【商检行业标准(SN)】 动物源性食品中二苯乙烯类激素残留量的检测方法 酶联免疫法

中华人民共和国出入境检验检疫行业标准SN/T1955—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 assay2007-08-06发布
中华人民共和国
国家质量监督检验检疫总局
酷码伤
2008-03-01实施
本标准的附录A和附录B为资料性附录：言
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：华人民其利国津出入境检验检疫局。本标雅尘要起草人；孙例、郑文杰、店丹舟、魏亚东，张宏伟。本标准系首欲发布的出入境检验检疫行业标准。SN/T1955—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
SN/T 1955—2007
本标准规定了动物源忙食品中二案乙烯类激素残留盘检验的酶联免疫测定方法。本标准川于鸡肉、鱼肉虾肉及鸡肝中已烷雌酚已烯酚残留乱的检测。2试样的制各和保存
2. 1试样的制备
从所取全部样品中取出有代表生样品约1飞，充分揽碎，握匀，亲用四分法，将样品分成两等份，装入洁净容露，加封并做标识。
2.2试样的保存
试样放置一20℃～18%℃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尔性联免疫反应。醇标极上已包被的雌抗体，可与已烷雅酚，已烯雌酚发生交联反应，标准液或样品中酵确酚好球根轧化游标记的单照抗原共间争夺此酚抗体上的结合位点，用酶标仪测量微孔溶液的吸光度值，筐舒浓度与殿死度置放反比.按绘制的校正帕线定冠计算。3.2试剂和材料
除有规定外，所用化学试剂到并分斯纯，或为更高级别。水为源蒸留水。3.2.1二苯乙烯类免疫测定试南盒（念览附录A）3.2.2权了基凹格醚：色谱纯，
三氯甲烷。
3.2.46mnl/1.磷酸：吸取58.
m做酸游于水求中，非定穿求
3.2.5乙醇。
3.2.68葡糖许酸酶SigmaG-0876)100mz
乙腔钓(CHCOONa·3H
乙酸销缓冲波(0.1mo1/L-pH=切称取13.G艺殿钠济解丁800ml水川氢氧化钠溶3.2.8
液调节pH值至5.0±0. 1,加水定穿至1000 mL。3,2.9氢氧化钝溶液(1umal/1.)：称取40g氢氧化钠溶1000ml.水中。印.
标推品：已烷雌粉标准品已烯雄龄标难品纯座均8%。维酚标准品溶液的配制：推确称取适整的己烷州龄和已矫脏配标雅品，用甲醇配制成1mg/mL标准贮备挤液.于4℃～8℃条件下保存，3.3仪器和设备
3.3. 1标仪：波长450 nm。
3.3.237℃=2℃培举箱。
3.3.3均质器，
SN/T 1955—2007
3.3.4天平。
3.3.5离心机：3000z/min。
3.3.6氮气吹干仪。
3.3.7振荡器。
3.3.8洗板机。
3.3.9国相举仪。
3.3.10微量加样器20μ,50100l200。3.3.11微量多通道加样器：200L3.3.12免疫亲和柱：50ng，5ml.。3.4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)试样丁离心管中，加人15ml.权丁基甲基醚，均质30s.涡旋振药3min。在2000z/min下离心10min，移取12niL醚层液休.在10℃氮气流下蒸发至于燥，用1mL三甲烷溶解下燥物，涡旋报药3min。加2mL1mol/L氢氧化钠，涡旋振菌3min在2000r/min下离心10min,移取上层波到另一试管t，保留1ml.水相。再加人1mL1mol/L氢氧化钠到三氯甲烷落被中，涡旋振荡3min，在2000r/min下离心10mit，移取上层提取液到另一试管中，保留1mL水相。合并两次提取液，加入200μL的6mol/L磷酸巾和2mL的氢氧化钠提取液后待疗化。3.4.1.2肝脏样品
