【商检行业标准(SN)】 肉及肉制品中已烯雌酚残留量检测方法 酶联免疫法

中华人民共和国出入境检验检疫行业标准SN/T 1956-2007
肉及肉制品中已烯雌酚残留量检测方法酶联免疫法
Determination of diethylstilbestrol residues in meat andmeat product-Enzyme linked immunosorbent assay2007-08-06发布
中华人民共和国
国家质量监督检验检疫总局
2008-03-01实施
本标准的附录A和附录B均为资料性附录。本标准出国家认证认川监督管理委员会提出开归口。本标准起草单位：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、郑文杰、唐丹舟、魏亚东、张宏作。本标准系百次发布的出入境检验检疫行业标准。SN/T 1956—2007
1范国
肉及肉制品中已烯雌酚残留量检测方法酶联免疫法
木标规定了肉及肉制品中已烯滩酸残留的酶联免疫测定方法。本标准适于鸡肉、猪肉、牛肉、羊肉、猪肉饼巾已烯雌酚残留杜的检测。2试样的制备和保存
2.1试样的制备
SN/T 1956—2007
从所服全部样品中出有管
代表性样品约！kg，充分搅碎，混每，采用四分法，将样品分成两等份，装人洁净容器，加封并做标识。
2.2试样的保存
试样放置20℃～～18亡条件下保存3测定方法
3.1方法提要
本方法的测定基础是竞争性确联免疫反应：已烯醛酚与已烯雕酚酶标记物共同竞争已烯惟酸抗体的结合位点，用酶标仪测量微孔溶液的吸范度值已烯雅耐淡疫与吸光度值成反比，按绘制的校正曲线定量计。
3.2试剂和材料
除另有规定外，所用化学试帮均为分析纯战为更高级别。水为查葬水。3. 2. 1
已矫雌酚酶联免疫定量测定试剂盒（参见附录A)叔丁基甲基醚：色谱纯
右汕醚。
二叙甲烷，
磷酸。
3.2.6盐酸。
伊醇：色谱绝。
磷酸二钠(Na.HPU2H.O
磷酸二氢钠(NaH,PO,·H,O)。
氯化钠。
兰羟甲基氨基甲烷。
3.2. 12磷酸盐凝冲液(67 mr1oL/I+PH=7.2):称瑕 9.61 g磷酸氢二钠.1. 79 g磷酸二氢钠和 8.7 g氛化钠溶辉于并足容至1.000mI.水t3. 2. 13 Tri:缓冲液(20 tnmol/L,pH=8. 5):称取 2. 42 g 三羟甲基兹基甲烷溶解于 700 mL 水中,加人 200 mL 甲醇,用 5 mol/I. 盐酸调节 pH 值至 8. 5,加水定容率至 1 000 ml.。3.2.14洗涤用磷酸盐缓冲液（20mmo1/L.pH=7.2)：称取2.85g磷酸氢二钠0.55g磷酸二氢钠，9 氯化钠和1mL吐温20,解并定容至1 000 mL水中，3.2.15氮氧化销溶液（1ma1/L)：称取0氢氧化钠溶子1000mL水中。SS/T1956—2007
3.2.16己烯雌酚标准品：标推品纯度学98%。3.2.17已烯雄酚标准品溶液的配制：推确称取适量的己稀雅标准品，用中醇配制成1mg/mL标准贮备溶液，于 4℃～8℃条件下保存。3.3仪器和设备
3.3. 1酶标仪：波长450 nm。
3.3.2高剪切分散器。
3.3.3涡旋振菌器。
天平。
离心机：3 000 r/rnin。
3.3.6氮气吹干仪。
洗板机。
固相举取仪。
3.3.9水浴锅：70℃±2℃。
3.3.10微量加样20 μl,50 ×L,100 μL,200 μL。3.3.11微航多通道加样器:50 μl.100 μl3. 3. 12RIDA Cr.柱: 10I mg,1 ml.3.4样品处理
3.4.1试样提取
