 l(体积比)混合使用。A.5 黯酸甲控孕翻抑制率标准曲线标准曲线00 90 80 70 、60 布鲁50 Z Z主4030 20 0 。- -0.5 。0.5 1 1. 5 罹度/(阳/U图A.1醋酸甲经孕回抑制率标准曲线5 S :Ii/T 1959-2007 附录B(资料性附录)不同样品中醋酸甲瓷孕丽的加标回收率表.1不同样品中醋酸甲瓷孕回的加标回收率样品添加水平!(用/kg)回收率/%1 60-80 猪肉2.5 64-76 5 66-76

11、 L i 100-110 羊肉80-84 82-86 /产170-100 鸡肉L. 2 61-88 J / 2, 13 66-86 俨l70-110 于CL1-hmME血牛肉61-81 72-78 /产飞、80-90 鱼/ / 15 72-76 707? J 1 f-飞飞V70- 100 F 2 8 Z.t 72-88 t 皇68-86 - 飞6 SN/T 1959-2007 Foreword Annex A of this standard is a normative annex. Annex B of this standard is an informative annex This

12、standard was proposed by and is under the charge of Certification and Accreditation Administra tion of the Peoples Republic of China. first tin飞e.可一一一一/ J ，-户/句SNjT 1959-2007 Determination of medroxprogesterone acetate residue in animal origin food -Enzyme linked immunosorbent assamethod 1 Scope Thi

13、s standard specifies the methods of determination by ELlSA method of medroxyprogesterone ac etate (MPAl residue in animal foods This standard is applicable to the screen determination of medroxyprogesterone acetate residue in p。此，bovine，chicken , lamb etc. and aquatic product such as fish and shrimp

14、 for import and export. Positive results should be confirmed by another method 2 Normative references The following standards contain provisions which. through reference in the text, constitute provi sions of this standard. Parties to agreements based on this standard are encouraged to investigate t

15、he possibility of applying the most recent editions of the standards indicated below. GB/T 6682 Analyse laboratary used water style and analyse method 3 Method of inspection 3. 1 Principle of determination Medroxyprogesterone acetate is distilled by ethyl acetate The basis of the test is the indirec

16、tly com petitive enzyme-linked immuno-react旧n3.2 Reagents and materials Unless otherwise specified , all regents are analytically pure. Water is redistilled water described by GB/T 6682-1992 3.2. 1 methanol. 3. 2. 2 ethyl acetate. 3.2.3 n-Hexane. 8 SN/T 1959-2007 3.2.4 Anhydrous Na, S04 Drying 4 h a

17、t 650C, cooling to room temperature in the desiccator, stored in sealed container before use. 3.2.5 syringe droven filter(O. 22m) 3.2.6 Medroxyprogesterone acetate detection kit . 3.2.6.1 96 wells microtiter plate (twelve 8-wells strips) coated antigen which OVA conjugated Medroxyprogesterone acetat

18、e 3.2.6.2 Anti-Medroxyprogesterone acetate antibody (concentrated solution ), Storage at 4C , di lute 50 times with antibody diluent buffer. The diluted antibody solution is useless after 8 h. 3.2. 6. 3 Goat anti-Rabbit IgG/HRP (concentrated solution ), Storage at 40C , dilute 50 times with conjugat

19、e diluent buffer. The diluted enzyme solution is useless after 8 h. 3.2.6.4 Wash buffer (concentrated solution) ,see Annex A. 3.2.6.5 Substrate A , see Annex A 3.2.6.6 Substrate B, see Annex A. 3.2.6.7 10 x antiboddiluent buffer(pH 7. 2) ,dilute 10 times with distilled water belore use. see Annex A.

20、 3.2.6.8 Enzyme conjugate diluent buffer (pH 7.4) ,see Annex A. 3.2.6.9 Stop solution ,2 mol/L H,S04 3. 2. 6. 10 Medroxyprogesterone acetate series standard solutions (6 concentration level and 1 blank). 3.2.6.11 Medroxyprogesterone acetate standard reserve, 1g/mL methanol, storage at 4C. AII soluti

21、on must be stored at low temperature (40C) never freeze and never be placed at room tem perature longer than 4 h, reach room temperature belore use. 1 ) Medroxyprogesterone acetate detect旧nkit is the commerc旧Iname which was provided by the clover Technol 。giesCo. Ltd. This informat旧nbeing given is c

