【商检行业标准(SN)】 进出口河豚中河豚毒素检验方法 ELISA法

中华人民共和国出入境检验检疫行业标准SN/T1569—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-FLISA method
2005-05-20发布
华人民共和国
国家质量监督检验检疫总局
2005-12-01实施
本标准的附录A为规范性断录，
本标准由区家认证认可监督管理委员会提出并归口本标雅起草单位：中华人民共和国迁宁出入境检验检疫局，本标雅主起草人：灵斌、秦成、于杰、十刚、赵守成、十敌、宋慧若。本标准系首次发布的出入境检验检疫行业标准。SN/T1569—2005
1范围
进出口河豚中河豚毒素检验方法ELISA 法
SN/T1569—2005
本标准规定了进出|「河豚中河毒素检验的样、制样和酶联免疫吸阴检测方法本标淮二进山口河豚鱼肉、支、肝、生殖器中河豚毒素的检测。2缩略语
TTXTetrodatoxini河豚毒素
ELisAEnzyine Linzed Irminunosorbet Assay翁联免疫吸附法A值Abstrptionval吸光度值
BAS-HCHO-TTX Bovine Serum Albumin-HCHU- Tetrodotoxin conjugete 牛白蛋白-甲醛-河豚穿索连接物
PBS-TPhosphateBuffered Saine-Tween磷酸盐吐湿-20洗液MeAh IIRPMonorlonalArtihodyIorscradlsh Pcroxidasc单克擎抗体辣根过氧化物酶标记物3抽样
3.1检验批
以同海区、同产地、同养殖场的河豚为·拾验批.同检验批的商品成具有相同特征，如品种、包装、标记、产池、愿格等.3.2抽样数量
根据各检验批的质景及件数，按表：确定抽样件数。如为散装的应均勾抽样。表1
抽样数量的确定
50~500
301--1 200
1 2013 200
3 201~-10 000
10 001~-35 00
O以上
3.3抽样方法
2·~25
6:-~-180
161~-500
501~-1 755
:730 以上
抽栏数量
箱（件）
按3.2规定的抽样箱数随机抽或，逐件并片，刘整体鱼每箱少打取究整个体3条作为原始样品，并详纽记录打收样品的特征（体色、纹，规格,体长、体重、有儿刺等）,装人盛样器内；戒品或半成品样品打取的每个小样不得少二200。加率后，标记。C℃~5℃条件下48h内送实验空。3.4样品的保存
如实验室不能立即检验，应分别尼标签注明米源、取样几期、品种及编弓，装塑料袋密封。抽样后样品应立即于一18C:以下冷冻保存。SN/T 1569—2095
4测定方法
4.1方法原理
范 TTX悔标记物与样品中的 TTX及标板上包被的TTX 发生免疫竞争反应，样品中的 TTX浓度与吸光度值成反比，按绘制的校止线定量计算烊品巾的TTX浓度，4.2方法提要
用:白蛋白-甲醛-河豚毒素连接物（I3SA-IICIIO-TTX)溶被包被标微孔板，然后人不同浓度的ITX标准游液(制作校正由线)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(McAh-IIRP)的混介获，加人底物液，显色，加人终正液，丁490 n:n处测定A值，根据A值得山样品中河琢母素的含量，
4.3试剂和材料
除方有规定外，所月试剂均为分析绰,水为蒸馏水。4.3.1乙酸缓冲液：pII=4,8(按除录A靶制）4.3.2酸站缓冲液(PS)：H7.2,0.05 mo1/T按附录 A配)4.3.3底物缓冲液：柠檬酸-磷酸盐缓冲液（获附录A配制）4.3. 4洗被(P13S-1):含 0,5%证温-20 的 0. 05nol/1. PIS(按附录 A 配制)。4.3.5MeAbHRP.Sigrme.公司牛产.4.3.6（PD底物液：按刚录A配制。4.3.7过氧化氢溶液;0.5 mol/1.。4. 3. 8终止滋:2 ml/L 硫酸-
4.3. 9 BSA-IICIIO-TTX:Sigima 公司生产4.3.13氢氧化钠溶液：［mnl/T.。4.4仪器和设备
4.4.1酶标分光度仪。
4.4.296孔嗨标板。
4.4.3均质露。
4. 4. 4天平：感量为 0. 1 rnE4.4.5 冰箱:0-~5℃利-15℃-—20℃。4.4.6培养箱：37℃+：℃。
4.4.7减压过滤置
4.5测定步骤
4.5.1试样制备
冷该样品装；封的塑料袋内丁流水卜急速解冻,整位用蒸馏水清洗后,用滤纸玻十，然后将鱼体分解成肌肉、肝脏、肠道、皮胺、卵巢（雌性为精束等部分·用蒸馅水洗去而污，再用滤红圾干、称量+分别将解剖后收得的样品剪穿，充分均质，冷冻的贼品或半成品样品装于密封的塑料袋内于流水小急速解冻。