称取 5. 0 g试样至离心管中,加人 10 nL 67 nmol/1. pH 7,2磷酸盐缓冲液,均质-历 8 mL 叔丁基甲基提取3.0R均质物，强烈振荡20min3000r/min离心10mini。转移出上清液，再用8切L叔基甲基醛重复提攻沉淀物，将两次提取的醚相合并，70℃水浴蒸发至下。用1mL70%甲醇溶解干煤的残留物，用3mI.石油醚洗涤甲醇溶液，均质155.30001/min短时离心，吸除石油醚。70℃水浴蒸于甲醇溶液,用 1 mL 二氯甲烷溶解.用 3 mL氢氧化钠(NaOH)溶液提取。用 300 μL 6 mol/I. 磷酸中和提取液。
3.4.2样品纯化下载标准就来标准下载网
用RIDAC柱纯化，流速均为1/s。用3mL无水F醇洗涤柱子,用2nL20IIol/1.pH8.5的Tris 缓冲液乎衡柱。将上述中和提取液加人柱中,用 2 mL 20 mmnl/I. pII 8. 5 的 Tris 缓液洗涤柱-用3mL40%中醇洗涤柱于，用正压去除残留的液体并且用空气或氮气吹2nin以干燥栏了。用1mL80%甲醇洗脱样品，用1mL80%甲醇稀释洗脱液，加入2mL蒸馏水进一步稀释，取20L进行分析。3.5酶联免症测定
3. 5. 1测定步骤>
已烯雌酚试剂盒中所有试剂的渴度均应回升至室温（20℃-~25℃）后方可使用。测定中吸取不同的试剂和样品溶液时应更换吸头。将测定用的微孔条捕人框架（标准液和样液，空白分别作平行试验测定），记录标准液和利样液的位置。分别圾取20L已烯耻酚标溶液，样品落液至各自的微孔，再加人50I.已稀释的已烯雄酚抗体至个微孔，持微孔板在台面上以圆周运动方式混勾后，然后用封口膜密封孔条以防溶液择发，于2℃～8℃避光孵存至少20h。微孔板恢复至室温，倒掉微孔中的液体，用洗涤缓冲液洗板探作3次，加人50μL已稀释的己烯雌骼酶标记物，充分混匀，室混孵有1h。倒微孔中的液体，洗板操作3次。加人50μL酵基质和50ul.发色剂至各微孔中，充分混勾，20℃~25℃处避光孵育30min。加入100L反应终止液至每个微孔中，充分混匀，在30min内测并记录每个激孔溶液450nm波长的吸光度值，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女骤的认可。如果其他产品的操作步骤有不同，需经实验评估后来川。
3.5.2空白试验
除不称取试样外,均接上述步骤进行。3.6 结果计算
按式(1)计算百分比吸光度值：
#—B×100%
B糖岛/H作
吸光度值－
B我品/标准
标谁品或样品的平均吸光度值%；空自孔的平均吸光度侦，
B：：零标难的平均吸光度值。
SN/T1956—2007
以百分比吸光按值为纵坐标，以已烯端酚标雄济液的浓度（ng/nL)的对数为横坐标，绘制出校正曲线（参见附录H）。每次试验均应罩新绘制校正明线。从标准山线上得到试样中相应的已烯难酚浓度后，结累按式(2)进行计算：
X EXVX1000
m × 1 000
x--样品中己烯雌酚的残留量，单位为微克每于克(ug/kg)；从标推曲线上得到的样品中已烯雌酚浓度，单位为纳克每毫升（n/mL)；V一一样品溶液的最经定容体积.单位为毫升(mI.);-样品落没所代表的歧终试择质量，单位为克（g)。也可以旧各种酶标仪的数处理软件进行计样，所得结累表示至一位小数。4检测限
方法的检山限为 0. 5 ug/kg。
5确证试验
如被测样品中已烯雌盼残留录的值大于限症要求时，应用其他力法进行确证。6回收率
回收率试验数拇参见随录B。
SN/T1956—2007
附景A
（资料性附录）
已烯雌酚酶联免疫定量测定试剂盒已烯雌酚酶联免疫定最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公词产品\，试剂盒包拆：耗架，95孔板：
标雅溶液，40%己烯雌甲醇溶液：0.0.025.0.05,0.10,0.2,0.4tg/mL；已烯雌酚酶标记物济液：用稀精缓计液以T化例稀释淡液，充分混分后使用：己焙雄酚抗体浓缩没：用称释缓冲液以上11比倒税释浓缩演，充分跟勾后使用；酶基质；
发色剂；
反应终止液：
稀释缓冲溶液。
已烯雌酚校正曲线
标涤法度/(ng/nl.)
