1、中华人民共和国出入境检验检疫行业标准SN/T 1569-2005 进出口河豚中河豚毒素检验方法ELISA法Inspection of tetrodotoxin in puffer fish for import and export ELISA method 2005-05-20发布2005-12-01实施中华人民共和国发布国家质量监督检验检疫总局前言本标准的附录A为规范性附录。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:中华人民共和国辽宁出入境检验检疫局。本标准主要起草人z吴斌、秦成、于杰、王刚、赵守成、王玫、宋慧君。本标准系首次发布的出入境检验检疫行业标准。SN/T 15
2、69-2005 I 1 范围进出口河豚中河豚毒素检验方法ELISA 法SN/T 1569-2005 本标准规定了进出口河豚中河豚毒素检验的抽样、制样和酶联免疫吸附检测方法。本标准适用于进出口河豚鱼肉、皮、肝、生殖器中河豚毒素的检测。2 缩略语TTX Tetrodotoxin 河豚毒素ELISA Enzyme Linked Immunosorbent Assay 酶联免疫吸附法A值Absorption value 吸光度值BAS-HCHO - TTX Bovine Serum Albumin-HCHO - Tetrodotoxin conjugate 牛白蛋白甲醒河豚毒素连接物PBS-T Pho
3、sphate Buffered Saline-Tween 磷酸盐吐温-20洗液McAb-HRP Monoclonal Antibody-Horseradish Peroxidase 单克隆抗体辣根过氧化物酶标记物3 抽样3. 1 检验批以同一海区、同一产地、同一养殖场的河豚为一检验批，同一检验批的商品应具有相同特征，如品种、包装、标记、产地、规格等。3.2 抽样敢量根据各检验批的质量及件数，按表l确定抽样件数。如为散装的应均匀抽样。表1抽样数量的确定批量抽样数量kg 箱(件)箱(件)50500 225 2 5011 200 2660 3 1 2013 200 61160 4 3 20110 0
4、00 161500 7 10 00135 000 5011 750 10 35000以上1 750以上16 3.3 抽样方法按3.2规定的抽样箱数随机抽取，逐件开启。对整体鱼每箱至少杆取完整个体3条作为原始样品，并详细记录抨取样品的特征(体色、斑纹、规格、体长、体重、有无刺等)，装入盛样器内;成品或半成品样品抨取的每个小样不得少于200g。加封后，标记。OOC50C条件下48h内送达实验室。3.4 样晶的保存如实验室不能立即检验，应分别用标签注明来源、取样日期、品种及编号，装塑料袋密封。抽样后样品应立即于一180C以下冷冻保存。SN/T 1569-2005 4 测定方法4. 1 方法原理抗-T
5、TX酶标记物与样品中的TTX及酶标板上包被的TTX发生免疫竞争反应，样品中的TTX浓度与吸光度值成反比.按绘制的校正曲线定量计算样品中的TTX浓度。4.2 方法提要用牛白蛋白-甲醒问豚毒素连接吻(BSA-HCHO-TTX)榕液包被酶标微孔板，然后加入不同浓度的TTX标准搭液(制作校正曲线)或样品提取液(检测样品)与单克隆抗体辣根过氧化物酶标记物(McAb-HRP)的混合液，再加入底物液，显色，加入终止液，于490nm处测定A值，根据A值得出样品中河豚毒素的含量。4.3 试剂和材料除另有规定外，所用试剂均为分析纯，水为蒸懵水。4.3.1 乙酸缓冲液:pH=4.8(按附录A配制)。4.3.2 磷酸
