克倫特羅、青霉素和慶大霉素抗體的制備與間接競(jìng)爭(zhēng)ELISA檢測(cè)克倫特羅殘留的初步研究.pdf

1、摘要克倫特羅具有典型的p一腎上腺素活性和對(duì)p2受體的選擇性親和力，它在獸醫(yī)臨床上被用作支氣管擴(kuò)張劑用于治療氣管炎和哮喘。但若在養(yǎng)殖業(yè)非法用作營(yíng)養(yǎng)再分配劑以提高瘦肉率，可造成其在動(dòng)物性產(chǎn)品中的殘留而危害消費(fèi)者健康。慶大霉素作用于多種革蘭氏陽(yáng)性和陰性菌，在獸醫(yī)臨床上，慶大霉素主要通過(guò)注射途徑給藥，用于呼吸道感染以及乳腺感染等。但該藥的排泄較慢，如果使用不當(dāng)，容易造成在動(dòng)物性產(chǎn)品中的殘留蓄積。青霉素G既能被青霉素酶滅活，也能被胃腸道的酸性環(huán)境

2、降解，獸醫(yī)臨床上多以注射方法用于革蘭氏陽(yáng)性敏感菌引起的感染性疾病(如奶牛乳腺炎等)的治療，青霉素G是牛奶中比較常見(jiàn)的殘留藥物之一，可以引起過(guò)敏反應(yīng)，抑制酸奶加工過(guò)程中乳酸菌的生長(zhǎng)。因此，建立針對(duì)這些藥物殘留的快速檢測(cè)方法對(duì)控制動(dòng)物性食品的衛(wèi)生質(zhì)量、保障消費(fèi)者健康具有比較重要的社會(huì)經(jīng)濟(jì)意義。r上述三種藥物為小分子半抗原，缺乏免疫原性。將半抗原與合適的載體蛋白偶上聯(lián)后，半抗原可以成為全抗原并具有較好的免疫原性。碳二亞胺(EDC)是一種同型雙

3、功能交聯(lián)劑，可以通過(guò)一步反應(yīng)形成穩(wěn)定的半抗原一載體復(fù)合物。免疫原EDC偶聯(lián)試劑盒可用于克倫特羅、青霉素和慶大霉素的偶聯(lián)。按照半抗原一載體EDC標(biāo)準(zhǔn)偶聯(lián)程序，我們偶聯(lián)了克倫特羅BSA、克倫特羅一OvA、青霉素BSA、青霉素一O、rA、慶大霉素一BSA、慶大霉索一KU。偶聯(lián)物4“C透析3天后，用BSA偶聯(lián)物加佐劑免疫新西蘭白兔。從第三次免疫后7天心臟采血。放血分離血清后，以間接ELISA測(cè)抗體效價(jià)，用OVA和KLH偶聯(lián)物包被酶標(biāo)板，1％酪蛋

4、白封閉1小時(shí)，一抗(倍比稀釋的相應(yīng)血清)和酶標(biāo)二抗(500x稀釋的羊抗兔IgG／HRP)分別在3TC反應(yīng)2小時(shí)。PBST洗3次后用0PD(鄰苯二胺)顯色，最后用2M硫酸終止反應(yīng)，酶標(biāo)儀測(cè)490nmOD值。根據(jù)OD值(Y一軸)和稀釋度(X～軸)的曲線，找出免疫血清曲線中的拐點(diǎn)及其對(duì)應(yīng)X軸的數(shù)值確定抗體效價(jià)。方案一中青霉素抗體效價(jià)為1600X，方案二中克倫特羅抗體效價(jià)為400。在方案三中，克倫特羅、青霉素和慶大霉素抗體效價(jià)分別為400X，4

5、00和200。在間接競(jìng)爭(zhēng)ELISA和硝酸纖維素膜試驗(yàn)中，我們同時(shí)比較了1％OVA、13％明膠、AbstractClenbuterolisasympathomimeticagent、vithprincipally6一adrenergicactivityandaselectiveactionon6receptorsItisusedinveterinarymedicineasbronchodilatatorandtocolyticIthasa

6、lsobeenillegallyappledinanimalfeedadditivesasarepartitioningagentwhichwould1eadtoresiudesintheanimalproductswiththepotentialoffood—bornepoisoningGentanaycinisactiveagainstmanygram—positiveandgramnegativebacteriaItisadmin

7、isteredbyintramuscularlyorintravenouslyforgeneralinfectionsororallyforgastro—intestinalinfectionItisalsousedintramammarilyforbovinemastiffsandtopicallyinocularandauricularinfectionsImproperuseofthedrugmayresultii3residua

8、lproblemsbecauseofitssloweliminationPenicillinGisinactivatedbypenicillinasesandisunstableingastricacidItisoftenusedforthetreatmentofinfectionsbysusceptiblebacteriathroughparenteralapplicationorintramammaryinfusionforbovi

9、nemastitisItsresideinmilkisofgreatconcerntotheconsumersforltspotentialofallergicreactionsaswellastothedairyindustryforitsinhibitoryeffectonthestarterculturesTherefore，developmentoffastmethodsfordetectionofresiduesofthese

10、drugsinthefoodsofanimaloriginsisofgreatpublichealthsignificanceandsocioeconomicimportanceTheabovethreedrugsarehaptensTheirimmunogenicitycanbeimporvedbycouplingthemtosuitablecarrierproteinmoleculesNethylN『3(dimethylamino)

11、propyllcarbodiimidehydrochloridefEDC)isabifunctionaIcouplingreagentItisemcientforone—stepcouplingtoformawidevarietyofhapten—carrierproteinimmunogensConiugationmayOCCUrateithertheC—orNterminalofthehaptenoratanycarboxylora

12、minecontainingsidechainsImjectImmunogenEDCConjugationKitsfromPiercearedesignedtosimpli白theproductionofclenbuter01BSA，clenbuterolOvA，penicillin—BSA，penicillin—OVAandgentamycinBSA，gentamycinKLHAfterDialysisfor3daysat46Cfor

13、desalting，theBSAconiugateswereimmunizedtorabbitsaftermixingwiththealuminumadjuvantBloodsamplesweretaken7daysafterthethirdvaccinationSerumsampleswerepreparedandstoredat20℃AntibodytitersweremeasuredbyindirectEnzymeLinkedIm

14、munosorbantAssay(ELISA)Theantigen(Ilapten一0VAorKLHconjugates)wasimmobilizedovemightat4etothepofystyrenemicrotiterplate(，n60undsiteswereblockedbyZ％caseinforJ6raf37℃Serialdilutionsofantisenlmsampleswereaddedtothewellsandal

15、lowedtobindfor2hrsat37℃followedbythreetimesofwashingGoatantirabbitHRPIgGconjugatef1：500)wasaddedandincubatedfor2hrsat37。CAtieranothercyclesofwashing，1OOuLofthesubstrateophenylenediaminesolutionwasaddedforcolordevelopment

16、at37℃for20minutesThereactionwasstopedbyadditionof50gLof2MH2S04Absorbalicewasmeasuredat49011121onamicroplatebasedELISAreaderAntibodytiterswereapproximatedbyplottingdilutionsoftheantiseraagainst0D490nmTheantibodytitersofpc

17、nicillin(Scheme1)andclenbuterol(Scheme2、are1600xand400respectivelywhilethetitersofpenicillin，clenbuterolandgentamycininScheme3are400X400and200respectivelyWealsocomparedtheblockingeriectsof1％ovoalbumin1—3％gelatin，1％bovine