称取2.5g(精确到0.1g)试样至离心管中，如人3mL乙酸销缓冲液(0.1mol/LpH5.0)，加人8000单位9的糖苷酸酶，均质约30。37℃±2℃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被。用杜错存缓冲液过柱，再用15mL柱洗涤缓冲液半衡柱子（流速≤3mL/min）。取全部中和后的样品提取液过柱（更力引流）。用5mL柱洗涤缓冲液洗涤柱子两次（流速≤2mL/min）。用5ml水洗涤粗了（流速≤2ml/min）。用3ml.乙醇/水（70/30.体积比）洗脱样品中可能存在的雌激索。此步的洗脱液用干净的试管收集质用于试剂盒检测。3.5酶联免疫测定
3.5.1操作条件
所有操作应在究温下（20℃～25℃）进行，雌酚试剂盒中所有试剂的温度均应回升至室温（20℃~25℃)后方而使用。
3.5.2测定步骤
将测定需用的微孔条插人框架（标准液，样液和空白分别作平行试验测定），记录标波和样液的位置，先吸取100μL已稀释的稀释缓冲溶液至酶标板各孔内，再分别吸取25叫L难爵标准溶液、样品游液至各白的微孔，持微孔板在台面上以圆周运动方式混幻后，然后用封口膜密封孔条以防落液挥发。将其置于20℃～25℃，避光游育1h。加人75aL已稀释的醇标记抗原至每个微孔，混勾，覆盖上封口膜，20℃～25℃避光孵育30min。倒掉微孔中的液体，用洗涤缓冲液洗板操作12次。加人125μI发色剂至每一微孔中，充分滤勾，20℃25℃避光解育20min。加入100叫L终止液至每个微孔中，充分混勺，在30 min内，测录并记录每个微孔溶获450 nm波长的吸光度值。3. 5. 3 空白试验此内容来自标准下载网
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不标准的使用者，并不表示对某一产品操作步骤的认可。如更其他产品的操作步操有不同,需经实验评估后采用。
3.5.4监控试验
每次测定均应一个添期雌酚标准的样品，添加浓度为相应产品的捡测限量。3.6结果表述
按式(1)计算百分比吸光度值：
吸光度值B5准/样%—
式中?
Ban×100%
Bi作品
一标摧品或样品微孔的平均吸光度值，%；-空白孔的平均吸光度值
Bu——零标准的平均吸光度值。SN/T 1955—2007
以百分比吸光度值(算术级)为纵坐标，以雌酚标准辫液的浓度（ng/ml.)(对数级)为横坐标终制出雌酚标准浓百分比吸光度值与雌酚浓度的控正也线（参见附录A）。每次试验均应再新绘制校正曲线。从标准工作曲线上得到试样中相应的雌翻浓度后，结果按式(2)进行计算；X=-eXVx1000
m × 1 000
式中：
样品中雌的残留量，单位为微克每千克(%/k）；c-从标准工作曲线上得到的样品中雌酚浓度，单位为纳克每毫升(ng/ml)：V.-样品落波的最终定容体积，单位为垒升（mL)；一样品落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鹅处理软件进行计算，所得结果表示至一位小数。4检测限
本方法的检出限已烧雌酚为0.5g/kg，已烯辉酚为1.0g/kg5确证试验
如被测样品中凹烷雌酚，已烯雌酚残留量的值火于检測限时应用仪器方法进行确证。6回收率
问收率试验数据参见附录B。
（2）
SN/T 1955—2007
A，1雌酶联免疫定试剂盒
附录A
（资料性附录）
雌酚酶联免疫测定试剂盒2
本标准酶联免疫别定步骤中使用的试剂盒为英国RAVDOX公司产品，试剂盒他括：糕架：95孔板；
标准溶液：0ng/ml..0.05ng/ml..0.25ng/ml.0.53pg/mL.l.0ug/ml.s.0ng/ml.；雕酚酶标记物溶液。按载剂意楚供前化例以酸称释释液针稀释，充分混句后使川：酶标稀释液；
发色剂：
反应终止液：
洗涤/释缓冲溶液。
雌酚免疫亲和柱
本标准净化步骤中使用的免痒象和赶为英国RAN公司童，储存在2℃～8℃。杜储存缓冲液，用双蒸水以119比例稀释燃缩波：非洗涤缀冲液，用双蒸水以
A.3雌标准校正曲线
4比例释释波琬液，
标准残获度ngmt.)