6、盐缓冲液CPB曰:pH=7.2,0. 05 mol/L(按附录A配制)。4.3.3 底物缓冲液:拧攘酸-磷酸盐缓冲液(按附录A配制)。4.3.4 洗掖(PBS-T):含0.5%吐温-20的o.05 mol/L PBS(按附录A配制)。4.3.5 McAb-HRP:Sigma公司生产。4.3.6 OPD底物液:按附录A配制。4.3.7 过氧化氢溶液:0.5mol/L。4.3.8 终止液:2mol/L硫酸。4.3.9 BSA-HCHO-TTX:Sigma公司生产。4.3.10 氢氧化铀榕液:1mol/L。4.4 仪器和设备4.4. 1 酶标分j(:;光皮仪。4.4.2 96孔酶标板。4.4.3 均
7、质器。4.4.4 天平:感量为0.1mg o 4.4.5 冰箱:05.C和15.C -20.C。4.4.6 培养箱:370C士1.C。4.4.7 减压过滤装置。4.5 测定步骤4.5.1 试样制备冷冻样品装于密封的塑料袋内于流水下急速解冻，整体鱼用蒸馆水清洗后，用滤纸吸干，然后将鱼体分解成肌肉、肝脏、肠道、皮肤、卵巢(雄性为精束)等部分，用蒸馆水洗去血污，再用滤纸吸干、称量，分别将解剖后取得的样品剪碎，充分均质。冷冻的成品或半成品样品装于密封的塑料袋内于流水下急速解冻。取上述选定的5.0g样品放入烧杯，加25mL 0.1%乙酸榕液，于沸水浴中搅拌10min。冷却至室温后，快速减压过滤，滤纸上的
8、残渣用20mL O. 1%乙酸溶液反复洗净。合井滤液，加入等体积乙酷振摇脱脂，静置待分层后，放出水层至另一分液漏斗中，再重复脱脂一次。然后将水层用1mol/L氢氧化铀调pH至6.57. 0后，放入50mL容量瓶中，用0.1%乙酸溶液定容至50mL，立即用于检测。难于过滤的皮、肝脏、卵巢分别用0.1%乙酸榕液处理后，经3000 r/min, 10 min离心，取上清液，再按上述方法定容至50mL。该提取液1mL相当于0.1g样品。SN/T 1569-2005 4.5.2 试样保存处理后的样品如不能及时检测，可取10g已均质待测样品加入10mL的0.1%乙酸溶液，置于40C保存。4.5.3 包被将
9、BSA-HCHO-TTX用O.05 mol/L PBS稀释至20g/mL，包被酶标板，每孔加100L，40C过夜，倒净孔内溶液，加3mg/100 mL的牛白蛋白于200C土lOC封闭30min，用PBS-T洗板3次，每次3 min，待用。4.5.4 酶标抗体浓度的选择将购置的酶标抗体用O.05 mol/L PBS稀释至100倍、500倍、1000倍和2000倍，与40g/mLTTX标准溶液等量混合，按4.5. 6检测A值。A值为O.3左右的酶标抗体浓度为酶标抗体的工作浓度。4.5.5 校E曲线的制备4.5.5. 1 用O.05 mol/L PBS稀释TTX标准储备液至5g/mL、O.5g/mL
10、、O.05g/mL、0.025g/mL、0.01g/mL，制成5个浓度的TTX标准溶液。4.5.5.2 取各浓度的TTX标准榕液100L，分别与100L已选定工作浓度的McAb-HRP充分提合，置40C过夜或370C士lOC2 h备用。4.5.5.3 取上述不同浓度的混合液LOOL，分别加入己包被的酶标板，置370C土lOC1 h。4.5.5.4 洗板:用PBS-T洗板5次，每次3min。4.5.5.5 显色:每孔加OPD底物液10OL，3盯70C置暗处3Om丑l1n4.5.5.6 终止显色:每孔加5切OL终止液。4.5.5.7 测定:于490nm处测定每孔的A值。4.5.6 样晶测定4.5.
