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抗原制备
AmericanJournalofAgriculturalandBiologicalSciences2
(2):
88-93,2007
ISSN1557-4989
©2007SciencePublications
CorrespondingAuthor:
WangJinyi,CenterofMicroTotalAnalysisandNanotechnology,CollegeofScience,Northwest
A&FUniversity,Yangling,Shaanxi712100,P.R.China.Fax:
00862987082520
88
RecentAdvancesintheSynthesisofArtificialAntigenandItsApplicationintheDetection
ofPesticideResidue
1,2TongDewen,1,2HeshengYangand1,2,3WangJinyi
1CenterofMicroTotalAnalysisandNanotechnology,NorthwestA&FUniversity,
Yangling,Shaanxi712100,P.R.China
2CollegeofAnimalScienceandTechnology,NorthwestA&FUniversity,Yangling,
Shaanxi712100,P.R.China
3CollegeofScience,NorthwestA&FUniversity,Yangling,Shaanxi712100,P.R.China
Abstract:
Recentadvancesintheresearchofartificialantigenhaveshownthatartificialantigenscan
bevaluableapproachforthetreatmentofsomediseasesaswellasthedetectionofpesticideresidues.
Bydirectly/indirectlycouplinghaptentoanappropriatedcarrier(macromolecule),artificialantigen
caninduceanimalstoproducehapten-specificantibody.Basedonthisprinciple,variousvaccineshave
beendeveloped.Moreimpotently,newanalyticalmethod,immunologicalanalysishasalsobeen
established.Comparingtheconventionaltechnologies,suchaschromatographicmethods,this
promisingmethodoffersanalternativewithhighspecificity,sensitivity,simplicityandsuitabilityfor
theanalysisofalargenumberofsamplesinashortperiodoftime.Inthisreview,wedescribethe
recentadvancesinthesynthesisofartificialantigenanditsapplicationinthedetectionofpesticide
residues.
Keywords:
artificialantigen,synthesis,pesticideresidue,analysis
INTRODUCTION
Beinganovelandpromisinganalyticaltechnique,
immunoassaywithhighspecificity,sensitivity,
simplicityandsuitabilityfortheanalysisofalarge
numberofsamplesinashortperiodoftime,has
exhibitedpotentialusageinthedetectionofpesticide
residues[1,2].Conventionalmethodsemployedto
detect/analyzethepesticideresidueare
chromatographictechniquessuchasgas
chromatography(GC)andhighperformanceliquid
chromatography(HPLC),which,however,aretime
consumingandrequiresophisticatedequipmentonly
availableinwell-equippedlaboratories[3,4].Inaddition,
theconventionalmethodsusuallyrequirealotof
complexpre-treatmentofsamples.Immunoassay,
however,caninsitudetectnano-gramscaletargeted
haptens[5].
Thecriticalcomponentofanimmunoassayisthe
productionofantibodiespresentingmaximum
specificityandsensitivityforthetargetedhapten.In
immunology,haptensdonotallowthemselvesto
induceanimmuneresponsebecauseoftheirlow
molecularweight(≤1000Da).Theyhavetobe
covalentlylinkedtoappropriatedcarriers,suchas
protein,toformanartificialimmunogenicconjugateto
indirectlyinduceBcelltoproliferate,differentiateand
producehapten-specificantibodies[6].
Thedesign,structure-modificationofhaptens,the
lengthofcouplingspacers[7]andtheselectionof
optimizedcarriersareveryimportantfactorsinthe
preparationofhapten-carrierimmunoconjugates,which
willdirectlyaffecttheproductionofhigh-quality
hapten-specificantibodies[8-10].Ifhaptenscontain
activegroupssuchas–COOH,-NH2,-OH,etal.,they
candirectlyreactwiththecarrierproteinstoformthe
desiredartificialantigens.Or,structuralmodification,
tointroduceactivegroupsatappropriatepositionsin
theirstructures,isrequired.Thecarrierproteins[8]such
asbovineserumalbumin(BSA),ovalbumin(OVA),
keyholelimpethemocyanin(KLH)andhumanserum
albumin(HSA),also,aremaincomponent.Different
carrierproteinscaninducedifferentimmuneresponse.
Generally,tooptimizespecificityofimmuneresponse,
severalhaptensandseveralcarrierproteinshavetobe
tested.Thespacercanbeeithergrafteddirectlyonthe
targetanalyteoronahaptenanalog.Otherwiseatotal
synthesisofthehaptenisnecessary.Herein,detailed
descriptionsonthesynthesisofartificialantigenandits
applicationinthedetectionofpesticideresiduesare
presented.
Am.J.Agri.&Bio.Sci.,2
(2):
88-93,2007
89
Designandstructuremodificationofhapten:
The
desiredhaptensshouldbethathapten-carrierconjugates
caninducespecificimmuneresponseandproducehigh
qualityhapten-specificantibodies.Whilehaptensbeing
designed/selected,thefinalchemicalstructureand
stereochemistryshouldbeidenticalorsimilarwiththe
originalhaptens[8].Ifhaptenscontainactivegroups
suchas–COOH,-NH2,theycanbedirectlycoupled
withthecarrierproteins.Otherwise,derivesofthe
haptensshouldbepreparedtointroducereactivegroups
intothestructure.Inaddition,thehaptensthemselves
shouldpossesscomplicatedchemicalstructures[9,10].
