Overview

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Ex/Em(nm)	490/None
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	CellBIOLOGy ReporterGeneEnzymes
Related	HydrolyticEnzymes

β-Lactamasesarealargefamilyofenzymescapableofhydrolyzingβ-lactams.β-Lactamringisthecommonelementinallbeta-lactamantibioticsincludingpenicillinderivatives,cephalosporins,monobactams,andcarbapenems.Throughhydrolysis,β-lactamasebreakstheβ-lactamringopen,thusdeactivatesthemolecule"santibacterialproperties.Bacteriafromclinicalandnon-clinicalsettingsarebecomingincreasinglyresistanttoβ-lactamantibioticsbysynthesizingβ-lactamase.Toovercomethisresistance,β-lactamantibioticsareoftengivenwithβ-lactamaseinhibitorssuchasclavulanicacid.Therefore,detectionofβ-lactamaseactivityisofcentralimportancetoassessbeta-lactamantibioticsaswellastopreventantibioticsresistance.AATBioquest"sColorimetricBeta-LactamaseActivityAssayKitoffersasensitivecolorimetricassayformeasuringβ-lactamaseactivityinbiologicalsamples.Theβ-lactamaseactivityisdetectedusingNitrocefin,whichchangescolorfromyellowtoreduponhydrolysisbyβ-lactamase.TheassaycanbeperformedusinganabsorbancemicroplatereaderbymeasuringtheODratioatthewavelengthof490nmto380nm.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Prepareβ-lactamasestandardstocksolution:

Add100µLofddH₂Ointothevialofβ-lactamasestandard(ComponentC)tomake50mU/mLβ-lactamasestandardstocksolution.

Note:Theunusedβ-lactamasestandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20^ºC.

2.Prepareserialdilutionsofβ-lactamasestandard:

2.1   Add10μLofβ-lactamasestandardstocksolution(50mU/mL,fromStep1)into990µL1×PBSbuffertogenerate500µU/mLβ-lactamasestandardsolution.

Note:Dilutedβ-lactamasestandardsolutionisunstable,andshouldbeusedpromptly.

2.2   Take200μLof500µU/mLβ-lactamasestandardsolutiontoperform1:2serialdilutionsinPBStogetapproximately250,125,62.5,31.3,15.6,7.8and0μU/mLserialdilutionsofβ-lactamasestandard.

2.3   Addserialdilutionsofβ-lactamasestandardandβ-lactamasecontainingtestsamplesintoa96-wellclearbottommicroplateasdescribedinTables1and2.

Table1Layoutofβ-lactamasestandardsandtestsamplesina96-wellclearbottommicroplate

BL	BL	TS	TS	….	….
Lac1	Lac1	….	….	….	….
Lac2	Lac2
Lac3	Lac3
Lac4	Lac4
Lac5	Lac5
Lac6	Lac6
Lac7	Lac7

Note:Lac=β-LactamaseStandards,BL=BlankControl,TS=TestSamples.

 

Table2Reagentcompositionforeachwell

β-LactamaseStandard	BlankControl	TestSample
SerialDilutions*:50μL	1×PBSBuffer:50μL	50μL

 *Note:Addtheseriallydilutedβ-lactamasestandardsfromapproximately500to7.8μU/mLintowellsfromLac1to   Lac7induplicate.

3.Prepareβ-lactamaseassaymixture:

Add50µLnitrocefinstocksolution(ComponentA)into5mLofComponentB,andmixwelltomakeβ-lactamaseassaymixture(ComponentA+B).

Note:Thisβ-lactamaseassaymixtureisenoughforone96-wellplate.Theβ-lactamaseassaymixtureisnotstable,preparefreshforeachuse.

4.Runβ-lactamaseassay:

4.1   Add50μLofβ-lactamaseassaymixture(fromStep3)toeachwellofβ-lactamasestandard,blankcontrol,andtestsamples(seeStep2.3)tomakethetotalvolumeof100µL/well.

Note:Fora384-wellplate,add25μLofsampleand25μLofβ-lactamaseassaymixtureintoeachwell.

4.2   Incubatethereactionatroomtemperaturefor30-60minutes,protectedfromlight.

4.3   MonitortheabsorbanceincreasewithanabsorbanceplatereaderatODratioof490/380nm.

References&Citations

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1. ChantemesseB,BetelliL,SolanasS,VienneyF,BollacheL,HartmannA,RocheletM.(2017)Anitrocefin-basedamperometricassayfortherapidquantificationofextended-spectrumbeta-lactamase-producingEscherichiacoliinwastewaters.WaterRes,109,375.
2. RocheletM,SolanasS,BetelliL,NeuwirthC,VienneyF,HartmannA.(2015)Amperometricdetectionofextended-spectrumbeta-lactamaseactivity:applicationtothecharacterizationofresistantE.colistrains.Analyst,140,3551.
3. HandaD,PandeyA,AsthanaAK,RawatA,HandaS,ThakuriaB.(2013)EvaluationofphenotypictestsforthedetectionofAmpCbeta-lactamaseinclinicalisolatesofEscherichiacoli.IndianJPatholMicrobiol,56,135.
4. KarsisiotisAI,DamblonC,RobertsGC.(2014)Complete(1)H,(1)(5)N,and(1)(3)CresonanceassignmentsofBacilluscereusmetallo-beta-lactamaseanditscomplexwiththeinhibitorR-thiomandelicacid.BiomolNMRAssign,8,313.
5. ThomasPW,SpicerT,CammarataM,BrodbeltJS,HodderP,FastW.(2013)Analteredzinc-bindingsiteconfersresistancetoacovalentinactivatorofNewDelhimetallo-beta-lactamase-1(NDM-1)discoveredbyhigh-throughputscreening.BioorgMedChem,21,3138.
6. PriyadharsiniRI,KavithaA,RajanR,MathaviS,RajeshKR.(2011)Prevalenceofbla(CTXM)extendedspectrumbetalactamasegeneinenterobacteriaceaefromcriticalcarepatients.JLabPhysicians,3,80.
7. VaidyaVK.(2011)HorizontalTransferofAntimicrobialResistancebyExtended-SpectrumbetaLactamase-ProducingEnterobacteriaceae.JLabPhysicians,3,37.
8. FarmerR,GautamB,SinghS,YadavPK,JainPA.(2010)VirtualscreeningofAmpC/beta-lactamaseastargetforantimicrobialresistanceinPseudomonasaeruginosa.Bioinformation,4,290.
9. SanchezPA,ToneyJH,ThomasJD,BergerJM.(2009)AsensitivecoupledHPLC/electrospraymassspectrometryassayforSPM-1metallo-beta-lactamaseinhibitors.AssayDrugDevTechnol,7,170.
10. YuanJ,HuangW,ChowDC,PalzkillT.(2009)Finemappingofthesequencerequirementsforbindingofbeta-lactamaseinhibitoryprotein(BLIP)toTEM-1beta-lactamaseusingageneticscreenforBLIPfunction.JMolBiol,389,401.

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