Goat AntiCanine Motilin Receptor

GoatAnti-CanineMotilinReceptor
                                                                                                  
Storage:2-8°C                                      Packagesize:96determinations  
 
PRINCIPLEOFTHEMETHOD
TheMTLRReceptorkitisasolidphasephasesandwichenzymelinkedimmunosorbentassay(ELISA).Samples,includingstandardsof knownMTLRReceptorconcentrationsandunknownsarePipettedintothesewells.Duringthefirstincubation,theMTLRReceptorantigenandabiotinylatedmonoclonalantibodyspecificforMTLRReceptoraresimultaneouslyincubated.Afterwashing,theenzyme(streptavidin-peroxydase)isadded.Afterincubationandwashingtoremovealltheunboundenzyme,asubstratesolutionwhichisactingontheboundenzymeisaddedtoinduceacolouredreactionproduct.TheintensityofthiscolouredproductisdirectlyproportionaltotheconcentrationofMTLRReceptorpresentinthesamples.
 
REAGENTSPROVIDEDANDRECONSTTTUTION

REAGENTS（Storeat2-8℃）	1×96WELLS	0.5×96WELLS	RECONSTTTUTION
96/48-wellsmicrotiterplates	1	0.5	Ready-to-use
Plastivcover	2	1	Ready-to-use
Standard:400pg/ml	1Vials (0.6ml)	0.5Vials (0.3ml)	Seereagentspreparationonpage3
Blankcontrol	1Vials (1.0ml)	1Vials  (0.5ml)	Ready-to-use
StandardDiluent	1Vials (4.0ml)	1Vials  (2.0ml)	Ready-to-use
Biotinylatedanti-MTLRReceptor	1Vials (6.0ml)	1Vials  (3.0ml)	Ready-to-use
Streptavidin-HRP	1Vials (8.0ml)	1Vials  (4.0ml)	Ready-to-use
WashingBuffer	1Vials (20ml)	1Vials  (10ml)	50×concentrate
Substrate A	1Vials (6.0ml)	1Vials  (3.0ml)	Ready-to-use
Substrate B	1Vials（6.0ml)	1Vials  (3.0ml)	Ready-to-use
StoppingSolution	1Vials (6.0ml)	1Vials  (3.0ml)	Ready-to-use
SampleDiluent	1Vials (12ml)	1Vials  (6.0ml)	Ready-to-use

 
 
MATERIALREQUIREDBUTNOTPROVIDED

• Distilledwater
• Pipettes:10ul、50ul、100ul、200ul、1000ul。
• Vortexmixerandmagneticstirrer.

 
SAFETY

• Forresearchuseonly
• AvoidanyskincontactwithH2SO4andTMB.Incaseofcontact,washthoroughlywater.
• Donoteat,drink,smokeorapplycosmeticswherekitreagentsareused.
• Donotpipettebymouth.

 
PROCEDURALNOTES/LAB.QUALITYCONTROL

• Whennotinuse,kitcomponentsshouldbestoredrefrigeratedorfrozenasindicatedonvialsorbottles.Allreagentsshouldbewarmedtoroomtemperaturebeforeuse.Lyophilizedstandardsshouldbediscardedafteruse.
• Oncethedesirednumberofstripshasbeenremoved,immediatelyresealthebagtoprotecttheremainingstripsfromedterioration.
• Coverorcapallreagentswhennotinuse.
• Donotmisorinterchangereagentsbetweendifferentlots.
• Donotusereagentsbeyondtheexpirationdateofthekit.
• Useacleandisposableplasticpipettetipforeachreagent,standard,orspecimenadditioninordertoavoidcross-contamination,forthedispensingofH2SO4andsubstratesolution,avoidpipetteswithmetalparts.
• Useacleanplasticcontainertopreparethewashingsolution.
• Thoroughlymixthereagentsandsamplesbeforeusebyagitationorswirling.
• Allresidualwashingliquidmustbedrainedfromthewellsbyefficientaspirationorbydecantationfollowedbytappingtheplateforcefullyonabsorbentpaper.Neverinsertabsorbentpaperdirectlyintothewells.
• TheTMBsolutionislightsensitive.Avoidprolongedexposuretolight,also,avoidcontactoftheTMBsolutionwithmetaltopreventcolourdevelopment.WarningTMBistoxicavoiddirectcontactwithhands.Disposeoffproperly.Ifadarkbluecolourdevelopswithinafewminutesafterpreparation,thisindicatesthattheTMBsolutionhasbeencontaminatedandmustbediscarede.Readabsorbanceswithin1houraftercompletionoftheassay.
• Whenpipettingreagents,maintainaconsistentorderofadditionfromwell-to-well.Thiswillensureequalincubationtimesforallwells.
• Respectincubationtimesdescribedintheassayprocedure.