Generally,mostofthesedesiredhaptensare
characterizedbythefollowingaspects[11]:
(1)amino
grouporcarboxylgrouporboth;
(2)aromatic
compounds.Asreportedpreviously[11],thepossibility
toproducehapten-specificantibodybytheartificial
antigenis1/3,ifthehaptencomposedofaromatic
compoundsorcontainaromaticrings.Or,the
possibilityis1/11;(3)highbranch;(4)heteroatom
rings,sincetheyareallhighlyimmuneactivitygroups.
Sometimethemetabolicintermediatescanalsobeused
asdesiredhaptenswhichcaninducebodiestoproduce
theoriginalhapten-specificantibodies.
Theotheroneveryimportantfactoristhelengthof
thespacer.Ifitistoolong,thehaptenscanoverlap
alongthespacerandchangetheirstereo-structures.Ifit
istooshort,thecarrierproteincancoverthehaptenand
cannotproducespecificantibody.Inaddition,the
spacershouldbenon-polar,or,theycanchangethe
distributionoftheelectricdensityofthehapten.
Selectionofcarrierproteinsforthesynthesisof
artificialantigen:
Theuseofcarrierproteinisnotonly
tosimplyincreasethemolecularweightofthehaptencarrier
conjugate;theycanalsoaffectthequalityand
quantityofimmuneresponses.Intheimmunologic
memoryofsecondaryimmuneresponsetheyplayan
importantrole.Inotherwords,secondaryresponseand
recallingresponsearealsodeterminedbythecarrier
proteins[10].
Proteinsusedascarriersforthepreparationof
artificialantigen,usually,arebovineserumalbumin
(BSA),ovalbumin(OVA),keyholelimpethemocyanin
(KLH),andhumanserumalbumin(HSA).Among
theseproteins,BSAisthepopularonebecauseofits
physicalandchemicalstability,notexpensive,easily
available,morelysineresiduesandmoreaminogroups.
Inaddition,BSAcanalsopresentexcellentsolubility
undervariouspHvalueandionicstrength.Itcanreact
withthetargetedhaptensinorganicsolventssuchas
pyridineandN,N-dimethylformamide(DMF),andthe
immuneconjugatecanwelldissolveinthereaction
mixtureafterreactionisdone.
Generally,thecarriershouldbeheterogenouswith
theexperimentalanimals,sinceitiseasiertoinduce
strongimmuneresponseandtoproducehigh-titerand
hapten-specificantibody.Moreheterogeneitycan
producehigherqualityantibody.Inspiringly,recent
researchresultsexhibitedthat:
(1)homologousproteincarrier
couldalsoinduceimmuneresponseandproduce
hapten-specificantibody[6];
(2)polypeptidesuchas
poly-L-lysinecouldalsobeemployedascarrier.
Comparingwiththeconventionalproteincarrier,poly-
L-lysinepossesseshigheraminodensityandthe
couplingreactionscarriedoutmoreeasily.Duetoits
simplystraightchainstructure,itsimmunogenicityis
alsolow[12,13].
Methodsforthecouplingofhaptenstocarrier
proteins:
Bindingthedesiredhaptenstothecarriersis
thecriticalstepinthesynthesisofartificialantigen.If
thehaptenspossessactivegroupssuchas–COOH,-
OH,and-NH2,asdescribedabove,theycandirectly
reactwiththecarrierprotein.Or,structural
modificationsarerequired[14].
Basedonthechemicalandstereo-structureof
haptens,varioussyntheticapproacheswereemployed:
(1)Carboxyl-containedhaptens,suchas
fluoroquinolones,polyethers,cephalosporinsand
peptides,canbecoupledwiththecarrierproteinsusing
N-hydroxysuccinimideactiveester/carbon-diimineand
Woodwardreagentprotocol.
Scheme1.WoodwardReagentProtocol[15]
(2)Amino-containedhaptens,suchas
sulfanilamides,aminoglycosides,β-lactams,
acheomycins,benzenimidazoles,benzenethylamines,
fluoroquinolones,canemployglutaraldehyde,
diisocyanate,halonitrobenzene,thiophosgenation,
diimineester,anddiazotizationprotocol.
N
O
CH
OH-HC:
C
O
NCHR-COOH
HN
O
OOR
H
CH
O
OR
H
O
NH2-Protein
H
O
NH
O
CHRNH
O
+Protein
Am.J.Agri.&Bio.Sci.,2
(2):
88-93,2007
90
Scheme2.DiisocyanateProtocol[16]
(3)Hydroxyl-containedhaptens,suchas
chloramphenicols,aminoglycosides,macrolides,
avermectins,steroidhormones,and
benzenethylamines,canbedirectlyconnectedto
thecarrierproteinsthroughsuccinicanhydrideor
azobenzoicacidprotocol.
Scheme3.SuccinicAnhydrideProtocol[16]
(4)Carbonyl-containedhaptens(ketoneoraldehyde),
suchasstreptomycins,acheomycins,macrolides,
fluoroquinolones,steroidhormones,usuallyuseaminoox-
aceticacidprotocol.
Scheme4.Amino-ox-aceticAcidProtocol[16]
(5)Mercapto-containedhaptenscanemploy
homogeneousorheterogeneousdifunctionreagentsto
synthesizetheimmunoconjugates.
Scheme5.SuccinicAnhydrideProtocol[17]
Purificationofartificialantigens:
Beforeimmunizing
animalsusingtheartificialhapten-proteinconjugateto
getthedesiredantibody,purificationisnecessarysince
theunreactedhaptenmolecul
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