【商检行业标准(SN)】 进出口蜂王浆中链霉素和双氢链霉素残留量测定方法酶联免疫法

中华人民共和国出入境检验检疫行业标准SN/T2059—2008
进出口蜂王浆中链霉素和双氢链霉素残留量测定方法
酶联免疫法
Determination of streptomycin and dihydrostreptomycinresidues in royal jelly for import and export-Enzyme-linked immunosorbent assay2008-04-29发布
中华人民共和国
国家质量监督检验检疫总局
2008-11-01实施
本标准的附录A为资料性附录。
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：中华人民共和国浙江出入境检验检疫局。本标准主要起草人：施伟良、张晓峰、朱振江、程洁、苏宗仁。本标准系首次发布的出入境检验检疫行业标准SN/T2059—2008
1范围
进出口蜂王浆中链霉素和双氢链霉素残留量测定方法
酶联免疫法
SN/T2059—2008
本标准规定了蜂王浆和王浆冻干粉中链每素和双氢链毒素残留量的酶联免疫测定方法本标准适用于蜂王浆和王浆冻干粉中链素和双氢链素残留总量的测定。2规范性引用文件
下列文件中的条款通过本标准的引用而成为本标准的条款。凡是注明日期的引用文件，其随后所有的修改单（不包括勘误的内容）或修订版均不适用于本标准，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。凡是不注明日期的引用文件，其最新版本适用于本标准。GB/T6682分析实验室用水规格和试验方法（GB/T6682—1992，neq3696：1987）3方法提要
本标准以酸性缓冲液来沉淀蜂王浆中蛋白质、提取残留的链霉素和双氢链霉素，然后以HLB柱净化。处理后样品中残留的链霉素和双氢链霉素与酶标记链霉素共同竞争结合链霉素抗体，同时链霉素抗体结合至包被有绵羊抗免IgG的抗体的微孔板上，通过洗涤除去未结合的链霉素和双氢链霉素和酶标记链霉素，然后加入底物显色，用酶标仪测定吸光度，根据吸光度值得出蜂王浆中链霉素和双氢链霉素的含量。
4试剂和材料
除去注明外，所有试剂均为分析纯，水为GB/T6682规定的一级水。4.1链霉素检测试剂盒（参见附录A)。4.2甲醇。
4.3SDB缓冲溶液：称取1.15g磷酸氢二钠0.2g磷酸二氢钾，0.2g氯化钾，30g氯化钠，0.5mL吐温-80，用水定容至1000mL，用磷酸/氢氧化钠调节pH至7.54.4庚烷磺酸钠缓冲液：称取10.1g庚烷磺酸钠[CHs（CH2).SO:Nal,11.4g磷酸钠（NasPO4·12H2O）用水溶解并定容至1000mL,用磷酸调节pH至2.0。4.5：10%甲醇：量取10mL甲醇（4.2）用水定容至100mL。4.6HLB小柱：Oasis（或相当产品）3mL（60mg）。4.7链霉素和双氢链霉素标准品：纯度大于等于98%。4.8链霉素和双氢链霉素标准品溶液的配制：称取0.25g链霉素或双氢链霉素，用甲醇定容至10mL，配制成25mg/mL的储存液，于一20℃条件下保存。5仪器
5.1酶标仪
5.28道移液器：10μL~100μL。5.3单道移液器：10μL~100μL20μL~200μL100μL~1000μL和2m~10mL。5.4混合振荡器。
SN/T2059—2008
5.5高速低温离心机：6000r/min。5.6固相萃取装置。
5.7氮吹仪。
5.8具塞试管：50mL
5.9电子天平：0.01g~100g。
5.10酸度计。
6试样的制备和保存
6.1试样的制备
原始样品总量不得少于200g，蜂王浆充分搅拌均勾后，将样品分成两等份：冻干粉采用四分法，将样品分成两等份。分好的样品装入洁净容器，加封并做标识。6.2试样的保存
试样放置一20℃～-18℃条件下保存7分析步骤
7.1提取
称取2.0g蜂王浆样品，置于50mL具塞试管中，加人8mL庚烷磺酸钠缓冲液（4.4），充分混匀：15℃条件下6000r/min离心10min直至清亮，取上层液备用。HLB小柱（4.6），依次用1mL甲醇（4.2）和1mL水活化，吸取1.5mL样品提取液上柱，然后3mL水洗，1mL10%甲醇（4.5）洗柱，去除残留的液体，氮气吹2min十燥柱子。然后以2mL甲醇（4.2）洗脱，将样品收集于十净塑料试管，氮气吹干。以3mLSDB缓冲液（4.3)溶解吹干残留物，用于ELISA检测。最后样品稀释倍数为10。王浆冻干粉则用水以1：2比例稀释.充分浸泡（2h以上）后，称取2.0g按照上述王浆前处理方法进行提取，最后以2mLSDB缓冲液溶解吹干残留物，最后样品稀释倍数为20。7.2测定条件1
