1、sm 中华人民共和国出入境检验检疫行业标准SN/T 1955一-2007动物源性食品中二苯乙烯类激素残留量检测方法酶联免疫法Determination of stilbenes residues in foodstuffs of animal origin Enzyme linked immunosorbent assay 2007-08-06发布2008-03-01实施盘叮)中华人民共和国发布国家质量监督检验检疫总同. _._ 目。吕本标准的附录A和附录B为资料性附录。本标准由国家认证认可监督管理委员会提出并归口。本标准起草单位2中华人民共和国天津出入境检验检疫局。本标准主要起草人g孙ffl
2、J、郑文杰、唐丹舟、魏亚东、张宏伟。本标准系首次发布的出入境检验检疫行业标准.SN/T 1955-2007 1 范围动物源性食品中二苯乙烯类激素残留量检测方法酶联免疫法本标准规定了动物源性食品中二米乙烯类激素残留量检验的联免疫测定方法。本标准适用于鸡肉、鱼肉、虾肉及鸡肝中己烧雌盼、己烯世Il:盼残饲量的价测。2 试样的制备和保存2. 1试样的制备自1 SN/T 1955-2007 从所取全部样品中取出有审二位ji:曰:约1kg.充分搅碎，混匀.采用四分法，将样品分成两等份，装入洁净容器，加封并做标识。L一卢一一一一一-;,) 2.2 试样的保存/ / 试样放i1-20C-18C条件下保存bfJ
3、/ / 3 测定方法/ / 3. 1 方法提要本方法的测定基础是竞争性i;n是兔疫反应。m标板t己也被的雌阶抗体，可与己统雌盼，己烯附盼发生交联反应，标准被或样品中、即盼匀辣粮过筑比如?标记的隙盼时原共同争夺雌酌抗体t的结合足点.Hl标仪测:fJ做孔溶液的吸光皮值，雌盼浓度与iJl!tj;度优陵反比，按绘制的校正曲线定是计算。3.2 试剂和材料/ i t 除另有规定外，所用化学试如JJ:)j :5Hfr纯，为更骂级别。水为1主蒸t留水。3.2.1 二苯乙烯类免疫测定试班以多月附录Al.3.2.2 叔丁基fjJ基础z色谱纯.1/ I / 3.2.3 三氯1f1J窍。I1 mo叫ol/L叫磷附限.
4、吸帆取呐m毗L阳阳主i附夜愤卜卉fh水扣刺川k扣川川r叶巾叫1护13.2.5 乙醇. f I J 3.2.6 卢-葡惦召E股主西邵j(Si咆gmaG-(吨j坞飞76).I I / 3.2.7 乙酸的(CH.CO川.3H， / I ._/ 3.2.8 乙酸的缓冲li(O.lmol!L.pH切1称取13.6g之酸纳济解于800mL水1, J日氢氧化的溶液i用1，pH值至5.0土0.1，加水定容至1000 mL。3.2.9 氢氧化的溶液(1mol!L)称取40日氢氧化的溶于1000 mL水中。3.2. 10 Fil字。3.2. 11 标准品=己统雌阶标准品，己烯i!tf:)标准品纯度均主主98%。3
5、.2. 12 雌盼标准品溶液的配制，/!I确称取适量的己烧雌盼和己烯蚓n吩标准品，用甲院配制成1 mg/mL标准贮备溶液.于.jC8C条件下保存。3.3 仪器和设备3.3. 1 酶标仪z波长450nm。3.3.2 37C士2C培养衍。3.3.3 均质器。SN/T 1955-2007 3.3.4 天平。3.3.5 离心机，3000r/mino 3.3.6 氮气吹干仪。3.3.7 振荡器。3.3.8 洗板机。3.3.9 回相萃取仪。3.3. 10 微量加样器，20L.50川，.100严L.200L。3.3. 11 微量多通道加样it，200Lo 3.3. 12 免疫亲和柱，50吨，5mL . 3.