11、6. 1 取毒素提取液100L加已选定工作浓度的McAb-HRP100L，充分混合，置40C过夜，或370C士lOC2 h备用。4.5.6.2 取上述混合液100L加入己包被TTX的微孔板，同时选择一孔加1g/mLTTX标准溶液为阳性对照，一孔加O.05 mol/L PBS为阴性对照，置370C:J:10C1 h。4.5.6.3 洗板:用PBS-T洗板5次，每次3mmo 4.5.6.4 显色:每孔加OPD溶液100L，370C置暗处30min。4.5.6.5 终止显色:每孔加50L终止液。4.5.6.6 测定:于490nm处测定每孔的A值。4.6 绘制校正曲线以百分比吸光度值(算术级)为纵坐标
12、，以TTX浓度(ng/kg)(对数值)为横坐标，绘制出TTX标准液百分比吸光度值与TTX浓度(对数值)的校正曲线。每次试验均应重新绘制校正曲线。4. 7 结果计算按式(1)计算试样中河豚毒素含量:E一-W D一-V C一X . ( 1 ) 式中:X 试样中河豚毒素的含量，单位为纳克每克(ng/g);C一一酶标板上测得的TTX含量，单位为纳克(ng)，根据工作曲线求得;D一一试样提取液的体积，单位为毫升(mL);E一一试样提取液的稀释倍数;V一一酶标板上每孔加入的样液体积，单位为毫升(mL);3 SN/T 1569-2005 W一一试样重量，单位为克(g)。5 结果与报告xx样品中河豚毒素的含量
13、:X X ng/g 注:阳性结果须用其他方法进行确证。6 测定低限、回收率6. 1 测定低限本方法的测定低限为1ng/g。6.2 回收率及回收的实验数据4 1 ng/g时，回收率为96.0%; 一-5ng/g时，回收率为95.2%; 一二10ng/g时，回收率为92.0%; 一-20ng/g时，回收率为90.1%; 一一50ng/ g时，回收率为105.5%。附录A(规范性附录)A.1 Z酸缓冲液0.2 mol/L乙酸铀59 mL 0.2 mol/L乙酸41 mL 将两种成分充分泪合，最终pH4.80A.2 PBS缓冲液氯化纳磷酸二氢饵磷酸氢二饵氧化饵水8 g 0.2 g 2.9 g 0.2
14、g 1 000 mL SN/T 1569-2005 将各成分榕解于蒸锢水中，用1mol/L氢氧化铀调至pH7.2。用蒸锢水加至1000 mL贮存冰箱。A.3 拧槽酸-磷酸盐缓冲液0.2 mol/L磷酸氢二铀(28.4 g/l 000 mL) 25. 7 mL 0.1 mol/L拧攘酸(21.014 g/l 000 mL) 24. 3 mL 水50mL 将各成分加入50mL蒸锢水中，pH值调至7.0，贮存于冰箱备用。A.4 0.5%吐温-20磷酸盐缓冲液0.5 mol/L磷酸盐缓冲液100 mL 吐温-200.5 g 将吐温-20溶解于0.5mol/L磷酸盐缓冲液中，最终pH7.2，贮存冰箱备用
15、。A.5 河豚毒素校正曲线校正曲线见图A.1。5 SN/T 1569-2005 6 XV 120 100 80 60 40 20 。2 3 4 5 6 7 8 9 10 11 1 gTl 固A.1河豚毒素校正曲线SN/T 1569-2005 Foreword Annex A of this standard is a regulative annex. This standard was proposed by Certification and Accreditation Administration of the People s Re public of China. This stan
16、dard was mainly drafted by Liaoning Entry-Exit Inspection and Quarantine Bureau of the Peoples Republic of China. This standard was mainldrafted by Wu Bin , Qin Che吨，YuJie, Wang Gang ,Zhao 5hou Cheng , Wang Mei ,50ng Hui Jun. This standard is a professional standard promulgated for the first time. 7
17、 SN/T 1569-2005 Inspection of tetrodotoxin in globefish for import and export-ELISA method 1 Scope This standard specifies the methods of sampling , sample preparation and inspection by ELlSA method of tetrodotoxin in puffer fish for import and export. This standard is applicable to inspect of tetro
18、dotoxin in puffer fishs muscle,skin ,liver and ovary (or stal的for import and export. 2 Abbreviation TTX Tetrodotoxin ELlSA Enzyme Linked Immunosorbent Assay A Absorption value BAS-HCHO-TTX Bovine Serum Albumin-HCHO-Tetrodotoxin conjugate PBS-丁PhosphateBuffered Saline-Tween 20 McAb- HRP Monoclonal An
19、tibody-Horseradish Peroxidase 3 Sampling 3. 1 Inspection lot Ea巾inspectionlot of puffer fish should come from the same sea , the same origination, the same breed factory. The characteristics of the cargo within the same inspection lot,such as type,packing , mark ,origin ,specification and grade etc.