图入。1雄懿标滋校正曲线
2）给马该信息是为了力便在标准的使用者，并不示对禁·产品的认可。如别共他产品具有相同的效患，需经实验评估后使用这些等效产品。
（资料性附录）
回收率
本方法中二花乙烯类激素添加浓度及回收率数：已烷雌粉：
涨加量为0.5/k时，平均回收率为85.5%~91.7%；一添加量为1.0g/kg时.平均回收率为81.2%～1051%：薪加质为5.0μg/kg时，平均回收率为87.6%～101.9%。己烯雉酚：
添加股为1.0μg/kg时，率均回收率为70.3%~91.1%：添加量为2.0Pg/kg吋，铲均回收率为76.5%～90%；-添加量为 10.0 μg/kg时,平均回收等为7范%～94.3%，SN/T 1955—2007
SN/T 19552007
Foreword
Annex A and B of this standard are informative annex.This standard was proposed by Certification and Accredditation Adiministration of the People's Re-public of china.
This standard was mainly drafted by Tianjin Entry-Exit Inspection and Quarantine Bureau afthe Peo.ple's Repubtic of china.
This standard was mainly drafted by Sun Li, Wenjie Zheng , Danzhou Tang , Yadong Wei, HongweiZhang.
This standard is professianal standard of Entry-exit inspection and quarantine promulgated for thefirst time.
SN/T 1955—2007
Determination of stilbenes residues in foodstuffs ofanimal origin --Enzyme linked immunosorbent assay1Scope
This standard specifies the determination by ELisA method of stilbenes residuses in foodstuffs afanimal origin.
This standard is applicable to the screen determination of stilbenes residuses in chicken meat, pork.fish, shtimp and chicken liver.2 Sarmple preparation and storage2.1 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 sample should be divided inta equat portions. Allsamples should be placede in a clean and dry container. then seal up. The label should be ladcled outside of each sample container.2.2 Storage of sample
After sampling,the test sample shauld be stored at - 20'℃ ~ - 18'c.3Method af determination
3.1 Principle af determinationThe basis of the testis the compeititve enzyme-linked immunoreaction. stilbene and stilbene enzymecanjugate compete for the limited anti-endosulfan antibody. The absorbance value of each well solu-tion is measured by microwell system. The stitbenes concentration is inversely proportional to theabsorption value, and compare with the calibration curve for quantitative maasurement.3.2 Reagents and materials
In this standard,all the chemical reagents should be A. R. grade unless it identified specialy,\water\ is doubled distilled water.SN/T 1955—2007
Enzyme immunoassay kit for the quantitative analysis of stilbenes(see Annex A).Tert-butylmethylether-chromatographic gradeChloraform.
6 mol/L phosphoric acid. Dissolve 58.8 mL phosphoric acid in 1oo mL distitled water.Ethano!
g-gtucurronidase (Sigma Gto876)Sodium acetate (CH,cooNa3H:O)-0. 1 mol/L Sodium acetate (pH= 5-0). Dissolve_13. 6 g Sadium acetate (3. 2. 7) in 800 mL3.2.8
distilled water, adjust to pH 5. o ±o. 1 1 mal/L. NaOH, the total volume is 1 0a0 mL3.2.9
1 mo1/L NaOH: Dissoive ko g Sodium hydroxide in 1 o00.m distilled water.Methanol.
The purity of hexestral ang-rethylstilbestrol is over gyPreparation of solution/Dissotve some DEs ang.HEx in mcihanol, and the concentration ofAt4℃-8℃.
thestoragesolutionsis1mg/my
Apparatus and equipment
Microwell system:45o
37'℃ ±2℃ incubator.
Homogenizer
Centrifuge: 3 000 r/min
Nitrogen Evaparator.
Thenrsto
3.3,8Washing machine.
