1、5 中华人民共和国出入境检验检疫行业标准SNjT 1956-2007 肉及肉制品中己烯雌酣残留量检测方法酶联免疫法Determination of diethylstilbestrol residues in meat and meat product-Enzyme Iinked immunosorbent assay 2007-08-06发布2008-03-01实施A吼一-w中华人民共和国发布国家质量监督检验检菇总同. 目Ij1=1 本标准的附录A和附录B均为资料性附录。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z中华人民共和国天津出人境检验检疫局。本标准主要起草人孙俐、郑
2、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 1956-2007 I 1 范固肉及肉制品中己烯雌酣残留量检测方法酶联免疫法本标准规定了肉及肉制品中己烯雌盼残留莹的院联免疫测定方法。本标准适用于鸡肉、猪肉、牛肉、羊肉、猪肉饼中己烯雌盼残留莹的检测。2 试样的制备和保存2. 1 试样的制备I I SN/T 1956-2007 从所取全部样品中取出有机表性样品约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。一-一一一丁户./ 2.2 试样的保存试样放置一20C一18C条件下3 测定方法甲事.-3. 1 方法提要I 1 本方法的测
3、定基础是竞争性rm免疫反应。己t拮据盼与己烯雌盼酶标记物共同竞争己烯雌盼抗体的结合位点，用ft标仪测量归Llrr浓的吸光度JT.TIj;ltiJIfIf附阳县13-k光度值成反比，按绘制的校E曲线定量计算。3.2 试剂和材料./ 1 I 除另有规定外，所用化学i咄均为分析时或为更高级别。水主l重蒸惚水。3.2. 1 己M雌盼酶联免疫定J1.ilj足试主Ij盒参见fl我A 3.2.2 叔丁基甲基脏z色谱t， 1、/ 1 3.2.3 石祖11脏。II I I I I 3.2.4 二氮甲炕。. J二i / i 3.2.5 磷般。飞I I I 3.2.6 盐般。飞/ I 3.2.7 lljW:包i普纯
4、。飞飞/ 3.2.8 磷自主氢二纳(Na2HF042H2orfJ3.2.9 磷股二氢纳(NaH，PO， H，O)。3.2.10 J;R化的。3.2. 11 三控甲基氨基甲烧。3.2. 12 磷股盐缓冲液(67mmol/L , pH=7. 2):称取9.61g磷酸氢二纳，1.79 g磷酸二氢纳和8.7g 氯化纳溶解于并定容至1000 mL水中。3.2.13 Tris缓冲液(20mmol/L , pH=8. 5)称取2.42g三经甲氨基甲:院溶解于700mL水中，加人200mL甲自草，用5mol/L盐股调节pH值至8.5，加水定容至1000mL。3.2. 14 洗rR用磷酸盐缓冲液(20mmol/
5、L, pH=7. 2):称取2.85g磷酸氢二纳，0.55g磷酸二氢纳，9 g氯化例和1mL吐温20，溶解并定容至1000 mL水中。3.2. 15 氮氧化纳溶液(1mol/U:称取40g氮氧化纳溶于1000 mL水中。SN/T 1956-2007 3.2.16 己烯雌盼标准品g标准品纯度二三98%.3.2.17 己烯雌盼标准品溶液的配制g准确称取适量的己烯雌盼标准品，用甲醇配制成1mg/mL标准贮备溶液，于4c 8C条件下保存。3.3 仪器和设备3.3. 1 酶标仪2波长450nm. 3.3.2 高剪切分散器。3.3.3 涡旋振荡器。3.3.4 天平。3.3.5 离心机，3000r/min。
6、3.3.6 氮气吹干仪。3.3.7 洗板机。3.3.8 固相萃取仪.3.3. 9 7)(自锅，70C土2C。3.3. 10 微量加样器，20L.50L.IOOL，200L.3. 3. 11 微量多通道加样器，50L.I00L。3.3.12 RIDA c18t宝，100mg.1 mL. 3.4 样晶处理3.4. 1 试样提取称取5.0g试样至离心管中，加入10mL 67 mmol/L pH 7.2磷酸盐缓冲液，均质.JH8 mL叔丁基甲基隧提取3.0g均质物，强烈振荡20min.3 000 r/min离心10min。转移出上r液，再用8mL叔丁基甲基隧重复提取沉淀物，将两次提取的隧相合并.70C
7、水浴蒸发至干。用1mL 70%甲部榕解干燥的残留物，用3mL石油酣洗涤甲部溶液，均质155.3000 r/min短时离心.吸除石油隧。70.C水浴蒸干甲院溶液，用1mL二氯甲烧溶解，用3mL氢氧化纳(NaOHl溶液提取。用300L6 mol/L磷般中和提取液。3.4.2 样晶纯化用RIDAC1B柱纯化，流速均为1d/s.用3mL无水甲醇洗涤柱子，用2mL 20 mmol/L pH 8.5的Tris缓冲液平衡柱。将上述中和提取液加入柱中，用2mL 20 mmol/L pH 8.5的Tris缓冲液洗涤柱，用3mL 40%甲M洗涤柱子，用正压去除残留的液体并旦用空气或氮气吹2min以干燥柱子。用1m
