1、s 中华人民共和国出入境检验检疫行业标准SN/T 1960一-2007进出口动物源性食品中磺肢类药物残留量的检测方法酶联免疫吸附法Screening method of sulfonamide residues in animal original food for import and export-Enzyme Iinked immunosorbent assay method 2007-08-06发布2008-03-01实施气龟咽?如雷叫硝)AMm企唰中华人民共和国发布国家质量监督检验检疫总局SN/T 1960-2007 前古同本标准附录A、附录B、附录C和附录D均为资料性附录。本标准由
2、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入境检验检疫局、天津科技大学、中华人民共和国湖北出入挠局检验检疫局。本标准主要起草人:郑文杰、王硕、张宏伟、胡小钟、吴廷晖、姚霞、张鸿雁、张杰、张骏。本标准系首次发布的出入境检验检疫行业标准a1 范围进出口动物源性食品中磺股类药物残留量的检测方法酶联免疫吸附法本标准规定了动物源性食品中七种磺胶类药物残留量的fj联免疫吸附测定方法。SN/T 1960一2007本标准适用于猪肉、鸡肉、猪肝、鸡蛋、鱼、牛奶中七种磺胶类药物残留量的筛选检测.2 规范性引用文件下列文件中的条款通过本标准叫引用而成为本标准的条款。凡是述日期的引用文
3、件，其随后所有的修改单(不包括勘误的内容)或修订I!J:&:均不运用于本标准，然而，鼓励根据本标准达成协议的各方研究是否可使用这些文件的最新版本。只品i卒注应且J胡启!L豆IJlJ文件，其f盖新哪本适用于本标准。GB/T 6682 分析实验室用水规格和试验方法。/ff 3 测定方法Jf /j/ 3. 1 方法提要/ /卢扩，哩霄用提取液提取样品中磺胶类药物iJ吕立i争性购股兔疫吸野I法进行检矶。3.2 试剂和材料除另有要求外，所有试剂均为分也t.*.1i.皿LI.J到础剧二础。3.2. 1 磺胶类药物酶联免疫吸附测定试剂盒1(参见附号3.2.2 丙隅。3.2.3 乙醇。3.2.4 磷酸氢二纳(
4、Na，HPO, . 12 3.3 仪器和设备3.3. 1 粉碎机。3.3.2 均质器.3.3.3 天平(精确到0.001gl。3.3.4 旋涡混合器。1)磺胶类药物前联免疫吸附测定试剂盒是由天津生物芯片技术有限责任公司提供的产品的商品名称.给出这一信怠是为了方便本标准的使用者.并不表示对该产品的认可.如果其他等效产品具有相同的效果.则可使用这些等效产品。SN/T 1960-2007 3.3.5 离心机(转速4000r/minl。3.3.6 旋转蒸发仪.3.3.7 酶标仪(测定波长450nml。3.3.8 洗板机。3.3.9 单道微量加样器20L，1001.200L。3.3. 10 多通道微量加
5、样器:200Lo3.4 试样制备取样品约500g，将其切碎后，经捣碎，混匀，均分成两份，装人洁净容器内，作为试样，密封，并标明标记。3.5 试样保存将试佯于一18(;以下冷冻保存.在制样的操作过程中，应防止样品受到污染或发生残留物含盐的变化。3.6 提取3.6. 1 猪肉、鸡肉、鱼称取4g(精确到0.01gl均质样品到玻鸦瓶中，加入20mL磷酸盐缓冲液，均质，剧烈i昆匀2min. 离心(250C, 4000 r/min , 10 minl，上消液用磷酸盐缓冲液稀fH倍后进行测定。猪肉、鸡肉提取液用磷酸盐缓冲液稀释:鱼肉提取液用1%牛血清白蛋白-磷股盐缓冲液稀择。3.6.2 蛋类称取4g(精确到
6、0.01gl均质样品到孩璃瓶巾，加入20mL磷酸盐缓冲液，轻t30 min，避免乳化.吸取50fL 昆匀后的洛液用磷酸盐缓冲液稀释4倍后进行测定。3.6.3 猪肝称取2g(精确到0.01gl均质样品样品到玻璃瓶中，加入20mL乙晖，均质，剧烈混匀2min.离心(25(; ,4000 r/min , 10 minl，吸取10mL上清液于50mL旋蒸瓶中，350C减压浓缩至干，残留物用10 mL磷酸盐缓冲溶解，滤纸过滤，吸取50L滤液用0.5%牛血清白蛋白磷酸盐缓冲浪稀释2倍后进行测定。3.6.4 牛奶量取20mL(精确到0.1mL)牛奶样品到离心瓶中，离心(25(;.3000r/min.l0 m
7、inl，除去上层脂肪后，加入20mL丙酬，手摇混匀，离心(25(;.3000 r/min.l0 minl.吸取50L上消液用磷自主盐缓冲液稀释20倍后进行测定.3.7 酶联免疫法检测3.7. 1 标准曲线的制备用磷酸盐缓冲浪稀释10mg/L的破胶药物标准溶液储备液至100g/L，用PBS对100g/L的磺胶药物标准溶液进行1 3梯度稀释，得到一系列不同浓度的0Ji1胶药物标准溶液000问/L.33.3g/L.11. 1g/L.3.7g/1.l. 2g/L.0.4g!Llo 3.7.2 样晶测定在酶标极中分别加入100L不同浓度的磺胶药物标准溶液和样品待测液;在空白和对照孔中分别加入100L的水
8、;除空白孔外，在每个孔中加人100L的酶标记物稀释液$空白孔中则加入100VL磷酸盐缓冲液g轻拍混匀，用粘股纸封住微孔以防溶液挥发，室温孵育60min.，弃掉酶标板中的液体，用洗涤液洗板三次。加入混合好的底物液150L.轻轻混匀，室温放置30min后，每个孔加入50VL的终止液，轻轻晃动酶标板，10mo内在fj标仪上i卖出每孔450nm吸光值。3.8 空白试验除不加试样外，均按上述测定步骤进行。2 SN/T 1960-2007 3.9 结果的计算3.9. 1 计算抑制率值按式()计算不同浓度磺胶药物对抗原抗体结合反应的抑制率zr. AA二卢1IC = 11一一直坐-l旦Ix100% L A对照
9、-A空白) 1 ( . . . . . . . . . . . . . . . . . . . . . . . . . . . . 式中-IC黄胶药物对抗原抗体结合反应的抑制率，%;A对照一一不加入磺胶药物标准液，仅加人酶标记稀释物和磷酸盐缓冲液，在450nm下测得的平均吸光度值gA样品质胶药物标准液或样液在450口m下的平均吸光度值;A空白不加入酶标记稀释物及磺胶药物标准液，仅加人水利磷酸盐缓冲液，在450nm下测得的平均吸光度值。3.9.2 绘制标准曲线以抑制率为纵坐标，磺胶药物浓度为横坐标(横坐标取对数真l度)绘制标准曲线.每次试验均应重新绘制标准曲线，参见附录D。3.9.3 结果计算从