7.2.1操作条件
所有操作应在室温下（20℃～24℃）进行，链霉素试剂盒中所有试剂的温度均应回升至室温（20℃～24℃）后方可使用。
7.2.2洗板条件
人工洗涤次数5次以上，每次加入洗涤液量为250μL；自动洗板可以预定5次。7.2.3酶标仪测定条件
酶标仪测定波长为450nm。
7.3测定步骤
7.3.1将测定需用的微孔板备齐并插人微孔架上，记录标准品及样品等在微孔架上的位置。7.3.2吸取100μL零浓度标准品于孔A1、A2；并吸取50μL零浓度标准品于孔B1、B2；分别吸取50μL链霉素标准溶液（浓度分别为：0.25、0.5、1.0、2.0、10.0、20.0ng/mL)于孔C1、C2H1、H2；分别吸取50uL样品提取液于其余微孔中。测定中吸取不同的试剂和样品溶液时应更换吸头。7.3.3分别吸取25μL.链霉素酶标记物溶液于除A1、A2外的每一个微孔，7.3.4分别吸取25uL链霉素抗体溶液于除A1、A2外的每一个微孔。7.3.5用封口膜封孔条，并持微孔板在台面上以圆周运动方式混匀。7.3.6将酶标板置于4℃避光温育1h。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步骤的认可。如果其他产品的操作步骤有不同，需经实验评估后采用。
SN/T 2059—2008
7.3.7、倒出孔中的液体，将微孔架反扣在吸水纸上反复拍打，以除去孔中过多的残液，但不能使微孔干燥。然后立即用洗涤缓冲液按7.2.2条件进行洗板。要注意不能使微孔干燥。7.3.8迅速加入100L底物溶液于每个微孔底部，然后持微孔板在台面上以圆周运动方式混匀后，于20℃～24℃避光温育30min。
7.3.9加入100uL反应终止液于每一个微孔，然后持微孔板在台面上以圆周运动方式混匀后，将微孔架置于酶标仪中，在450nm处测量吸光度（应在加入反应终止液后30min内读取吸光度）。7.4平行试验
按以上步骤，对同一标准溶液、同一样品溶液均应进行平行试验测定。7.5空白试验
除不加人试样外，均按上述步骤进行。7.6阳性质控
每次测定均应做一个添加链霉素和双氢链霉素标准品溶液（4.8)的监控样品测定，以确定实验过程的操作准确性。
8结果计算
从标准品和样品的吸光度（OD)值中，减去空白孔A1、A2的平均OD值。标准品和样品的OD平均值除以零标准（B1、B2)的平均OD值，再乘以100。零标准为100%（最大百分比吸光度值），其他OD值为最大吸光度值的百分数，
以吸光度的百分比值（B/B。）为纵坐标（%）.链毒素标准溶液浓度（ng/mL）的对数值为横坐标，绘制标准工作曲线。从标准工作曲线上得到试样中相应的链霉素浓度后，结果按式（1)进行计算：X=cXVX1000
m×1000
式中：
X—样品中链霉素和双氢链霉素的残留总量，单位为微克每千克（ug/kg）；..(1)
从标准工作曲线上得到的样品中链霉素和双氢链霉素浓度，单位为纳克每毫升（ng/mL）；V一样品溶液的最终定容体积，单位为毫升（mL）；m样品溶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据处理软件进行计算。所得结果表示至一位小数9确证试验
如被测样品中为阳性结果时，应用其他方法进行确证。10本方法测定低限和回收率
10.1方法测定低限
10μg/kg。
10.2回收率
本方法中链霉素和双氢链霉素添加浓度及回收率的试验数据：a）链霉素
添加量为10g/kg时，回收率为89%114%—添加量为20μg/kg时，回收率为83.5%~109.5%；一添加量为200μg/kg时，回收率为86.4%~101.6%；b）双氢链霉素
添加量为10μg/kg时，回收率为79%~108%；添加量为20ug/kg时，回收率为82%~107.5%—添加量为200ug/kg时，回收率为79.2%～102.8%。3
SN/T2059—2008
A.1试剂盒组成
附录A此内容来自标准下载网
（资料性附录）