8、L 80%甲醇洗脱样品，用1mL 80%甲M稀释洗脱液，加入2mL蒸馆水进一步稀释，取20L进行分析。3.5 酶联免疫部l定3.5.1 测定步骤己烯雌盼试j)lj盒中所有试剂的温度均应回升至室温(20C25.C)后方可使用。视定中吸取不同的试剂和样品溶液时应更换吸头.将测定需用的微孔条插入框架(标准液和样液、空白分别作平行试验测定).记录标准液和样液的位置.分别吸取20L己烯雌盼标准溶液、样品溶液至各自的微孔，再加入50L己稀释的己烯雌盼抗体至每个微孔，持微孔板在台面上以困周运动方式混匀后，然后用封口膜密封孔条以防溶液界发。于2.C8.C避光孵育至少20h.微孔板恢复至室温，倒排微孔中的液体，
9、用洗涤缓冲液洗板操作3次。加入50L已稀释的己烯雌盼酶标记物，充分i昆匀，室温孵育1h。倒掉微孔巾的液体，洗板操作3次。加人50L酶基质和50L发色j)lj至各微孔中，充分j昆匀.20C25.C暗处避光孵育30min. IJII人100L反应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。2 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。3.5.2 空自试验除不称取试样外，均按上述步骤进行。3.6 结果计算按式(1)计算百分比吸光度值2吸光度值二h吨主且x100% 式中3B
10、，I标准一一标准品或样品的平均吸光度值，%;B空白一一一空白孔的平均吸光皮值3B。一一零标准的平均吸光度值。SN/T 1956-2007 . ( 1 ) 以百分比吸光度值为纵坐标，以己烯雌盼标准溶液的浓度(ng/mL)的对数为横坐标，绘制出校正曲线(参见附录B)。每次试验均应重新绘制校正曲线。从标准曲线上得到试样中相应的己烯雌盼浓度后，结果按式(2)进行计算gx cV100。一二m X 1 000 式中zx一一-样品中己烯雌盼的残留茧，单位为微克每千克(g/kg); C一一从标准曲线上得到的样品中己烯雌!li浓度，单位为纳克每毫升(ng/mL); V 样品溶液的最终定容体积，单位为毫升(mL)
11、; m一一样品溶液所代表的最终试样质量，单位为克(g)。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为o.5g/kgo 5 确证试验如被视样品中己烯雌盼残留莹的值大于限量要求时，应用其他方法进行确证。6 回收率回收率试验数据参见附录Bo( 2 ) 3 SN/T 1956-2007 附录A(资料性附录)己烧雌盼酶联免疫定量测定试J盒A.1 己烯雌盼酶联免疫定量测定试剂盒本标准酶联免疫测定步骤中使用的试剂盒为德国r-biopharm公司产品21，试剂盒包括:a) 框架，96孔板;b) 标准溶液，40%己烯雌盼甲醇溶液:0，0.025，0.05，0.10
12、，0.2，0.4ng/mL; c) 己烯雌盼酶标记物溶液:场雨骨1!7OClXT11比例稀ft浓缩液，充分i昆匀后使用;d) 己烯雌盼抗体浓缩液z用手E释绥lr夜以1 11比例稀释浓缩液，充分混匀后使用;e) 隅基质pf) 发色剂;g) 反应终止液gh) 稀释缓冲溶液-A.2 己烯雌酣校正曲线100 90 / / / X(J笔与000000000 87654321 线曲酬正L校mh! jfgE 如/雌昆/局MJ己标1A 图2)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的认可.如果其他产品具有相同的效果，需经实验吓估后使用这些等效产品.4 附录B(资料性附录)回收率本方法中己烯雌盼添
13、加浓度及回收率数据z添加量为0.5g/kg时，平均回收率为85.2%97. 5%; 一一添加世为1.0 flg/kg时，平均回收率为82%l04.2%;-添加最为5.0flg/kg时，平均回收率为84.5%94. 8%。SN/T 1956-2007 3 SN/T 1956-2007 Foreword Annex A and B of this standard are informative annex. This standard was pr。口。sedby Certification and Accredditation Adiministration of the Peopfes Re
14、. pubfic of china. This standard was mainfy drafted by Tianjin Entry.Exit fnspection and Quarantine Bureau of the Peo. pfes Repubfic of china This standard was mainfy drafted by Sun Li, Wenjie Zhen日，Danzhou Tang , Yadong Wei , Hongwei Zhang. This standard is professionaf standard of Entry-exit inspe