10、标准曲线上读取样液抑制率所对应的荫胶药物浓度(c)按式(2)计算试样中的政胶药物残留量X=cXR ( 2 ) 式中zx一一某种磺胶药物残留量，单位为微克每千克(f1g/kg), C一二根据样品孔的抑制l率查得试样中磺胶药物浓度，单位为微克每升(f1g/U，R一一某类样品换算系数(分别为z猪肉:20，鸡肉:20，猪肝:20，鸡蛋:20，牛奶:40，鱼:20)。4 检测低限、回收率4. 1 检测低限本方法检W1!J低限参见附录B。4.2 回收率七种破胶类药物添加浓度和囚收率的实验数据参见附录C，实验结果为阳性应用仪器方法进行确证。3 SN/T 1960一20074 附录A(资料性附录)磺肢类药物酶
11、联免疫测定试剂盒本标准酶联免疫测定步骤中使用的试剂盒为天津生物芯片技术有限责任公司产品2，试剂盒包括za) 96孔酶标板gb) 磺胶药物酶标记物溶液，c) 底物液A，于4(;保存，使用时需达到室温gd) 底物液也使用前室温避光保存.使用时.按底物液Az底物液巴为32 (体积比)混合使用FU 反应终止液;fr一p g) h) 洗涤液2使用时，用1待测物f 磺胶二甲异昭瞠礁胶哩哗磺胶对甲氧暗院磺胶甲氧嗦，股毗昵飞磺胶甲二哇磺胶氯达嗦v E者I 5 4卡可/J / ./ 咛/d俨/ / 附隶、)IC(;1 f资料性附录I水1 5稀释:/:1;二离(g/kg)I鸡鸡蛋;:;/ 2.5 5 1 / 10
12、 37.5 40 I /1O 32 I 40 主LZJ/525J挖. 52.5 75 75 60 75 直1牛奶2, 5 4 5 8 10 16 37.5 60 40 64 52.5 84 75 120 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，需经实验评估后使用这些等效产品，SN/T 1960-2007 附录C(资料性附录磺胶添加浓度和平均回收率表C.1磺肢添加浓度和平均回收率平均回收率/%待测物添加浓度/(阳/kg)猪肉鸡肉猪肝蛋类牛奶鱼50 76.6 99.8 13 1. 5 103.7 104.9 81. 5 硕胶二甲异口罩哇- 98
13、.8 96.7 11 1. 9 95.7 200 81. 6 90.0 吕4.9113.6 119.0 80.3 50 120.0 102.0 165.0 110.5 115.9 98.5 磺胶哩哇100 1211 89.6 /.)82.4 105.8 113.6 105.2 200 ./ 严俨108.8l/ ol/5 88.5 122.0 104.0 86.0 50/ 82.5卢 135日139.9 125.4 11 1. 2 77.6 磺胶对甲氧暗睫J苦。80 118. 0 113. 1 114.7 124.4 83.6 200 77.9 95.6 31. 3 12 1. 5 130.9
14、75.7 50 99.5 132.4 34. 7 122.1 117.8 119.5 E盖胶甲氧嗦-106 吉7的一rTs:5 才。7.810 1. 3 119.2 99.2 20鸟，回6卢122民109.9 116. 8 123.5 81. 0 j50 9.9 133.3 r 125.3 130.6 124. 1 72.9 磺胶毗咬100 115.8 114.4 128.2 84.9 ,-? 250/A I 88.7 ;( 104 /03.1 108. 1 130.4 10 1. 7 122. 112.4 139.3 107.5 139.4 磺胶甲二略100. j 12页7123.7 116
15、. 9 118.8 91.4 02.4 117.2 112.8 95.5 _50 131.6 18. 1 1125.3 128.5 120.9 磺胶氯达嗦,.-_.-l/113.0 叫92;.z，/104.3 120.6 114.5 118. 9 200 119.8 98.2 105.7 119. 1 100.7 120.6 5 SN/T 1960-2007 附录(资料性附录)七种磺肢类药物标准曲线D 磺眩药物悻准曲线F t;:; 1-/1/ w / l/ 11 七V l7 J! 120 60 40 20 100 80 汉、MZH军1 000 100 10 0 o. 1 酷度I(ng/mL)
16、-+一磺眩二甲异晤哩，-一磺肢喔睡一舍一睛眩对甲氧晴嗖E-)确肢毗哇z._破眩甲氧嘻g-硕践甲二睡z-+-磺眩扭达嘻.七种磺胶类药物标准曲线图D.16 SN/T 1960-2007 Foreword Annex A , B, C and 0 of this standard are informative annexes This standard was proposed by and is under the charge of Certification and Accreditation Administra tion of the People s Republic of China
17、 This standard was drafted by Tianjin Entry-Exit Inspection and Quarantine Bureau of the People s Republic of China. , Tianjin University of Science and Technologuy, Hubei Entry-Exit Inspection and Quara1tine Bureau of the People s Republic of China. This standard was mainly drafted by Wenjie Zheng
18、, Shuo Wang , Hongwei Zhang , Xiaozhong Hu , Yanhui Wu , Xia Yao , Hongyan Zhang , Yan Zhang , Jun Zhang. This standard is a professional standard for entry-exit inspection and quarantine promulgated for the first time. 7 SN/T 1960-2007 Screening method of sulfonamide residues in animal original foo