链霉素试剂盒（荷兰EURO-DIAGNOSTICA公司产品）23预包被抗体的96孔板：12条×8孔A. 1. 1
链霉素标准溶液：00.25、0.5、1.0、2.0、10.0、20.0和100ng/mL。A.1.24
链霉素酶标记物冻干粉：根据链霉素试剂盒中说明，可用稀释缓冲液配制成链霉素酶标记物A.1.3
溶液。
抗链霉素抗体冻干粉：根据链霉素试剂盒中说明，可用稀释缓冲液配制成抗链霉素抗体溶液A.1.4
底物TMB溶液。
稀释缓冲液。
洗涤浓缩液：可用水10倍稀释后使用。A.1.8
反应终止液。
试剂盒应在4℃～8℃避光条件下保存，溶解后的酶标记物和抗体溶液需一15℃条件冻存。A.2标准校准曲线
标准校准曲线见图A1。
17.126lnx+65.95
R2—0.9954
Ang/mL)
图A.1链霉素标准曲线
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。
Foreword
AnnexAofthis standardare informativeannexs.SN/T2059—2008
This standard was proposed by Certification and Accreditation Administration of the People'sRepublic of China.
This standard was mainly drafted by Zhejiang Entry-Exit Inspection and Quarantine Bureau of thePeople's Republic of China
This standard was mainly dafted by Wei-liang Shi,Feng-xiao Zhang,Zhen-jiang Zhu,Jie ChengZhong-ren Su
This standard is professional standard of Entry-Exit inspection and quarantine promulgated for thefirsttime.
SN/T2059—2008
Determination of streptomycin and dihydrostreptomycinresiduesinroyal jellyforimportandexportEnzyme-linkedImmunosorbentassayScope
This standard specifies the methodsof determination by ELisA method of streptomycinanddihydrostreptomycinresiduesinroyal jellyandroyaljellypowder.This standard is applicable to the screen determination of streptomycin and dihydrostreptomycin residues in royal jelly and royal jelly powder.2Normativereferences
The following normative documents contain provisions which, through reference in this text, con-stitute provisions of this standard. For dated references, subsequent amendments to,or revisionsof, any of these publications do not apply. However parties to agreements based on this stan dardare encouraged to investigate the possibility of applying the most recent editions of the normativedocuments indicated below.For undated references,the latest edition of the normative documentreferredtoapplies.
