01237769567ELASA.docx
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01237769567ELASA
Enzyme-LinkedImmunosorbentAssay
生31卢文
2003012377
※BACKGROUND
TheELISAisthenew-styleenzymeimmunoassaytechniquedevelopedfromimmuno-enzymatictechniques.Whichhasbeenadaptedforuseindeterminingtheamountofspecificproteinsintissuesamples,inblood,orinurine.
※PRINCIPLEOFTHEEXPERIMENT
AnELISA(enzyme-linkedimmunosorbentassay)allowsforrapidscreeningandquantificationofthepresenceofanantigeninasample.Proteinsinasampleareadsorbedtoaninertsurface,usuallya96-wellpolystyreneplate.Thesurfaceiswashedwithasolutionofaninexpensivenonspecificprotein(oftencaseinfromnonfatdrymilkpowder)toblockproteinsintroducedinsubsequentstepsfromalsoadsorbingtothesesurfaces.Thesurfaceisthentreatedwithasolutioncontainingtheprimaryantibody—anantibodyagainsttheproteinofinterest.Unboundantibodyiswashedawayandthesurfaceistreatedwithasolutioncontainingantibodiesagainsttheprimaryantibody.Thesesecondaryantibodieshavebeenlinkedtoanenzymethatcatalyzesareactionthatformsacoloredproduct.Afterunboundsecondaryantibodyiswashedaway,thesubstrateoftheantibody-linkedenzymeisadded.Productformation(monitoredascolorintensity)isproportionaltotheconcentrationoftheproteinofinterestinthesample.
※AIMOFTHEEXPERIMENT
Understandtheprincipleoftheenzymelinkedimmunosorbentassay.
Masterthemethodandoperationoftheenzymelinkedimmunosorbentassay.
※REAGENTSANDAPPARATUS
Reagents
1.Coatingbuffer(Carbonate-Bicarbonatebuffer,pH9.6):
Na2CO30.15g,
NaHCO30.293g,addH2Oto100ml(pH9.6).
2.Phosphatebufferedsaline(PBS):
NaCl8g,
KCl0.2g,
KH2PO40.24g,
Na2HPO4.12H2O2.9g,addH2Oto1000ml(pH7.4).
3.Washingbuffer(PBS-T):
PBS+0.05%Tween20.
4.Blockingsolution:
0.5%bovineserumalbumininPBS.
5.Firstantibody:
Rabbit-anti-humanIgGantiserum.
6.Peroxidaseconjugatedsecondaryantibody:
Goat-anti-rabbitIgG-HRP.
7.Substrate:
substratebuffer(fresh):
0.1mol/LCitricacid(2.1g/100ml),6.1ml.
0.2mol/LNa2HPO4.12H2O(7.163g/100ml),6.4ml.
AddH2O12.5ml,dissolve10mgo-Phenylenediamine,andadd40μlof30%H2O2beforeuse.
8.Stoppingreagent:
2mol/LH2SO4.
Equipment
Microplatereader
Microtiterplate
Pipette
※PROCEDURE
A.AntigenCoating
1.Prepareanantigensolutionattheappropriateconcentrationincarbonate-bicarbonatebuffer(coatingbuffer),e.g.humanIgG(0.025mg/ml)incoatingbuffer(antigensolution).
2.Pipette0.2mloftheabovesolutiontoeachwellofthemicrotiterplate.
3.Incubateat37ºCfor30min.,(orincubateovernightat4ºC).
Removethecoatingsolution.WashthreetimeswithPBS-T.
Blockingstep:
0.5%BAS-PBS(0.2ml/well)at37ºCfor1h(anadditionalblockingstepmayberequiredtoblocknon-specificbinding).
●Haveonecontrastwithoutblocking.
B.PrimaryAntibodyReaction
1.Dilutetheprimary(first)antibodiesinPBS-T,e.g.Rabbit-anti-humanIgGantiserum.(add750mlPBS-Ttoeachwell,thenadd750mlfirstantibody(1:
200)tothefirstwell.Sogetfirstantibody1:
400.Pipette750mlofthesolutioninthefirstwelltothesecondwell,getthefirstantibody1:
800.continuediluteasthesameway,wecangetaseriesofdilutefirstantibody.)
2.Add0.2mlofthedilutedfirstantibodytoeachwell.Negativecontrolshouldbeincluded.
3.Incubateat37ºCfor2h.
4.WashthreetimeswithPBS-T.
C.ApplicationofSecondaryAntibody
1.DilutetheperoxidaseconjugatedsecondaryantibodyinPBS-T,e.g.Goat-anti-rabbitIgG-HRP(1:
20000).Add0.2mlofthissolutiontoeachwell.
2.Incubateat37ºCfor45min.
3.WashthreetimeswithPBS-T.
D.SubstratePreparation
Duringthelastincubationandimmediatelybeforeuse,dissolveo-PhenylenediamineinCitricacid-sodiumhydrogenphosphatebuffer,add30%H2O2beforeuse.
E.Development
1.Add0.2mlofthefreshlypreparedsubstratetoeachwell.
Orange-yellowcolorshoulddevelopinpositivewellsafter15min.
2.Stopreactionwith50μlperwellofpreferredstoppingreagentandreadat490nminamicroplatereader.
※RESULT
Table1Dilutionrateofthe1stantibodyinthewellsoftheplate
1
2
3
4
5
6
7
8
9
10
11
12
A
B
1/400
1/800
1/1600
1/3200
1/6400
1/12800
1/25600
1/51200
No
No
C
1/400
1/800
1/1600
1/3200
1/6400
1/12800
1/25600
1/51200
1st.