20、 ,should be the same. 3. 2 Ouantity of sample taken Table 1 Determination of Ouantity of Sample Taken Number of packages in each inspection lot Number of packages to be taken kg packages packages 50-500 2-25 2 501-1 200 26-60 3 1 201-3200 61-160 4 3201-10000 161 -500 7 10 001-35 000 501-1 750 10 Mor
21、e 35 000 More 1 750 16 8 SN/T 1569-2005 3.3 Sampling procedure A number of packages specified in 3. 2 are taken. Select at least 3 from each package as the originate sample. Record in detail the characterizes of samples (color of body, spot and grain , tpe，length， weight , with or without thorn etc.
22、 ) , put into container , seal , mark and deliver to the laboratory at 0C -5 C within 48 h. 3. 4 Storage of sample If the samples can t be inspected at once , the label should be labeled outside of each sample contain er. The label indicates origin ,the date of sampling ,the type,the sample number,a
23、nd sealed in plastics bags. After sampling , the test sample should be stored below一180Cimmediately. 4 Method of inspection 4.1 Principle of method The basis of the test is the competitive enzyme-linked immuno reaction between tetrodotoxin en zyme conjugate and TTX in samples , TTX coated on microti
24、tre plate. The tetrodotoxin concentration in the sample is inversely proportional to the absorption value , and compare with the calibration curve for quantative measurement. 4.2 Abstract of method Microtitre plate is coated by BAS-HCHO-TTX conjugate. The plates are washed three times and each time
25、is 3 min. The different densities of TTX standard or sample solution which is mixed with McAb HRP are added and react with TTX coated on microtitre plate. The plates are washed five times and each time is 3 min. The stop reagent is added after coloring. The absorbance value of each well solu tion is
26、 measured by microwell system at 490 nm. The TTX concentration in the sample is according to the absorption value. 4. 3 Reagents and materials In this standard ,all the chemical reagents should be analyticallpure， water is distilled water. 4.3. 1 Acetate acid: pH = 4. 8Caccording to Annex A). 4.3.2
27、PBS: pH=7.2 ,0.05 mol/LCaccording to Annex A). 4.3.3 Substrate buffer: Citric acid-Phosphate Buffered Saline buffer Caccording to Annex A). 4.3.4 Washing solution (PBS-T): 0.05 mol/L PBS including 0.5% Tween 20 Cacrding to Annex A). 9 SN/T 1569-2005 4.3.5 Anti-TTX -McAb-HRP: purchased from Sigma. 4.