22、onvenient for the user of the standard a门dis not the confirm of 50me products protocol. Other products protocol should be confirmed by experiments if any difference 9 SN/T 1959-2007 3.3 Apparatus and equipment 3.3. 1 Homogenizer. 3.3.2 CentrifugeCprecision O. 001 gl. 3.3.3 Vibrator. 3.3.4 Microwel1

23、system. 3.3.5 Rotary evaporator. 甲3.3.6 3.3.7 3.3.8 3. 4 Preparation of Sample 3, 4. 1 Sampling procedure wds vyonu ，SJMYJIi，因a泪i叫咱jmmwmySJ Ffff相盯h泸:1 j叫:JJffwidmm ，mm一副uia屯2，.E-EA吨，hymi町e泪hrd1.。可引hn由eBoqauihuzte1a uhumm 2ty m咐咱川hMrun opco ammVM 白UM2叮川4koa af;, 3A剖kediately and deliver to laboratory

24、 3.4.3 Sample extraction Weigh 5 9 Cprecision 0.01 gl of minced sample in a 50 mL glass centriluge tube. Add 5 9 anhydrous Na,SQ, and 20 mL ethyl acetate. Homogenize by tissue homogenizer 1 min-2 min.mix 5 min. Centri fuge for 10 min at 4 000 r/min , RT. Transfer 4 mL 01 supernatant in a clean glass

25、 tube , dry out solvent by evaporation Cunder nitrogen ,at 40C 3.4.4 Sample purify Resuspend the residue with 1. 5 mL 80% methanol. Add 1 mL n-Hexane. mix 1 min , centriluge lor 2 min at 4 000 r/min. take the n-Hexane out ,and repeat 2times. Dry out solvent by evaporation Cun 10 SN/T 1959-2007 der n

26、itrog凹，at40 C), Sus口endthe residue with 5% 2 mL methanol/antibody diluent buffer and 0.22m filter with syringe droven filter, the solution is ready for analysis 3.5 ELlSA determination 3.5. 1 Preparation of working solutions 3.5. 1. 1 Anti-Medroxyprogesterone acetate antibody The antibody solution p

27、rovided is concentrated solution. The diluted antibody has a limited stability, 3. 5. 1. 4 Wash buffer 。ilute1 10 with distilled 3.5.2 Detection of sample 也J j / L十9mll. j j 2 , 、.i. / Insert a sufficient amount of m出ioti!_t!ellsinto the !1j;Jowell holder for all standards and samples to be run in d

28、uplicatei. Record standard and sample positions. Add 100L of six kinds of prepared medroxyprogesterone acetate standard solution and sample solution to separate duplicate wells. Add 100L antibody diluent buffer to separate duplicate wells as blank and maximum binding. Add 50L diluted anti-medroxypro

29、gesterone acetate antibody solution into each well except the blank well.and add 50L antibody diluent buffer into blank.flap lightly and mix thoroughly. Sealing all wells with ad hesive paper to avoid solution evaporating. incubate for 30 min at 37 C. Pour the liquid out of the wells and wash the we

30、lls three times with PBST. Add 100L of diluted enzyme conjugate (used within 8 hl to the bottom of each well.flap lightly and mix thoroughly. Sealing allwells with adhesive paper to avoid solution evaporating. incubate for 30 min at 37 C. Pour the liquid out of the wells and wash the wells three tim

31、es with PBST. Add 100L of substrate solution into each well (Take care the substrate Solution in the dark 1 flap lightly and mix thoroughly and incubate for 3 0 min at room 11 SN/T 1959-2007 temperature. Add 50L of stop solution into each well. flap Iightly and mix thoroughly. Measure and record the

32、 absorbance value of each well solution at 450 nm and 650 nm within 10 min 3. 5. 3 Blank test The operation of the blank test is the same as that described in the mention of determination but without the addition of sample. 3.5.4町、onitortest In course of each detection the sample added medroxyproges

33、terone acetate standard should be dealed with the test. The added concentration is the same as the detect limit of corresponding prod uct 3. 6 Calculating the result and express 3.6. 1 Calculating inhibition value in percentages Calculate the inhibition value of the combination of each MPA standard

34、and antigen-antibody. the MPA standard and sample inhibition value are calculated in percentages according to the formula C 1) : r_ A.n1-Ahl，寸IC = 11-二旦旦. m， I x 100% L AXlI1ttO，-Ab酣where ICthe inhibition value of MPA to the response of antigen and antibody. %; A=，rnlO ng/mL MPA standard mean absorb