15、ction and quarantine promufgated for the first time. 6 SN/T 1956-2007 Determination of diethIstilbestrol residues in meat and meat product一Enzymelinked immunosorbent assay 1 Scope This standard specifies the determination by ELlSA method of diethlstilbestrol residuse in meat and meat product. This s
16、tandard is applicable to the screen determination of diethlstilbestrol residuse in chicken meat. pork, beef. mutton and breaded pork. 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 h
17、omogenized thoroughly. At least each sample should be divided into equal portions. AII samples should be placede in a clean and dry container to maintain the sample completeness and traceability. then change into a bigger clean container to seal up and transport. The label should be labeled out side
18、 of each sample container. 2. 2 Storage of sample After sampling. the test sample should be stored at - 20t - -1St. 3 Method of determination 3. 1 Principle of determination The basis of the test is the competitive enzyme.linked immunoreactions. Diethylstilbestrol and diethylstilbestrol enzyme conju
19、gate compete for the limited anti- diethylstilbestrol antibody. The ab sorbance value of each well solution is measured by microwell system. The diethylstilbestrol concen tration is inversely proportional to the absorption value. and compare with the calibration curve for quantitative measurement. 7
20、 SN/T 1956-2007 3. 2 Reagents and materials In this standard, all the chemical reagents should be A. R. grade unless it identified special忡，water is doubled distilled water. 3.2.1 Enzyme immunoassay kit for the quantitative analysis of diethylstilbestrol(see Annex A). 3.2.2 Tert.butylmethylether:chr
21、omatographic grade. 3.2.3 Petroleum ether 四111/r d/ / / f 3.2.4 Dichloromethane 3.2.5 Ph05phoric acid. 俨3.2.6 HCI. 3. 2. 7 Methanol: Chromatogram grJ 3. 2. 8 Na, HPO, 2H,O 3. 2. 9 NaH, PO, . H, O. 3.2. 10 Sodium chloride. 3.2.11 )山川):Dis肮Diss阳s臼sdis剖till怡ed!wa剖tel咽.F 3.2.13 叩4忆阳C1.阳川-占扣b川buf旷f川Omm叫m
22、ol/L吨E亏?吧85引5队P吵?怜卢白灿Ive川v四e2守俨目y俨(hydrox叫Y阳川me阳e创时th川mlno。nem ap口rox.700mL dis引til川11怡edwa剖ter，m以x飞怦战江200(mLm、e时th怕al:l且i守呻a均JUs创tpH 8. 5 with 5 mol/L HCI ,and fill up to 1 000 mL with distilled water 3.2.12 3.2.14 Washing buffer(20 mmol/L,pH = 7.2): Dissolve 2.85日Na，HPO, 2H, 0 , O. 55 9 NaH, PO, .