19、d for import and export-Enzyme linked immunosorbent assamethod 1 Scope 2 Normative references 3 3.2 Reagents and materials In this standard. all the chemical reagents should be analytically pure and water is the first.degree water as GB/T 6682 described. 3.2.1 Sulfonamide detection kit (see Annex Al
20、. 8 1) Sulfonamide detection kit is the commerc旧Iname which was provided by the Tianjin Biochip Technologies Co. , Ltd. This information being given is convenient for the user of the standard and is not the confirm of some products protocol. Other products protoI should benfirmed by experiments if a
21、ny difference SN/T 1960-2007 3.2.2 Acetone 3.2.3 Ethanol. 3.2.4 Na,HPO, . 12H,O. 3.2.5 NaH, PO, . 2H, O. 3.2.6 NaCI. 3.3. 1 Muller. 3.3.2 Homogenizer. 3. 3. 3 8alance 0. 001 9 l. 3.3.4 Vortex mixer (4 0 3.3.5 Centrifuge. 3.3.6 Rotary evaporator. 3.3.7 Microwell system (450 nml. 3.3.8 Plate-washing m
22、achine. 3.3.9 Pipettes: 20L. 100L. 200L. 3.3.10 Multichannel pipette: 200L. 9 SN/T 1960-2007 3.4 Preparation of test sample The combined primary sample is reduced to 500 g. which is blended. homogenized thoroughly. and then divided into two equal portions. Each portion is placed in a clean container
23、 as the test sample. which is sealed and labeled. 3. 5 Storage of test sample The test sample should be stored below -18C. In the course of sampling and sample preparation. precaution should be taken to avoid the contamination or any factors which may cause the change of residue content 3. 6 Sample
24、extraction 3.6.1 Pig muscle. chicken muscle and fish 4 9 sample was added 20 mL PBS. thoroughly mixed for 2 min. and then filtrated by using filter paper or centrifuged at 4 000 r/min for 10 min. Take 50L filter or supernate. diluted 4 folds. Pig muscle and chicken muscle were diluted with PBS; fish
25、 was diluted with 1% BSA before ELlSA anal ySls. 3.6.2 Egg 4 9 sample was added 20 mL PBS. gently mixed for 30 min to avoid foam and emulsion and take 50L sample. diluted 4 folds with PBS directly before ELl SA analysis. 3. 6. 3 Pig liver 2 9 sample was added 20 mL ethanol , and then thoroughly mixe
26、d for 2 min. centrifuged at 4000 r/min for 10 min. The solvent of 10 mL of the upper liquid was removed under reduced pres sure. The residues were dissolved in 10 mL PBS and then filtered. 50 IL filtrate was diluted 2 folds with 0.5% BSA-PBS before ELlSA analysis. 3.6.4 Milk 20 mL milk was centrifug
27、ated for 10 min at 3 000 r/min. After the fat layer was discarded. 20 mL of acetone was added and shaked by hand. then centrifugated for 10 min at 3 000 r/min. 50L upper clear liquid was diluted 20 folds before ELlSA analysis. 10 SNjT 1960-27 3.7 ELlSA determination 3.7. 1 Manulacture 01 calibration