6、4 试样提取和净化3.4. 1 提取3.4. 1. 1 肌肉样品称取2.5g(精确到0.1g)试样于离心管中，加入15mL叔丁基甲基脏，均质30s，涡旋振荡3min。在2000 r/min下离心10min，移取12mL 盖层液体，在400C氮气流下蒸发至干燥。用1mL三氯甲烧溶解于燥物，涡旋振荡3min。加2mL 1 mol/L氢氧化钩，涡旋振荡3mino在2000 r/min下离心10 min，移取上层液到另一试管中，保留1mL水相。再加入1mL 1 mol/L氢氧化纳到三氯甲炕溶液中，涡旋振荡3min，在2000 r/min下离心10min，移取上层提取液到另一试管中，保留1mL水相。合并
7、两次提取液，加入200L的6mol/L磷酸中和2mL的氢氧化纳提取液后待净化。3.4. 1. 2 肝脏样品称取2.5g(精确到0.1g)试样至离心管中，加入3mL乙酸纳缓冲液(0.1mol/L;pH 5.0).加入8000单位葡糖背酸酶，均质约3050 37C土2C培养2h.以下按3.4.1.1提取步骤操作。3.4.2 净化以免疫亲和柱净化样品提取液。用柱储存缓冲液过柱，再用15mL柱洗涤缓冲液平衡柱子(流速;3 mL/min)。取全部中和后的样品提取液过柱(重力引流入用5mL柱洗涤缓/ljt液洗涤柱子两次(流速;2mL/min)。用5mL水洗涤柱子(流速;2mL/min)。用3mL乙院/水(
8、70/30.体积比)洗脱样品中可能存在的雌激素。此步的洗脱液用干净的试管收集后用于试剂盒检测.3.5 酶联免疫测定3.5. 1 操作条件所有操作应在室温下(20C25C)进行，雌盼试lJJ盒中所有试剂的温度均应回升至室温(200C 250C)后方可使用。3.5.2 ilm定步骤1)将测定需用的微孔条插入框架(标准液、样液和空白分别作平行试验测定).记录标准液和样液的位置。先吸取100L已稀释的稀释缓冲溶液至酶标板各孔内，再分别吸取25L雌盼标准溶液、样品溶液至各自的微孔，持微孔板在台面上以圆周运动方式混匀后，然后用封口膜密封孔条以防溶液挥发。将其置于20C25C .避光孵育1ho加入75L已稀
9、释的酶标记抗原至每个微孔，混匀，覆盖上封口膜，200C25C避光孵育30mn.倒掉微孔中的液体，用洗涤缓冲液洗板操作12次。加人125L发色开rJ至每一微孔中，充分混匀.20C25C避光孵育20min。加入100L终止液至每个微孔中，充分混匀，在30min内，测量并记录每个微孔溶液450nm波长的吸光度值。3.5.3 空白试验2 除不称取试样外，均按上述步骤进行。1)给出该信息是为了方便本标准的使用者，并不表示对某一产品操作步骤的认可。如果其他产品的操作步骤有不同，需经实验评估后采用.SN/T 1955-2007 3.5.4 监控试验每次测定均应做一个添加雌盼标准的样品，添加浓度为相应产品的检
10、测限量。3.6 结果表述按式(1)计算百分比吸光皮值2吸光度值=BIO吨之旦X100 . ( 1 ) 式中gB标准/时标准品或样品微孔的平均吸光度值，%; B空白一一空白孔的平均吸光度值;B。零标准的平均吸光度值。以百分比吸光度值(算术级)为纵坐标，以雌酷标准溶液的浓度(ng/mU(对数级)为横坐标，绘制出雌盼标准液百分比吸光度值与雌盼浓度的校正曲线(参见附录A)。每次试验均应重新绘制校正曲线。从标准工作曲线上得到试样中相应的雌盼浓度后，结果按式(2)进行计算z式中x=-羊卫兰1000m X 1 000 X一一样品中雌盼的残留量，单位为微克每千克(Ig/kg); C-从标准工作曲线上得到的样品
11、中雌盼浓度，单位为纳克每毫升(ng/mU;V一样品溶液的最终定容体积，单位为毫升(mL), m 样品溶液所代表的最终试样质量，单位为克(g)o也可以用各种酶标仪的数据处理软件进行计算，所得结果表示至一位小数。4 检测限本方法的检出限己烧雌盼为0.5g/kg，己烯雌盼为1.0g/kgo5 确证试验如被测样品中己炕雌盼、己烯雌盼残留量的值大于检测限时，应用仪器方法进行确证。6 回收率回收率试验数据参见附录Bo( 2 ) 3 SN/T 1955-2007 附录A(资料性附录)雌盼酶联免疫测定试剂盒2A.1 雌盼酶联免疫测定试剂盒本标准酶联免疫测定步骤中使用的试Jl1J盒为英国RAlDOX公司产品，试
12、)11企包括:a) 框架，96孔饭;b) 标准溶液，0ng/mL.O. 05 ng/mL.O. 25 ng/mL, O. 53 ng/mL.l. 0 ng/mL.5. 0 ng/mL; d 雌盼酶标记物济液。按Y11J忘记川现urrn协商乔汉时J稀释，充分混匀后使用，d) 阪标稀f液se) 发色齐IJ; 。反应终止液gg) 洗涤/稀释缓冲溶液。A.2 雌lIfr免疫亲和柱A.3 雌酣标准校正曲线4 100 90 8 6 主: 50 40 30 20 10 。0.05 0.24 0.51 0.99 如准ii;ITttt&/(nKmL) 图A.1雌盼标准校正曲线2)给出该信息是为了方便本标准的使用
13、者，井不表示对某一产品的认可。如果其他产品具有相同的效果.需经实验l于估后使用这些等效产品.附录B(资料性附录)回收率本方法中二苯乙烯类激素添加浓度及回收率数据己饺雌盼z添加Lt为0.5g/kg时，平均回收率为85.5 % 94. 7 % ; 一一添加是为1.0g/kg时，平均回收率为81.2%-105.4%;添加最为5.0g/kg时，平均回收率为87.6%101. 9%。己烯雌盼:-., 添加最为1.0g/kg时，在均囚收率为70.3%91.1%;1 添加盐为2.0g/kg时，fF均囚收率为76.5%-90%;1 添加的10.0内时:fEJ1ill胖芳7%94.3凡l毡，,7 W 4倍 J