28、 3. 6 OPD substrate: purchased from Sigma. 4.3.7 Peroxide solution: 0.5 mol/L. 4. 3. 8 Stop reagent: 2 mol/ L H2 S04. 4.3.9 BAS-HCHO-TTX: purchased from Sigma. 4.3. 10 NaOH solution: 1 mol/L. 4.4 Apparatus and equipment 4.4. 1 Microwell system. 4.4.2 96-well polystyrene microtitre plate. 4.4.3 Homog
29、enizer. 4. 4. 4 Balance: O. 1 mg. 4.4.5 Refrigerator: OoC _50C and一150C- - 20oC. 4.4.6 Inculcator: 37 C :t 1 oC. 4.4.7 Reduce pressure and filter system. 4. 5. 1 Sample preparat ion Frozen sample in sealed plastics bag is rapidly refrozen under flowing water. after washed by distilled water and abso
30、rbed by filter paper , the fish is divided into muscle , liver, intestines,ovary(or stalk) , etc. wash out blood ,absorb off and weight. Take samples from the above , cut off with a clean scis sors and homogenized respectively. Weigh 5. Og of the test sample into a container ,add 25 mL of 1 % acetat
31、e acid solution, vibrate continually 10 min in boiling water bath. Freeze to room temperature , reduce pressure and filter rapidly , wash remains on the filter paper with 20 mL of 1 % acetate acid re peatedly. Then mix filter-solution , add the same volume ether vibrating in order to drop fat. After
32、 let stand to separate , put the lower aqueous phase into the other funnel , repeat it again. Adjust pH of the aqueous phase with 1 mol/L NaOH solution to 6. 5-7. 0, blew into 50 mL vessel , fix volume with 1 % acetate acid solution into 50 mL, inspect at once. Deal the skin , liver and ovary with 1
33、 % acetate acid solution, centrifuge 3 000 r, take the clean-up solution, fix volume to 50 mL according to the above method for 10 min. The extraction is-equal to O. 1 9 samples. 10 SN/T 1569-2005 4.5.2 Sample conservation If the 4. 5. 1 samples can t be inspected at once , they may be taken 10 9 in
34、to 10 mL of 1 % acetate acid solution and stored at 4C . 4. 5. 3 Coating BSA-HCHO-TTX was diluted in 0.05 mol/L PBS into 20g/mL and coat enzyme-marked plate. Add 100L to each well and incubate overnight at 40C. Pour the liquid out of the wells and seal with 3 mg/100 mL BSA for 30 min at 200C :f: 1 o
35、C. Wash p!ate in PBS-T solution for 3 min and repeat three times. 4. 5.4 Choosing of concentration of enzyme-marked antibody The enzyme-marked antibody was diluted in 0.05 mol/L PBS into 100 times,500 times , 1 000 times, 2 000 times and mix with 40g/mL TTX standard solution respectively. A value is
36、 measured accord ing to 4. 5. 6. The concentration of enzyme-marked antibody while A value is about O. 3 is the concen tration of enzme-marked antibody we need. 4. 5. 5 Manufacture of calibration curve 4.5.5.1 The TTX standard solution was diluted in 0.05 mol/L PBS into five kinds of prepared TTX st
37、andard solution-5g/mL、0.5g/mL、0.05g/mL、0.025g/mL、0.01g/mL.4.5.5.2 Take 100L of the above TTX standard solution , mix appropriate dilutions of anti-TTX -McAb-HRP respectively and incubate overnight at 40C or 2 h at 37C :f: 1 oC. 4. 5. 5. 3 Take mixture of the above different concentration , add to en
38、zyme-marked plate respec tively and incubate for 1 h at 37C士1oC. 4.5.5.4 Washing plate: wash plant in PBS-T solution for 3 min and repeat five times. 4.5.5.5 Coloring: add 100L OPD substrate solution to each well and incubate for 30 min at 37 C 士1oC in darkness. 4. 5. 5. 6 Stopping color: add 50L of