23、 H,O,9 9 NaCI and 1 mL Tween 20 in 1 000 mL distilled water. 3.2.15 1 mol/L NaOH:Dissolve 40 9 sodium hydroxide in 1 000 mL distilled water. 3.2.16 The purity of diethylstilbestrol is over 98%. 3.2.17 preparation of solution:Dissolve 50me DES in methanol ,and the concentration of the stor. age solut
24、ions is 1 mg/mL. Then store at 4(; _80C. 8 SN/T 1956-2007 3.3 Apparatus and equipment 3.3.1 Microwell system:450 nm 3.3.2 Homogenizer 3.3.3 Shaker. 3.3.4 Balance. 3.3.5 Centrifuge:3 000 r/min 3.3.6 trogen Ev叩仁3.3.7 Washing machine. 3.3.8 SPE cartridge. 3.3.9 Water bath boiler: 70t士3.3.10 Pipettes:20
25、L.50L.100L.200L 3.3.11 3.3.12 3.4. 1 Sample extraction tant into a glass centrifuge tube. Repeat the extraction with 8 mL Tert-butylmethylether. Combine the ether phase. the organic phase evaporated with nitrogen gas in water bath at 70t. Dissolve the residue in 1 mL70% methanol. wash the methanolic
26、 solution with 3 mL petroleum ether. homoge nize for 15 s. centrifuge 3 000 r/min shortly. aspirate and reject petroleum ether supernatant. The methanolic solution is then evaporated to dryness under a gentle stream of nitrogen. The residue is dissolved in 1 mL dichlormethane and extracted with 3 mL
27、 NaOH. Neutralize the extract with 300L 6 mol/ L phosphoric acid 3.4. 2 Sample cleanup The extract is further purified as following by means of RIDA C18 columns. The column is rinsed with 9 SN/T 1956-2007 3 mL of methanol and equilibrated with 2 mL 20 mmol/L tris-HCI. The sample solution pass though
28、 the column at a rate of 1 dro口/s，followed by rinsing with 2 mL of 2 mL 20 mmol/L tris-HCI and 3 mL 40% methanol in wter. The fluid residue is removed by positive pressure, the column is dried for 2 min by floating it with air or nitrogen. Then elute sample with 1 mL 80% methanol in water and di lut
29、e the eluent with 1 mL 80% methanol in water, further dilute this solution with 2 mL doubled dis tilled water and employ 20 fL L in the assay. 3.5 ELlSA determination 3.5.1 Test procedure Bring all reagents and the microtiter plate to room temperature before use(20(; -25(;). Pipette tips should be c
30、hanged by using for different reagents and sample solutions 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 20 fL L of six kinds of diethylstil bestrol standard solution and sampl
31、e solution to separate duplicate wells. Add 50L of diluted anti diethylstilbestrol antibody solution to each well , flap lightly and mix thoroughly, sealing all wells with adhesive paper to avoid solution evaporating , incubate for 20 h at 2(; -8(; in darkness. The microtiter must be brought to room
32、 temperature. Pour the liquid out of the wells and wash the wells three times. Add 50L of diluted DES enzyme conjugate to the bottom of each well , mix gently and incubate for 1 h at 20(; -25(;. Pour the liquid out of the wells and wash the wells three times. Add 50L of substrate and 50L of chromoge
33、n to each well , flap lightly and mix thoroughly and incubate for 30 min at 20(; -25(; in darkness. Add 100L of stop solution to each well , flap lightly and mix thoroughly. Measure and record the absorbance value of each well solution at 450 nm within 30 min 3.5.2 Blank test Follow the sample extra