14、d / 一J / 专乒SN/T 1955-2007 5 SN/T 1955-2007 Foreword Annex A and B of this standard are informative annex. This standard was proposed by Certification and Accredditation Adiministration of the Peop怡sRe public of china. This standard was mainly drafted by Tianjin Entry-Exit Inspection and Quarantine B
15、ureau ofthe Peo ples Republic of china. This standard was mainly drafted by Sun Li, Wenjie Zheng , Danzhou Tang , Yadong Wei , Hongwei Zhang. This standard is professional standard of Entry-exit inspection and quarantine promulgated for the first time. 6 SN/T 1955-2007 Determination of stilbenes res
16、idues in foodstuffs of animal origin一Enzmelinked immunosorbent assa 1 Scope This standard specifies the determination by ELlSA method of stilbenes residuses in foodstuffs of animal origin. This standard is applicable to the screen determination of stilbenes residuses in chicken meat. pork. fish. shr
17、imp and chicken liver. 2 Sample preparation and storage 2. 1 sampling procedure The representative sample should be taken out. and total weight is not less than 1 kg. Sample defa. ted and homogenized thoroughly. At least each sam口leshould be divided into equal portions. AII samples should be placede
18、 in a clean and dry container. then seal up. The label should be ladeled out side of each sample container. 2. 2 Storage of salple After sampling. the test sample should be stored at - 20t - -18t. 3 Method of determination 3. 1 Principle of determination The basis of the test旧thecompeititve enzyme.l
19、inked immunoreaction. stilbene and stilbene enzyme conjugate compete for the limited anti.endosulfan antibody. The absorbance value of each well solu. tion is measured by microwell system. The stilbenes concentration is inversely proportional to the absorption value. and compare with the calibration
20、 curve for quantitative measurement. 3. 2 Reagents and materials In this standard. all the chemical reagents should be A. R. grade unless it identified specially. . wa. ter is doubled dist川edwater. 7 SN/T 1955-2007 3.2.1 Enzyme immunoassay kit for the quantitative analysis of stilbenes(see Annex Al.