己烯雌酚校正曲线
2)给出该信息是为了方使本标准的使用者，并不表示对某一产品的认可。如单其他产品且有相同的效果,需经实验评估后使用这些等效产品。
（资料性附录）
回收率
本方法中已烯酚添加浓度及回收率数据：添加量为0. 5 μg/kg时,平均向收率为 85. 2%~97. 5%添加量为1.0μg/k时，平均回收率为82%~-104.2%；添加抵为 6. 0 μg/kg时,平均回收率为 81.55%～94.8%。SN/T1956—2007
SN/T1956—2007
Foreword
Annex A and B of this standard are informative annex.This standard was proposed by Certification and Aceredditation Adiministration of the Peopte's Re-public of china.
This standard was mainly drafted by Tianjin Entry-Exit Inspectian and Quarantine Bureau of the People's Republic of china.
This standard was rmainly drafted by Sun Li, Wenjie Zheng , Danzhou Tang , Yadong Wei, HongweiZhang.
This standard is professional standard of Entry-exit inspection and quarantine pramulgated far thefirst time.
SN/T 1956—2007
Determination of diethylstilbestrol residues in meat andmeat product-Enzyme linked immunosorbent assayScope
This standard specifies the determination by ELisA method of diethlstilbestrol residuse in meat andmeat product.
This standard is applicable ta the screen determination of diethlstitbestral residuse in chicken meat,pork, beef.mutton and breaded pork.2 Sample preparation and storage2. 1sampling procedure
The representative sample should be taken out, and totat weight is not less than 1 kg. Samnple defa.ted and homogenized thoroughly. At [east each sample should be divided into equal portions. Afrsamples should be placede in a clean and dry container to maintain the sample completeness andtraceability, then change into a bigger clean container to seal up and transport. The label should belabeled out side of each sample container.2. 2 Storage of sample
After sampling, the test samplo should be stored at - 2o'c - 18' .3Method of determination
3. 1Principle of determinationThe basis of the test is the competitive enzyme-linked immunoreactions. Diethylstilbestro! anddiethylstilbastrol enzyme conjugate compete for the limited anti- diethylstilbestrol antibody. The ab-sorbance value of each well solution is measured by microwell system. The diethylstitbestro! concen-tration is inversely proportional to the absorption value, and compare with the calibration curve forquantitative measurement.
SN/T 1956—2007
3.2 Reagents and materials
In this standard, all the chemical reagents should be A. R. grade unfess it identified specially,\water\is doubled distilled water,
Enzyme immunoassay kit for the quantitative analysis of diethylstilbestrol(see Annex A).Tert-butylmethylether:chromatographic grade.Petro[eurm ether.
Dichloromethane
Phosphoric acid.
Methanal ; Chromatogram
Na, HPO, - 2H,O.
NaH, POr + H,Q.
Sodium chloride.
Tris-(hydroxymethyl)-arinomethdne :Dissplve 9. 61 g 'NaHPO, ? 2H,O, 1. 79 g NaH, PO4Phosphate buffer(67 mnaol/Lr3.2. 12
- Ha and 8.7 g Sodium chloride oo mt distiriedlwate3. 2. 13
Tris-HCl-buffer(20 mmol/ L
=g.5),Dissolve2.+2g.tris-(hydroxymethyl)-aminometha-nein approx. 700 mL distilled water,mixwith,200'mL methahal-adjust pH 8, 5 with 5 mol/L HC1,andfill up to 1 00o mL with distilled water.3. 2. 14
Washing buffer(20 mmol/L.pH= 7, 2) : Dissolve 2. 85 g Na2 HPO4 - 2H,O.0. 55 g NaH,PO, H,,9 g NaCt and 1 mL Tween 20 in 1 000 mL distilled water.3. 2. 15
1 mol/L NaOH:Dissolve 40 g sodium hydroxide in 1 c00 mL distilled water.The purity of diethylstilbestrol is over 98%.preparation af solution:Dissolve same DEs in methanol,and the concentration of the stor-age splutions is 1 mg/mL, Then store at 4'℃ ~8'℃.8
3. 3Apparatus and equipment
3.3.1Microwell system:450 nm.Homogenizer.