GB/T 6682 Water for analytical laboratory useSpecification and test methods.(GB/T 66821992,neqiso3696：1987)
Principle
The microtitre based ElA kit consists of 12 strips, each 8 wells,precoated with sheep antibodies torabbit IgG.A specific antibody(rabbit anti-Streptomycin),enzyme labelled Streptomycin (enzymeconjugate)and Streptomycin standard or sampleare added to theprecoated wells followed bya sin-gle incubation step.The specific antibodies are bound by the immobilised antibodies and at the sametime free Streptomycin/Dihydrostreptomycin (present in the standard solution or sample)and en-zyme conjugated Streptomycin compete for the Streptomycin antibody binding sitea (competitiveenzyme immunoassay).After an incubation time of 1h,the non-bound(enzymelabelled)reagentsare removed in a washing step. The amount of Streptomycin enzyme conjugate is visualised by theaddition of a chromogen substrate （tetramethylbenzidine,TMB). Bound enzyme conjugate trans-forms the colorless chromogen intoa coloured product.The substrate reaction is stopped by photo-metrically at 450 nm. The optical densityis inversely proportional to the Streptomycin/Dihydro-streptomycin concentration in the sample6
4Reagentsandmaterials
SN/T2059—2008
Unless otherwise specified, all the chemical reagents should be A. R. grade.\water\is the first gradewaterprescribedbyGB/T6682.
Enzyme immunoassaykitforthe quantitativeanalysis of streptomycin.（seeannexA）Methanol.
SDB:Dissolvein1000mLdistlledwater1.15gNa2HPO4;0.2gKH2PO40.2gKCl;30gNaCl0.5 mL Tween-80(pH=7.5).
Heptane sulfonic acid sodium salt buffers:Dissolve in 1 o0o mL distlled water 10.1gCH3(CH2)6SO3Na,11.4g NaaPO4:12H2O,adjust pH to 2.0 usingH3PO4.4.5
10%Methanol:Dissolve10mLmethanol in90mLdistilledwater.HLBcolumns:Oasis,3mL(60mg).
StreptomycinandDihydrostreptomycinstandard（Lyophilized):Purity>98%The preparation of Streptomycin and dihydrostreptomycin standard solution(25 mg/mL):dissolve0.25gStreptomycinordihydrostreptomycinstandard in10mLmethanol.5
Apparatus and equipment
ELiSAreaderequippedwitha450nmfilter.10uL~100μLmultichannelmicropipettewithsuitabletips.variable volume precision micropipette with suitable tips:10 μL~100 μL,20 μL~200 μL,100μL1000μLand2mL~10mL.
Sakervotex
Centrifuge: 6 000 r/min.
SPEcartrige.
Nitrogen evaproator.
Testtubewithplug:50mL.
SN/T2059—2008
Electronicbalance:0.01g~100g.pHmeter.
Preparation and storage of sample6.1
Preparation of sampleextraction.The whole primary sample should be more than 20o g.For royal jelly samples:Divide it into twoequal parts aftermixwell.Forroyal jellypowdersamples:Divide it intotwo equal partsbyquarta-tion.Thenkeepthepreparedsampleintotwosamplebottles,sealand label.6.2
Storage of sample.
Store sampleat-20℃～-18℃.
Teststeps
Extraction
Weigh 2.0 g of royal jelly into 50 mL tube with plug.add 8 mL 1-Heptane sulfonic acid sodium saltbuffers(4.4).centrifuge6000 r/min at 15℃ for10min.This extract isfurtherpurifiedas followingby means of HLB columns(4. 6).The columns is rinsed with 1 mL methanol(4,2) and equilibratedwith1mLdistilledwater,1.5mLsamplesolutionispastthoughthecolumn,followedbyrinsingwith3mLdistilled waterand 1mL10%methanol(4.5).The fluid residus isremovebypositivepres-sure,the column is dried for 2 min by floating it with air or nitrogen. Then elute sample with 2mLmethanol(4. 2) and collect it into a clean plastic tube, blew with nitrogen until to dry. Dissolve theresidue with 3mL SDB.It is ready for ELiSAassay.For royal jelly powder,it should be dip in water(1:2)at least2h,thenweigh2.0gforextractionaccordingtoroyal jellymethod.Dissolvetheresiduewith2mLSDB
Testcondition1)
Operationcondition
All theoperationshouldbedoneatroomtemperature(20℃~24C).Thereagentsshouldbebrougheuptoambienttemperature（20℃~24℃）beforestartingthetest.The information supplying herein is just for the convenience usage for the users of this standard and not for1)
some certain product's authorization analysis procedure.The testing process of another equal kits maybe dif-ferso it shouldbeused afttesting.evalution andverification.8
7.2.2Theworkconditionof washmachine.SN/T2059—2008
Manualwork:All thewellsmustbewashedmanuallymorethan5times.Eachtimeinject25oμLwashsolutionwithmultichannelmicropipette,Automatismwork:All thewellsshouldbewashed5timesbythewashmachine.