21、 3.2.2 Tert-butylmethylether-chromatographic grade. 3.2.3 Chloroform 3.2.4 6 mol/L phosphoric acid. Dissolve 58.8 mL phosphoric acid in 100 mL distilled water. 3.2.5 Ethanol. 3.2.6 3.2.7 -glucuronidase (Sigma口876). / -/ Sodium acetate (CHJCOQNa-v3HiQ)-，-;-甲-一3.2.8 0.1 mol/L Sodium acetate (pH = )， D
22、issolve) 3. 6 9 Sodium acetate (3.2.7) in 800 mL 在-/distilled water. adjust to pH 5. O:t O. i,V1ith 1 mot/LNOH. the total volume is 1000 mL. 3.2.9 1 mol/L NaOH , Dissolve 3.2. 10 Methanol. 3.2.11 3.2. 12 Preparation of solution the storage solutions is 1 mg/ 3. 3 Apparatus and equipment 3.3. 1 Micro
23、well system, 450 3.3.2 37t :t 2t incubator. 3.3.3 Homogenizer 3.3.4 Balance. 3.3.5 Centrifuge, 3000 r/min 3.3.6 Nitrogen Eva口orat。3.3.7 Shaker. 8 distilled water. / SN/T 1955一20073.3.8 Washing machine. 3.3.9 SPE cartridge. 3.3.10 Pipettes, 20L.50L. 100L. 200L 3.3.11 Multichannel pipette, 200L. 3.3.
24、12 Immunoaffinity columns , 50 ng. 5 mL The extract is further purified as followed by means of immunoaffinity columns. Allow column stor. age buffer to flow through and then equilibrate with 15 mL diluted column wash buffer at a rate of 3 mL/min. Load total volum of extraction onto the column and a
25、llow to flow through under gravity. Wash the column twice with 5 mL diluted column wash buffer at a rate of 2 mL/min. Then wash the column with 5 mL doubled distilled water at a rate of 2 mL/min. Eluted by the application of 3 mL 70% ethanol/30% water and collecting the eluate in a clean glass test
26、tube. The sample is now ready for appl icat旧nto the stilbene ELl SA microtitre plate 9 SNjT 1955-2007 3.5 ELlSA determination 3.5.1 Test condition AII the procedures should be done at room temperature (20C -25t). 8ring all reagents and the mi crotiter plate to room temperature before use (20t -25C)
27、. 3.5.2 Test procedure Insert a sufficient amount of microtitre wells into the microwell holder for all standards and samples to be run in duplicate. Record standard and sample positions. Add 100L the diluent buffer to each wells. Add 25L of six kinds of stilbene standard solution and sample solutio
28、n to separate duplicate wells. Flap lightly and mix thoroughly. sealing all wells with adhesive paper to avoid solution evap rating. incubate for 1 h at 20t -25t in darkness. Add 75L of diluted enzyme conjugate to the bottom of each well. mix gently and incubate for 30 min at 20t -25t. Pour the liqu
29、id out of the wells and wash the wells twelve times. Add 125L of one shot substrate to each well. flap lightly and mix thoroughly and incubate for 20 min at 20t -25t in darkness. Add 100L of stop solution to each well. flap Ii日htlyand mix thoroughly. Measure and record the absorbance value of each w
30、ell solution at 450 nm within 30 min. 3. 5. 3 81ank test Follow the sample extraction and test procedure without sampling. 3. 5. 4 Control test Each test shuld determinate a fortified sample. The fortifying concentrations of stilbene is the limit of determination. 3.6 Calculation and express Calcula
31、te absorbance value in percentage according to the formula (1) : Bt.m/I. - B absorbance value in percentage% = LJSl旦出世L一旦哩x100% ( 1 ) Bo where B矶时rd/sam川，-meanabsorbance value of standards and samples. %; Bb叫meanabsorbance value of blank; 10 Bo-Zero standard mean absorbance value. 1) Gives this info
32、rmation is in order to the user who facilitate this standard.does not express to 50me product 58-quence of operation approval. The testing process of another equal kits maybe a little d1ffer , 50 it should be used after testing. evaluation and verification. S:I/T 1955-2007 Make the absorbance value
33、in percentage (arithmetic grade) as ordinate. and stilbenes concentration (g/kg) (Iogarithmic grade) as abscissa. plot the calibration curve of the absorbance value in per centage and stilbenes standard concentration (see Annex A). A new calibration curve should be pre pared for each determination.