Ag
D
1/800
1/400
1/1600
1/3200
1/6400
1/12800
1/25600
1/51200
Ab.
E
F
G
H
Antigen:
HumanIgG(5μg/well).(Block:
BSA)
Firstantibody:
Rabbitanti-humanIgGantiserum(1/400-1/51200).
Secondaryantibody:
Goatanti-rabbitIgG-HRP(1:
20000).
*TheD2&D3wellsabovearetreatedreversedbecauseofthecarelessness.
Table2OD490nmofeachwell(withblocking)
1
2
3
4
5
6
7
8
9
10
11
12
A
0.002
0.003
0.002
0.003
0.003
0.002
0.003
0.003
0.002
0.003
0.002
0.002
B
0.002
0.668
0.518
0.320
0.304
0.114
0.097
0.042
0.019
0.005
0.002
0.002
C
0.002
0.468
0.568
0.468
0.279
0.121
0.089
0.057
0.023
0.003
0.002
0.003
D
0.001
0.564
0.671
0.388
0.251
0.181
0.099
0.057
0.026
0.003
0.002
0.002
E
0.002
0.003
0.002
0.003
0.003
0.003
0.004
0.003
0.003
0.003
0.002
0.001
F
0.002
0.002
0.002
0.003
0.003
0.004
0.004
0.004
0.003
0.003
0.003
0.003
G
0.002
0.002
0.003
0.004
0.003
0.004
0.003
0.003
0.002
0.004
0.002
0.001
H
0.002
0.003
0.003
0.003
0.002
0.004
0.003
0.002
0.003
0.003
0.002
0.002
※THEANALYSISOFTHERESULT
Let’sconsidertheLangmiouradsorbancetheory.Whentwokindsofmoleculesaremixedinasolution,theyinteractwitheachotherrandomly.AssumemoleculeAistightlyboundtoasolidface,justlikeantigenboundtothewellsurfaceasperformedinthisexperiment.MoleculeBrepresentsthefirstantibodymixedwithA.Asweknow,therateofBbindingtoAisproportionaltotheamountsofunboundA.
①
raistheassociationrate,orthebindingrate.θistheratioofboundAtototalA,thus,1-θistheratioofunboundAtototalA.
TherateofthebindingofBtoAisalsoproportionaltotheconcentrationofB,ashighconcentrationincreasestheprobabilitythatBmoveclosedtoandbindA.
②
CBistheconcentrationofB.
Assummarizedform①and②,weconcludetheassociationrateequation
Kaistheassociationcoefficient.
ThedissociationoftwoAandBhappenssimultaneouslywiththeassociation.ThedissociationrateisnotrelatedtoCB,becauseonlytheBwhichisboundtoAthatattributestothedissociation.AsmoreAisboundwithB,themorechancethatBdissociatewithA,weconcludethedissociationrateequationbelow:
rdrepresentsthedissociationrate,kdrepresentsthedissociationcoefficient,andθrepresentstheratioofboundAtototalA.
Whenthesteadystateisattained,theassociationrateandthedissociationrateequate.
③
④
⑤
Fromequation③,④,and⑤,wededucethefollowingequationabouttherelationshipbetweenθandCB:
ThisisknownasLangmiouradsobenceeqution.BecausetheamountsofantigenwhichisrepresentedbyAinthetheoryaboveareequalinallELISAwells,andthefirstantibodyrepresentedbyBattributestoA490onlyafteritbindstoantigen,thereislinearrelationshipbetweenA490andθ.ThustheequationoftherelationshipbetweenA490andfirstantibodyconcentrationispredictedas:
※
Kisthecoefficientwhichisaconstantvalueaskaandkd.
TheplotoftheA490-CBequationisshownbelow:
WecanusetheregressiontogetthevaluesofKandka/kd:
WhereRisthelinearrelativitycoeffient.
So:
A490--CBequation
TheplotoftheA490-CBequationisshownbelow:
Thisplotisquitefittheexperimentdata.Andwecandrawthefollowingconclusions:
1.Whenthefirstantibodyconcentrationisextremelylow,thatistheCBisveryclosed0,theA490-CBequationisconvertedto:
TheequationalsorevealsalinearrelationshipwithaslopeofK(ka/kd).
2.WhenCBisverylarge,theequationisconvertedto:
Asweknow,whenantigensbindenoughantibodies,theybecomesaturated,afterwhichtheboundantibodiesortheA490willreachaconstantvalueevenexcessantibodiesareadded.fromthispoint,thenewequationalsofitsthefactconsiderablybecauseA490reachaconstantvalueKasexpected.AndourexperimentalcurveofA490versusfirstantibodyconcentrationshowsatendencytoahorizontallinewhichrepresentsthesaturatedstatus.
※FURTHERDISCUSSION
Thecontrolgroupalsohassomeabsorbance.itismuchlowerthantheformerdilutionwell.Justasweanticipate.Thecontrolgroupalsohassomeabsorbanceanditislittlehigherthanthebackground.Soifwetreattheresultstrictly,weshoulddisposethecontrolabsorbancefromeachdatum.
※RENFERENCE
BingbinYU.TheTechniqueofExperimentalBiochemistry.TsinghuaUniversity,2001.
David·L·NelsonMichael·M·CoxLehningerPrinciplesofBiochemistryThirdedition
Physicalchemistry:
principlesandapplicationsinbiologicalsciences3rded.UpperSaddleRiver,N.J.:
PrenticeHall,c2002.
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