28、 cu阿eDilute the prepared standard solution with P8S into 100g/L. 33. 3g/L， 11.1g!L. 3.7g!L. 1.2g!L.0.4g!L 3. 7. 2 Detection 01 sample Insert a sullicient amount 01 microtitre wells into the microwell holder lor all standards and samples to be run in duplicate. Record standard and sample posit旧ns.add
29、 100L 01 six kinds 01 prepared sullonamide standard solution and sample solution to separate duplicate wells. Add 100L double water as blank. Add 100L 01 diluted sullonamide enzyme conjugate (used within 8 h) to the bottom 01 each well and add 100L P8S in blank , Ilap lightly and mix thoroughly. Sea
30、ling all wells with adhe sive paper to avoid solution evaporatin日，incubate lor 60 min at room temperature. Pour the liquid out 01 the wells and wash the wells three times with P8ST. Add 150L 01 substrate and chromogen to each well Take care the substrate solution in the dark) Ilap lightly and mix th
31、oroughly, and then incubate lor 30 min at room temperature. Add 50L 01 stop solution to each well , Ilap lightly and mix thoroughly. Measure and record the absorbance value of each well solution at 450 nm within 10 min. 3. 8 81ank test The operation 01 the blank test is the same as that described in
32、 the method 01 determination but without addition 01 sample. 3.9 Calculating the result and express 3.9. 1 Calculating inhibition value in percentages Calculate the inhibition value of the combination 01 each sulfonamide standard and antigen-antibody, the sulfonamide standard and sample inhibition v
33、alue are calculated in percentages according to the lormula (1) : r _ A一，.，1.- A hln.寸IC = 11-一旦卫凹1x 100% L Acontro - A blank J ) 1 ( . . . . . . . . . . . . . . where IC -the inhibition value 01 sullonamide to the response 01 antigen and antibody. % ; AcontroOg/L sullonamide standard mean absorbanc
34、e value in percentages: Am.-sullonamide standard or sample solution mean absorbance value; Ab础Og/Lsulfonamide standard solution and enzyme conjugate mean absorbance value. 11 SNjT 1960-2007 3. 9. 2 Plot calibration curve Make the inhibition value in percentages (arithmetic grade) as ordinate. and su
35、lfonamide concentra tion as abscissa. plot the calibration curve of the inhibition value in percentages and sulfonamide standard concentration (see Annex D). A new calibration curve should be prepared for each determi nation. 3. 9. 3 Calculating the result The sulfonamide c咀ncentrationof the test sa
36、mple solutin can be read corresponding to the percent age inhibition value of each sample frj!i白;.-.I.h+;民. ,. .J画画， :1 -;:.:J sulfonamide in the test sample (吕/LIwhere X一theresidue of sulfonamide (问/同主码:与吨c-the residual of sulfonamide read R-the sample convertion factors ( milk: 40; fish: 20). 4 4.