取「述选定的5.0 样品激烧杯.加25 ImL 0._%乙酸溶液于沸水浴捞拦10 Ilin，冷却个室温后，快速减压过滤瀛纸上的凝湾用2010.1为乙酸落液反复洗学。合并滤液：加人等体积三避派摇脱脂，静背待分层后，放山水层至另分液漏斗巾，再重复说脂次，然后将水层用［氢氧化钠调凋 pll 牟 6. 5~-7. 0后,激人 50 ImL容量瓶中,用 0. 1%乙酸溶液定窄牟 50 rmL,文即用于检测炸于过滤的度、肝脏、卵巢分别月 0. 1%之酸溶液处理后，经 3 000 t/mitn,1C mir: 离心,取 1:清液,再按「:述方法定容至5℃l该提取液 mT.相当于！.1样品。2
4.5.2试样保存
SN/T 1569—2005
处后的详品如不能及时检测，可取I已均质待测样品近人ImT.的(.「为乙酸溶液，置士4℃保存。
4.5.3包被
将BSA-HCHO-TTX用0.C5 nol/L PBS 稀释至 2C μ/mL,他被酶标板,每孔严 1C0 #L，℃过夜,倒净孔内溶被，加 3 rig/[G0 ml.的牛白蛋白丁 20℃士1封闭 30 mir,用 P13s-T洗板 3 次，每次3 tmi,待用。
4.5.4酶标抗体浓度的选择
将离置的标抗体用0.G5mol/LPBS稀释牟10C倍、5c0倍.1000倍和2000倍，与4Cg/mlTTX标准溶液等量混合,按 4. 5. 6 检测 A 值。A 估为 0. 3左右的酶标范体浓变为酶标抗体的二作浓度。
4.5.5校正曲线的制备
4.5.5.1用0.05Inol/LPHS释释TTX标准储备液至5g/inL、C.5ug/1rL、0.C5μg/=nL、C. 025 μ/rrL,0. 01 μg/nL.制成5 个浓度的 TTX标准溶液。4.5.5.2收务浓度的TX标准液:00 ul.,分别与130μl.已选定1作浓度的McAb-HRP充分混合，置℃过夜或37℃11℃2h备用。4.5.5.3取上述不同浓度的混个液100m1.,分别加入已包被的薄标板，置37=1℃1h4. 5. 5. 4 洗板:用 PrS-T 洗板 5 次,每次 3 rin。4.5.5.5最色：每孔加0PD底物液100μL.37℃骨暗处3CLin4.5.5.6终止显色：每码加50μL终上液，4.5.5.7测定：=490 nm处测定每孔的A值4.5.6样品测定
4. 5.6.1取带素提最液[G0 I.加已选定上作浓度的 McAh-1IRP 100L.,充分混介，置1C:过夜,或37±1%2h备角，
4.5.6.2取上述混合液100L加人已包被TTX的微孔板，同时选择-孔加1/mLTTX标准溶液为阳件对照,一孔加 0.05 mo/L PBS为明性对照-置 37℃+1℃ 1 h。4. 5. 6. 3洗板:用 PBS-I 洗板 5 饮,每次 3 nin。4.5.6.4晟包衔乳加PD溶液10cμ，37℃置暗处30rmnin4.5.6.5终正显色：每孔加50u1.终上液。4. 5. 6.6测定：一 493 mm处测定衔孔的A值：4.6绘制校正曲线
以自分比玻北度值（算个级）为坐称.以TIX度（/（对数值)为横坐标，绘剖TIX弧液百分比破光估与TIX浓博（对数估)的校止曲线：每饮试验均避重新绘制校止曲线4.7 结果计算
按式()计算试栏中河豚毒素含量：X=
戏中：
试样户测豚市素的含量.单位为纳克每克（n/）：酶标板上测得的 TTX含量，单位为纳克（ng）.根据T作曲线求得；D
试样提取被的休积，单位为毫(mI)；F
试样提版液的稀释倍数；
翼际极[得扎近人的详液体积，单位为毫于（t，;-(1
SN/T 1569—2005
试样意呆，单位为克(g)。
结果与报告
样品中河球串素的含量：×X/
注：旧性结果氛用其他方法进行确证。测定低限、回收率
6.1测定低限
本方法的测定低限为二ng/g
2回收率及回收的实验数据
11g/g时，四收率为96.0%；
5ng/g时.网收率为95.2%：
10ng/g时，-改率为92.6%：
20 ng/g时，向改率为90.「%；
-50ng/g时，回改率为105.5%
A.1乙酸缓冲液
0.2 mol/L乙酸钠
0.2 mol/LZ酸
将两种戒分充分混合，最终pII4.8A.2PBS缓冲液
氯化钠
磷酸二氨钾
磷酸氧二钝
瓦化镇
附录A
（规范性附录）
41 znL
l 900 rl.
SN/T 1569—2005
将各戏分溶解于蒸馏水中,用1 mol/L氢氧化钠词至pH7.2，用蒸水至二0C0 mL:存冰A,3柠檬酸磷酸盐缓冲液
0. 2 mol/1. 磷酸氢一钠(28. 1 g/1 000 ml.). 1 m01/1. 柠檬酸(21. 012 g/1 000 m1)水
25. 7 ml.