Centrifuge:3 000 r/min.
Nitrogen Evaporator.
Washing machine.
SPE cartridge.
Waterbathboiler:70℃±
Pipettes:20μL50μL.100uL,200μLMultichannel pipetre:50 μL,100fl3. 3. 12
RIDA Cie columns: 100 m
3.4 Sample extraction and cleanjup3.4.1 Sample extraction
SN/T1956—2007
Weigh 5. a g of the test sample intd,a centrifug tube, add 1o mL 67,mmol/L pH 7. 2 phosphate buff.er, vibrate for 1 min, take 3. 0 g of-the above sample , with S ml Tert-butylmethylether, vibrate2 min strongly. Centrifuge at room temperature 3 o00 r/mih-for 10 min. Remove the ether superna-tant into a glass centrifuge tube. Repeat the extraction with 8 mL Tert-butylmethylether, Cormbinethe ether phasg, the organic phase evaparated with nitrogen gas in water bath at 7o. Dissolve theresidue in 1 mL70% methanol. wash the methanolic solutian with 3 mL petroleum ether, homoge-nize for 15 s, centrifuge 3 000 r/min shortly: aspirate and reject petroleum ether supernatant. Themethanalic solution is then evaporated to dryness urnder a gentle stream of nitrogen. The residue isdissolved in 1 mL dichlormethane and extracted with 3 mL NaOk. Neutralize the extractwith 300 μ L 6 mol/L phosphoric acid.3.4.2Sampie cieanup
The extract is further purified as followingbymeans of RIDA Cre columns.The column is rinsed with5
SN/T 1956--2007
3 mL of methanol and equilibrated with 2 ml 20 mmol/L tris-HCl. The sample solution pass thoughthe column at a rate of 1 drop/s, followed by rinsing with 2 mL of 2 mL 20 mmol/L tris-HCl and 3 ml40% methanol in water. The fluid residue is removed by positive pressure, the column is dried for2 min by floating it with air or nitrogen. Then elute sanple with 1 m 80% methanol in water and di.lute the eluent with 1 ml 80% methanol in water, further dilute this solution with 2 mL doubled dis-tifed water and emptoy 20 pL in the assay.3.5 ELISA determination
3.5.1Testprocedurel)
Bring all reagents and the microtiter plate toroom temperature before use(20C~25'c).Pipette tipsshould be changed by using fordifferent reagentsand sample solutions.Insert a sufficient amount of microtitre wells into the microwell holder for all standards and samplesto be run in duplicate. Record standard and sample positions. Add 20 μL of six kinds of diethylstil-bestral standard solution and sample solution to separate duplicate wells. Add 50 μL of diluted anti-diethylstilbestrol antibody solution to each well, flap lightly and mix thoroughly, sealing all wellswith adhesive paper to avaid solutian evaporating, incubate for 20 h at 2℃ -8'c in darkness. Themicrotiter must be brought to room temperature. Pour the liquid out of the wells and wash the wellsthree times. Add 50 μL of dilutad DEs enzyme conjugate to the bottom of each well, mix gently andincubate for 1 h at 20'C -~25', Pour the liguid out af the wells and wash the wells three times. Add5o μL of substrate and 50 μL of chrornogen to each well, flap lightly and mix thoroughly and incubatefor 30 min at 20℃ ~25'℃ in darkness. Add 100 μL of stop solution to each well, tlap lightly and mixthoroughly.Measure and record the absorbance value of each well solution at 450 nm within 30 min.3.5.2 Blank test
Follow the sample extraction and test procedure withaut sampling.3.6resultsexpression
Calculateabsorbancevalue in percentage according to the formula(1):absorbance value in percentagewhere
e-Baank×100%
B,.andard:cam
Bstondkr/saomean absorbance value of standards and samples. % Bunkmeanabsorbancevalue of blank;B,:Zero standard mean absorbance value.t)
1) Gives this information is in order to the user who facilitatg this standard,does nat express to some product seuenceof operation approval. The testing process of another equal kits maybe a little differ,so it should be used after tes-ting : evaluation and verification.1c
小提示：此标准内容仅展示完整标准里的部分截取内容，若需要完整标准请到上方自行免费下载完整标准文档。