SPE cartridge
Pipettes: 20 μL, 50 μL, 100 μL,200 μL3. 3, 11 Mulitichannel pipette: 200 μL.3. 3. 12
Irmmunoaffinitycotumns:50ng:5mL.3. 4Sarnple extraction and cleant3. 4. 1Sample extraction
3.4. 1. 1 Tissue samptes
SN/T 1955—2007
Weight 2, 5 g (precision 0. 1 g)of the-test sampte intb a centtrifuge tube, add 1s mL of Tert-butylm-ethylether. Homogenise the sanie for aproximiately- 3QwGythenyortex for 3 min. Centrifuge for10 min at 2 0oa r/min, Remove j2 mt af ths ether layer. Evapurate to dryness at 40℃ in a waterbath with air stream. Dissolved In 1 mL chlorofarm and vortex iof3 min, Add 2 mL 1 mol/L NaOHand vortex for 3 min. Centifugekor-1o-min'at'2:0o0-r-min:Rcmove and keep 1 mt of the agueousTayer. Add a further 1 mL 1 mol/L NaOH to the chlaroformaver. Repeat this step and combine thef by adding 20D geL 6 mol/L phosphoric acid to theextracts. Neutralise the pH of the Naot extracr2 mL portion. Apply the total vghime of, the ngutralised fraction th the column after equilibration.3, 4. 1.2 Liver samples
Weight 2. 5 g (precision 0. 1 g of the, test Aample ihto a ceftrifugei tube, Add 3 mL of sodium acetate buffer to the sample. Add'g aoo units of &-cluclronidase, ondf Fomogenise for 30 s, Incubate for2 h at 37'℃ ±2'℃. Then follow th3. 4. 2 Sample cleanup
e tissue samplg extraction procedures 3. 4. 1. 1.The extract is further purified as fallowed by rmeans of immunoaffinity columns. Allaw calumn stor-age buffer to flow through and then equilibrate with 15 mt filuted column wash buffer at a rate af3 mL/min. Load total volum of extraction onto the colurmr and ailow 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 thecolumn with 5 mL doubled distilled water at a rate of 2 mt/min. Eluted by the application of 3 ml.70% ethanol/30% water and collecting the eluate in a clean glass test tube. The sampte is naw reacdyfor application to the stilpene ELISA microtitre plate.9
SN/T 1955—2007
3.5 ELISA determination
3. 5. 1 Test condition
All the procedures should be done at room temperature (20'C ~25'c ). Bring all reagents and the mi-crotiter plate to room temperature before use (20℃ 25'c) :3. 5.2 Test procedurel>
Insert a sufficient armount of microtitre wells into the microwell holder far all standards and samplesto be run in duplicate. Record standard and sample positions, Add 1oo μL the dituent buffer to eachwells. Add 25 ul of six kinds of stilbene standard solution and sample solution to separate duplicatewells, Flap lightly and mix thoroughly, sealing all wells with adhesive paperto avoid solution evapo-rating. incubate for 1 h at 20 ~25C in darkness.Add 75 gμl of diluted enzyme conjugate to thebottom of each well, mix gently and incubate for 3D min at 20'C~25'c. Pour the tiguid aut of thewells and wash the wells twelve times, Add 125 μL of one shot substrate to each well, flap lightlyand mix thoroughty and incubate for 20 min at 20c ~25℃ in darkness. Add 100 μL of stop solutiorto each well, flap lightly and mix thoroughly. Measure and record the absorbance value af each wellsolution at 450 nm within 30 min.3.5.3Blank test
Followthe sample extraction and test procedure without sampling3.5.4Control test
Each test shuld determinate a fortified sample. The fortifying concentrations of stilbene is the limitof determination.
3. 6 Calculation and expressCalculate absorbance yalue in percentage according to the formula (1) :a-Bmiark×100%
Baedaeri:t
absorbance value in percentage% = B.
Bstenars?anpe --maan absorbance value of standards and samples,% ;Buaik mean absorbence value of blank:B。-Zero standatd mean absorbance value.1
1) Gives this information is in order to the user who facilitate this stardard,does not express to some product se-quonce of operation approval. The testing procass of another equral kits maybe a littfe difertso it should be uscdafter testing,evaluation and verification.10
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