35、ance value, A臼叩陆一MPAstandard or sample solution mean absorbance value, Ab，kmean absorbance value of 0 ng1mL MPA standard solution and antibody solution 3.6.2 Plot calibration curve C 1 ) Make the inhibition value in percentages Carithmetic grade) as ordinate.and MPA concentrationC loga rithmic grade

36、) as abscissa. plot the calibration curve of the inhibition value in percentages Csee annex A). A new calibration curve should be prepared for each determination 3.6.3 Calculating the result The MPA concentration of the test sample solution can be read corresponding to the percentage inhi bition val

37、ue of each sample from 2. 6. 2 calibration curve and namely equal to the residue content of MPA in the test sample 12 X=cxR where X一theresidue of MPA in the test sample.g/闸，c-the residual of MPA read from inhibition value，g/L; R-the sample dilution factor, is 2 in this method. 4 Limit of determinati

38、on and recovery 4. 1 Li mit of determination The Iimit of determination of this method is 1g/kg 4. 2 Recovery SN/T 1959-2007 . ( 2 ) The experimental data of these MPA concentration spiked in the pork sample and recoveries are as Annex B. The positive result should be confirmed by the equipment meth

39、od. 1.1 SN/T 1959-2007 Annex A (informative Annex) Preparation of working solutions A. l 10Xantibody diluent buffer(pH 7.2) 100 mmol/L Phosphate Buffer-9% NaCI.pH 7. 2 Na,HP04 12H,O 12.89 9 Dilute 1 : 10 with distilled water beftfre use_ 1 ) / / ter and adjljsf pH to 7. 2 and fix volume to O. 5 L. 7

40、 NaH, P04 H,O 啊!飞。1-Qunu j -句d2习一一NaCI Resolve all types of ingredients into distilled A.2 Enzyme conjugate diluent bufftr (pH 7. 4) 10 mmol/L Phosphate Buffer-D. 9% NaCI.pI:L7. 4 Na,HPO, . 12H,O NaH,P04 H,O NaCI Resolve all types of ingredients into A. 3 10 x Washing buffer (PBST) Na,HP04 12H,O 2.9

41、 9 KH,P04 0.2 9 KCI 0.2 9 NaCI 8g Tween-20 11 0.5盯lLS:-I/T 1959-2007 Fix volume to 100 mL.stored 3-4 week at room temperature. Dilute 1目10with distilled water before use. A. 4 Substrate solution Substrate solution A: (adjust pH to 5.0): Na, HPO, 12H, O 1.841! C; H,O, H, O 0.51 9 冒川 r句.，四-时_.，.，.，饲%H

42、,O, I幻5L 1 Resolve all ty阳of町edientsir川istilledwate.!)仙，1叫umet100 mL and store in refrigerate (4t ). Substrate solution B: 3.3 ,5.5.tetramethy.benzidine(TM C; H,O, H,O EDTA DMSO Precautions: store the solution at room temperature in dark place. 15 SNjT 1959-2007 A.5 MPA inhibition value standard cur

43、ve 16 100 90 W .: 80 至，70 C 60 且!: 50 亏40, 2 30 至2010 0 1 0.5 o 0.5 1 1. 5 ConcentratlOn/ (g/I) Figure A. l-MPA standard concentration Sample pork 阳mpchicken beef fish shrimp Annex B (informative annex) The recovery of MPA indifferent samples Table B. l-the recovery of MPA indifferent samples Level/

44、 (g/kgl 2 , 5 5 1 2 , 5 5 1 2 , 5 5 1 2 , 5 5 1 2 , 5 5 1 2 , 5 5 SN/T 1959-2007 Recovery/% 60-80 64-76 66-76 1口。-11080-84 82-86 70-1 84-88 66-86 70-110 84-84 72-78 80-90 72-76 70-72 70-100 72-8日68-86 17 中华人民共和国出人攻检验检疫行业标准动物源性食品中醋酸甲范孕嗣残留量的检测方法酶联免疫法SJjT 1959-2007 中网标准出版社出版北京复兴门外三里河北街16号邮政编码:100045网址电话:6852394668517548 中国标准出版社秦皇岛印刷厂印刷吁，nu -ilili-lnU MMMMMMMHHHHHnJ EEEEE配EEEEmE配酌-HH川tH川川!UUUUUUUrDEBE-QU i-mmmmmmmTt l/ 啤啤啤啤啤M川HHHHHS | 4峰开本880X1230 1116 印张1.5字数35千字2007年11月第一版2007年11月第一次印刷印数1-2 000 号岳书号，1550G6. 2-18276 定价13.00元