将各成分加人50 mL蒸谱水中pH估调至7,0,贮存于冰箱备用。A.40.5%吐温-20磷酸盐缓冲液
0.5mol/1.磷酸盐缓冲液
吐温-20
将吐温-2C溶解于 0.5 rn1o1/L磷酸盐缀冲液中,最终pII7.2,存冰箱备压。A.5河豚毒素校正曲线
校正曲线见图A.丨：
SN/T 1569—2005
河豚毒素校正曲线
Foreword
AnnexA of this standard isa regulativeannexSN/T 1569—2005
This standard was proposed by Certification and Accreditation Administration of the People's Re-publicof China.
This standard was mainly drafted by Liaoning Entry-Exit Inspection and Quarantine Bureau of thePeople's Republic of China
This standard was mainly drafted by Wu Bin,Qin Cheng, Yu Jie, Wang Gang,Zhao Shou Cheng, WangMei,SongHuiJun
This standard is a professional standard promulgated for the first time.SV/T1569—2005
Inspectionoftetrodotoxininglobefishforimport and exportELISA methodScope
This standard specifies the methods of sampling, sample preparation and inspection by ELisA methodof tetrodotoxin in puffer fish for import and export.This standard is applicable to inspect of tetrodotoxin in puffer fish's muscle, skin,liver and ovary (orstalk) for import and export.2
Abbreviation
TTX Tetrodotoxin
ELISA Enzyme Linked Immungsorbent AssayAAbsorption value
BAS-HCHO-TTXBovine Serum Albumin-HCHO-Tetrodotoxin conjugatePBS-TPhosphate Buffered Saline-Tween 20McAb-HRP Monoclonal Antibody-Horseradish PeroxidaseSampling
3. 1 Inspection lot
Each inspection Jot af puffer fish should corre from the sarie sea, the sarre origination, the sarnebreed factory. The characteristics of the cargo within the same inspectian lot,such as type,packing:mark.origin,specification and grade etc. ,should be the same,3.2
Quantity of sample taken
Determination of Quantity of Sample TakenTable 1[免费标准bzxz.net
Number of packages in each inspection lotkg
50~500
501~~1 200
1 201 ~ 3 200
3 201 ~ 10 000
10 001~35 00D
More 35 000
packages
26 ~ 60
61~160
161 ~ 500
501~1750
More 1 750
Number of packages to be takenpackages
3.3Sampling procedure
SN/T 1569—2005
A number of packages specified in 3, 2 are taken, Select at least 3 from each package as the originatesample. Recard in detail the characterizes of samples (colar of body, spat and grain, type, length,weight, with or without thorn etc. ) , plit into container, seal,mark and deliver to the [aboratory at O'c5℃within48h.
3.4Storage of sample
[f 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,and sealed in plasticsbags. After sampling, the test sample should be stored below - 18'c immediately.4Method of inspection
4. 1Principle af 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 microtitre plate, The tetrodotoxin concentrationin the sample is inversely proportianal to the absorption value, and campare with the calibrationcurve for quantative measurement.4. 2 Abstract of method
Microtitre plate is coated by BAS-HCHO-TTX conjugate. The plates are washed three times and eachtime 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 andeach time is 3 min. The stop reagent is added after coloring. The absorbance value of each well solu.tion is measured by microwell system at 490 nm, The TTX concentration in the sample is according tathe absorption value.
4.3Reagents and materials
In this standard,all the chemical reagents should be analytically pure,\water\ is distilled water.4. 3. 1 Acetate acid: pH=4.8(according to Annex A)4. 3. 2PBS: pH=7. 2,0. 05 mol/L(according to Annex A).4.3.3 Substrate buffer: Citric acid-Phosphate Buffered Saline buffer (according to Annex A).4. 3, 4 Washing saluticn (PBS-T) : 0. a5 mol/ L PBS including 0. 5% Tween 20 (according to Annex A).SV/T1569—2005
4.3. 5 Anti-TTX -McAb-HRP: purchased from Sigma,4.3. 6 OPD substrate. purchased from Sigma.4. 3.7 Peroxide solution: 0. 5 mol/L3Stop reagent: 2 mol/ L H, sO4.4.3.8
BAS-HCHO-TTX: purchased from Sigma4.3.9
4. 3. 10 NaOH solution: 1 mol/L.4. 4 Apparatus and equipment4.4.1Microwellsystem
4.4.296-well polystyrene microtitre plate.Homogenizer.
Balance:0.1mg
4,4. 5 Refrigerator: 0℃ ~5℃ and -15℃ ~- 20℃.Inculcator; 37℃ ±1℃
4.4.7Reduce pressure and filter system.4.5.15
Sample preparation
Frozen sample in sealed plastics bag is rapidly refrozen under flowing water, after washed by distilledwater and absorbed by filter paper, the fish is divided inta 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 inta a cantainer,add 25 mL of 1%acetate acid solution,vibrate continually 1o 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 letstand to separate,put the lower aqueous phase into the other funnel, repeat it again. Adjust pH of theaqueous phase with 1 mol/L NaCH 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 % acetate acidsolution,centrifuge 3 000 r,take the clean-up solution, fix volume to 50 mL according to the abovemethad for 10 min. The extraction is egual to 0. 1 g samples.10
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