39、 stop - reagent to each well. 4. 5. 5. 7 Measuring: the absorbance of each well is read at 490 nm. 4. 5. 6 Inspection of sample 4.5.6. 1 Take 100L of extraction of TTX, mix appropriate dilutions of anti-TTX -McAb-HRP and incubate overnight at 40C or 2 h at 37C土1oC. 11 SN/T 1569一20054.5.6. 2 Take mix
40、ture of the above different concentration, add to enzme-marked plate, at the same time select one well added 1g/mL TTX standard solution as a positive reference and 0.05 mol/L PBS as a negative reference and incubate for 1 h at 370C土lOC.4.5. 6. 3 Washing plate: wash plant in PBS-T solution for 3 min
41、 and repeat five times. 4. 5. 6. 4 Coloring: add 100L OPD substrate solution to each well and incubate for 30 min at 370C 土lOCin darkness. 4.5.6.5 Stopping color: add 50L of stop- reagent to each welL 4.5.6.6 Measuring: the absorbance of each we is read at 490 nm. 4. 6 Plot calibration curve Make th
42、e absorbance value in percentages (arithmetic grade) as ordinate, and TTX concentration (Iogarithmic grade) as abscissa , plot the calibration curve of the absorbance value in percentages and TTX standard concentration. A new calibration curve should be prepared for each determination. 4. 7 Calculat
43、ing the result Calculating concentration of TTX in samples according to formula (1) : FE -w D一-v pu一X 、-Jat- 飞. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . where: X一一-concentrationof TTX in sample ,ng/g; c-一-concentrationof TTX attained by calibration curve , ng; D -volume of
44、 sample extraction,mL; E-一-dilutedtimes of sample extraction; V一一volumeof sample solution added into microtitre plate, mL; W一一-sampleweight, g. 5 Resolution and report Concentration of TTX in X X samples: x x ng/g. Note, Positive reslts shOuld benfirmed by another method. 12 6 Limit of detemination
45、and recovery 6. 1 Limit of detemination The limit of this method is 1 ng/kg. 6. 2 Recovery 一一-At1 ng/kg,the recove叩is96. 0%; At 5 ng/I唱，therecovery is 35. 2%; 一一-At10 ng/kg,the recove叩is92. 0%. 一一-At20 ng/kg,the recovery is 90.1 %; 一一-At50 ng/kg,the recove叩is105.5%. SN/T 1569-2005 13 SN/T 1569-2005
46、Annex A (Regulative Annex) A. 1 Acetate acid buffer 0.2 mol/L Nasium acetate O. 2 mol/ L Acetate acid Mx the two ingredents and the fnal pH is 4. 8. A. 2 PBS buffer NaCI KH2同)4K2HP04 KCI distilled water 59 mL 41 mL 8g 0.2 9 2.9 9 0.2 9 1 000 mL Resolve all types of ingredients into distilled water,
47、adjust pH with 1 mol/ L NaOH to 7. 2, fix volume to 1 000 mL and store in refrigerate. A. 3 Citric acid-Phosphate Buffered SaJine buffer 0.2 mol/L Na2HP04 (28.4 g/1 000 mL) 0.1 mol/L Citric acid (21.014 g/1 000 mL) distilled water 25.7 mL 24.3 mL 50 mL Add all tpes of ingredients to 50 mL distilled
48、water and the final pH is 7. O. Store n refrigerate. A.4 0.5% Tween-20 Phosphate Buffered Saline buffer O. 5 mol/L Phosphate Buffered Saline buffer Tween-20 100 mL 0.5 9 ResolveTween-20 into O. 5 mol/L Phosphate Buffered Salne buffer and the fnal pH s 4. 8. Store in refrigerate. A.5 Tetrodotoxin caJ
49、ibration curve The curve see Fig A. 1. 14 SN/T 1569-2005 120 80 60 40 20 100 XV85去SA11 10 9 8 7 6 5 4 3 2 。1 gTTX (Tetrodotoxm) tetrodotoxin calibration curve Fig. A. 1 的DON-gmFH军中华人民共和国出入境检验检疫行业标准进出口河豚中河豚毒素检验方法ELISA法SN/T 15692005 中国标准出版社出版发行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* ，晤印张1.25 字数26千字2005年8月第一次印刷开本880X1230 1/16 2005年8月第一版9峰定价12.00元如有印装差错曲本社发行中心调换版权专有侵权必