37、 1 Limit of determination The limit of determination of this The positive result should be confirmed by the equipment method. 12 ( 2 ) SN/T 1960-2007 Annex A (Informative Annex) Sulfonamide detection kit Sulfonamide detection kit is provided by the Tianjin Biochip Technologies Co. , Ltd. It includes
38、, a) 96-microwell plate, b) Sulfonamide enzyme conjugate, c) Substrate solution A, d) Substrate solution B, store (he solution at room temperatu叫indark place, Substrate A , substrate B, 32 1, e) Stop solution, / / f) 0.5% and 1% BSA-PBS,store / / / / / / / g) Phosphate Buffered Saline (PB) , store a
39、t 4C. dilute five time with distilled water, 由自土Li mit dete t of Table B.l一Limidetecfqij:zyme Iil1ked jpmunosorbent assay Analytes 飞l,.imit d 时t忖g盼)pig muscle 队由iclcenmu回le，/live / j egg fish sulfisozole 2.5 、气、2c5-/2 L.,-j 2.5 2.5 sulfathiazole 5 5 4 5 5 sufameter 10 10 8 10 10 sulfamethoxypyridazi
40、ne 37.5 37.5 30 37.5 37.5 sulfapyridine 40 40 32 40 40 sulfamethizole 52.5 52.5 42 52.5 52.5 sulfachlorpyridazine 75 75 60 75 75 milk 4 8 16 60 64 64 120 2) This information beng given is convenient for the user of the standard and is not the confinn of $ome products protocol. Other products protoco
41、l should be confirmed bV experiments if any difference 13 SN/T 1960-2007 Annex C (Informative Annex) Addition and average recovery of sulfonamides Table C. l-Addition and average recovery of sulfonamides Fortification level/ Sample Analytes (g/kg) pig muscle chicken muscJe pig liver ogg 50 76.6 99.8
42、 131.5 103.7 sulfisozole 100 81.3 92.3 98.8 96. 7 200 81.6 9口。84.9 113.6 50 120.0 102.0 65.0 110.5 sulfathiazole 100 103.1 89.6 82.4 105.8 2 108.8 101.5 88.5 122.0 50 82.5 135.8 139.9 125.4 sufameter 100 80.0 118.0 113. 1 114.7 200 77.9 95.6 131.3 121.5 50 99.5 132.4 134.7 122.1 sulfamethoxypyridazi
43、ne 100 79.3 115.5 107.8 101.3 200 68.2 122.5 109.9 116.8 50 89.9 133.3 125.3 130.6 sulfapyridine 100 87.4 115.8 113.0 114.4 200 88.7 104.2 103.1 108. 1 50 84.9 122.8 112.4 139.3 sulfamethizole 1口。87.6 103.7 96.6 123.7 200 74.1 91.4 102.4 117.2 50 137.6 118.1 125.3 128.5 sulfachlorpyridazine 100 113.
44、0 92.7 104.3 120.6 200 119.8 98.2 105.7 14 milk fish 104.9 81.5 111.9 95. 7 119.0 80.3 115.9 98.5 113.6 105.2 104.0 86.0 111. 2 77.6 124.4 83.6 130.9 75.7 117.8 119.5 119.2 99.2 123.5 81.0 124. 1 72.9 128.2 84.9 130.4 101.7 107.5 139.4 116.9 118.8 112.8 95.5 120.9 142.2 114.5 118.9 100.7 120.6 SNjT
45、1960-2007 Annex 0 (Informative Annex) Standard curve of sulfonamide 占2/ /1/ 在/;附f的但才1主r mrt1llliLllli 111111111 120 60 40 20 100 80 这ZGZE-521 000 100 10 0 O. 1 Concentration / (ng/mL) -+- sulfisozole; -t昏-sulfathiazok: _.比_sufamdcr + 号码-sultp、ridinc-神-sulfamctho巧pyn由Llne;-唱-sulfamethizo1c; -+- sulfachlorpyridazine. Figure D. 1 Standard curve of sulfonamide 11111111111 中华人民共和国出入境检验检疫行业标准迸出口动物源性食品中磺胶类药物残留量的检测方法酶联免疫吸附法S)I!T 1960 -2007 峰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1舌，6852394668517548 中国标准出版社秦皇岛印刷厂印刷 开本880X12301/1 6 印张1.25 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口00* 书号，155066.2-18241 定价12.00元