7.2.3The work condition of microplate reader.Themicroplatereadershouldbeusedata45onmfilter.7.3Determination
Predeterminean assay layout,recording blank,standard and sample positions,taking into7.3.1
account that all have to be run in duplicate7.3.2Pipette100μL of zero standard in duplicate(wellsA1.A2);Pipette50μLof zero standard induplicate(wells B1,B2); Pipette 50 μL of each standard dilutions(0.025.0.05,0.1,0.2,0.5 and 2 ngmL)in duplicate(wellsC1.C2to H1H2);Pipette50μLof each sample solution in duplicate into theremainingwellsof themicrotitreplate.when inject differentreagentsand samplesolutions,tipsshould be replaced.
Add25μLofconjugate(Streptomycin-HRPO)intoallwells,exceptwellsA1andA2.Add25μLofantibodysoltion intoall wells,exceptwellsA1andA2Seal themicrotitreplate and shake the plategently with rotating motion for few secondstoIncubatefor1h in the dark at4℃.Discardthesolutionfromthemicrotitreplateandwash5timeswithrinsingbufferfollowingtheconditionof7.2.2
Pipette100μL of substratesolution into eachwell.Incubate30min.at roomtemperature7.3.8
（20℃～24℃）
Add 1oo μL of stop solution into each well.Read the absorbance values immediately at7.4Parallel test
Following theabove steps,the same standardsand sample solutions also should be measuredat thesame time for parallel test.9
SN/T2059—2008
7.5Blank test
The test should be done following the above steps except for adding samples7.6Positivemonitoring
Analyzea recoveriesfortified atmaximumresidue limit(MRL)level using blank sampleeachtest8
Calculating theresult
Subtract the mean optical density (OD) value of the blank wells A1 and A2 from the individual OD.of the wells containing the standards and the samples. the OD. values of the standards and the sam-ples(mean values of the duplicates)are divided by the mean OD.value of the zero standard (wellsB1andB2)andmultiplyby100(B/Bo);thezerostandard isthusmadeequal to100%(maximalabsor-bance)and the other OD values are quoted in percentages of the maximal absorbance.Enter the B/Bo values calculated for each standard in a semi-logarithmic system of coordinates a-gainst the standard Streptomycin concentration; draw the standard curve; Interpolate the corre-sponding concentration from the calibration curve; Calculate the concentrations of Streptomycin inthe samples according to formula(1):Where
×= C×V×1 000
m×1000
X-theconcentrationsof Streptomycin/Dihydrostreptomycin inthe samplesunit inμg/kg;c-the concentrationsgotten fromthe semi-logarithmic systemof coordinates unit in ng/mL;Vthefinalvolumeof thesamplesolutionunit inmL;mthefinal sampleweightofthesamplesolutionunit ing（
Other data processing software can also beusedto calculatethe concentrationsof Streptomycin/Dihydrostreptomycin.Theresults indicatetoonedecimal.ConfirmatoryTest
If sample result is positive, it should be confirmed with confirmation method.10
Limit of determination and recovery10.1Limitof determination
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。