34、ction and test procedure without sampling. 3.6 results expression Calculate absorbance value in percentage according to the formula (1) , 酬。阳ncevalue in阳m时e=LE古二旦出100%. ( 1 ) where B叫时!=由mean absorbance value of standards and samples , %, Bb1ook-mean absorbance value of blank, Bo-Zero standard mean
35、absorbance value 1) Gives this information i5 in order to the user who facilitate this standard. does not express to 50me product sequence 。foperation approval. The testing process of another equal kits maybe a little differ. 50 it should be used after te5-ting .evaluation and verification 10 SN/T 1
36、956-2007 Make the absorbance value in percentage as ordinate, and diethylstilbestrol concentration (ng/mL) as abscissa , plot the calibration curve of the absorbance value in percentage and diethylstilbestrol standard concentration (see Annex B). A new calibration curve should be prepared for each d
37、etermi nation. Read corresponding to the concentration of each sample from calibration curve and calculate according to the formula (2): where X=CXVX1000 -一mXl000 X-The diethylstilbestrol residues content of the test sample，g/kg; c-The diethylstilbestrol concentration value of sample read from calib
38、ration curve , ng/mL; V-The total volume of sample solution , mL; m-The total sample quality of sample solution , 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
39、of this method is O. 5g/kg. 5 confirmation If the diethylstilbestrol residue is over the limit of determination , it should be confirmed byanother method. 6 Recovery See Annex B. 11 SN/T 1956 2007 Annex A (Informative Annex) Enzyme immunoassay kit for the quantitative analysis of diethylstilbestrol
40、A. 1 Enzyme immunoassay kit for the quantitative analysis of diethylstilbestrol The r-biophann kit of Gennany is used in this standard 3.5 ELlSA determinat旧n，Each test kitntains， a) Microtiter plate with 96 wells; i;，;/ b) DES standard solution ,0 ，Ot.o2坚，0队.05.冶0.10，0.2，0.4n斗g/m L DS in 40% met甘怕h丁
41、anol，r陌ead to use; c) 叫g归a挝t叫呻川DE阳lZy而矶lat古古云J步，/d) Anti-DES-antibody, The anti-DES an_.tiddy is dilute(i11 in buffer; ,y ，/ / e) Substrate; XJ笔也000000 54321 f) Chromogen; g) Stop solution, h) Buffer. A. 2 0.025 0.05 0.1 0.2 0.4 Stan由rdconcentration!(ng/mL) Figure A. 1一Diethylstilbestrolcalibration
42、curve 2) Gives this information is in order to the user who facilitate this standard.does not express to some product sequence 。foperation approval. The testing process of another equal kits fhaybe a littJe differ. so it should be used after tes ting ,evaluation and verification 12 SN/T 1956-2007 An
43、nex B Clnformative Annex) Recovery The experimental data of these fortifying concentrations of diethylstilbestrol and recoveries are as follows: At 0.5g/kg. the recovery is 85.2% -97.5%; At 5.0g/kg， the recoverylis 84.5% -94. 8%. 7/ / 13 中华人民共和国出人搅检验检疫行业标准肉及肉制品中己烯雌盼残留量检测方法酶联免疫法S:-.I/T 19562口87 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网址电话，6852394668517548 中国标准出版社秦皇岛印刷厂印刷骨开丰880X1230 1/16 印张1.25 字数24于字2007年11月第一版27年11月第一次印刷印数1-2000 书号，155066.2-18238 定价12.00兀