34、Read corresponding to the concentration of each sample from calibra tion curve and calculate according to the formula (2) , where x-CXV 1000 -二mX1000 x-The stilbenes residues content of the test sample. unit isg/kg， c-The stilbenes concentration value of sample read from calibration curve. unit is n
35、g/mL, V一Thetotal volume of sample solution. unit is mL, m-The total sample quality of sample solution. unit is g. ( 2 ) Also may use each kind of the data processing software to carry on the computation. Obtained result expression to a decimal. 4 Limit of determination The limit of determination of
36、this method is O. 5 1g/kg for hexestrol and 1.0g/kg for diethylstilbes trol. 5 confirmation If the stilbenes residue is over the limit of determination. it should be confirmed banother method. 6 Recovery See Annex B. 11 SN/T 1955-2007 Annex A Clnformative Annex) Enzyme immunoassay kit for the quanti
37、tative analysis of stilbenes2) A. 1 Enzyme immunoassay kit for the quantitative analysis of stilbenes The RANDOX kit of UK is used in this standard 3. 5 ELlSA determination. Each test kit contains: a) Microtiter plate with 96 wells: bl Standard solut町、:0ng/mL.ct05ng ml.O. 25 ngJmL.O. 53，ng/r礼，1.0ng!
38、mL.5. 0 ng/mL; j J c) Conjugate: The enzyme conjugate is diluted injugate buffer. For details of dilute consult se口arate conjugate diluents procedure shee / / / d) Conjugate diluent; /. /r e) One shot Substrate; f) Stop solution; 日)Diluent IWash Buffer. A. 2 Immunoaffinity columfts Column wash buffe
39、r.dilute 1 part Column storage buffer.dilute 1 part column wash buffer with 4 parts double distilled water 12 2) Gives this informaton is in order to the user who facilitate th旧standard，doesnot express to some product se quence of operation approval. The testing process of another equal ki恬maybea li
40、ttle differ. 50 it should be used after testing. evaluation and verification S:-.IjT 1955-2007 A.3 stilbenes calibration curve 100 90 80 70 60 m x足乓40 。/ 5 J 俨- 啕唱-JJf/ / 13 SN/T 1955-2007 Annex 8 Clnformative Annex) Recovery The experimental data of these fortifying concentrations of stilbenes and
41、recoveries are as follows: For hex: 一At0.5凹/kg，therecovery is85.5%-94. 7%, At 1. 0g/kg， the recovery is 81.2% -105.4% , -At 5.0g/kg.the recovery is 87.6%-101.9%. For diethylstilbestrol: 一At1.0g/kg. the recovery is 70. 3% -91.1 %, 一At2.0g/阁，therecovery is 76啕5%-90%，At 10.0月几g.therecovery is 75. 6%-94. 3%. 14 中华人民共和国出人境检验检疫行业标准动物源性食品中二苯乙烯类激素残留量检测方法酶联免疫法SN/T 1955-2007 善中国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阿址电话，6852394668517548 中国标准出版社秦皇岛印刷厂印刷* 开本880X12301/16 印张1.25 字数25千字2007年11月第一版2007年11月第一次印刷印数1-2000等书号，155066.2-18237定价12.元