 
SPECIMENCOLLECTIONPROCESSINGANDSTORAGE

• Serum---Avoidanyinintentionalstimulationofthecellsbytheprocedure.Usepyrogenendotoxinfreecollectingtubes.Serumshouldberemovedrapidlyandcarefullyfromtheredcellsafterclothing.Forthat,afterclothing,centrifugeatapproximately1000×gfor10minandremoveserum.
• Plasma---EDTAcitrateandheparinplasmacanbeassayed.Spinsamplesat1000×gfor30minremoveparticulates.Harvestplasma.
• Cellculturesupernatants---Removeparticulatesandaggregatesbyspinningatapproximately1000×gfor10min.
• Storage---Ifnotanalyzedshortlyaftercollection,samplesshouldbealiquoted(250-500ul)toavoidfreeze-thawcyclesandstoredfrozenat-70℃.Avoidmultiplefreeze-thawcyclesoffrozenspecimens.WhenpossIBLe,avoiduseofbadlyhemolyzedorlipemicsera.Iflargeamountsofparticlesarepresent,thisshouldberemovedpriortoassaybycentrifugationorfiltration.
• Recommendation---Donotthawbyheatingat37℃or56℃.Thawatroomtemperatureandmakesurethatsampleiscompletelythawedandhomogenousbeforeassaying.

 
PREPARATIONOFREAGENTS

• Standards:Standardhavetobereconstituledwiththevolumeofstandardbufferdiluentindicatedonthevial.Thisreconstitutionproducesastocksolutionof400pg/mlMTLRReceptor.Allowstandardtostandfor5
• minuteswithgentleswirlingpriortomakingdilutions.Serialdilutionsofstandardmustbemadebeforeeachassysandcannotbestored.

 

400pg/ml	（6 Standard）	Originaldensity50ul。
200pg/ml	（5 Standard）	100ul 6Standard +100uldiludent
100pg/ml	（4 Standard）	100ul 5Standard +100uldiludent
50pg/ml	（3 Standard）	100ul 4Standard +100uldiludent
25pg/ml	（2 Standard）	100ul 3Standard +100uldiludent
12.5pg/ml	（1 Standard）	100ul 2Standard +100uldiludent
0pg/ml	BlankControl	50ul。

 

• Washingbuffer50×concentrate: Dilute50timesindistilledwater.

 
ASSAYMETHOD

• Beforeuse,mixallreagentsthoroughlywithoutmakingfoam.
• Determinethenumberofmicrowellstripsrequiredtotestthedesirednumberofsamples,plusappropriatenumberofwellsneededforrunningblanksstandards.Eachsample,standardandblankshouldbeassayedinduplicate.Removesufficientmicrowellstripsfromthepouch.
• Add50ulofstandarddiluenttostandardwellsB1,B2, C1,C2, D1,D2, E1,E2, F1,F2.Reconstitutestandardvialwiththeappropriatevolumeasdescribedinthechapterreagentspreparation.Preparation.Pipet100ulofstandardintowellsA1andA2(seeplateschemebelow).Transfer50ulfromA1andA2toB1andB2wells.Mixthecontentsbyrepeatedaspirationsandejections.Takecarenottoscratchtheinnersurfaceofmicrowells.RepatthisprocedurefromthewellsB1,B2towellsC1,C2andfromwellsC1,C2toD1,D2andsooncreatingtwoparallelrowsofMTLRReceptorstandarddilutionsranging，Add50ulofstandarddiluenttotheblandwells.
• Dilutesamples1:1distribing50ulofsampleinto50ulofdilluent,Add50ulofdilutedsampletowells..
• Add50ulofdilutedbiotinylatedanti-MTLRReceptortoallwells.
• Coverwithaplatevoverandincubatefor1hourat37℃.
• Removethecoverandwashtheplateasfollows:⑴　aspiratetheliquidfromeachwell,⑵　dispensse0.3mlofwashingsolutionintoeachwell.⑶　Aspirateagainthecontetofeachwellafter0.5minute.⑷　Repeatsteps⑵and⑶threetimes.
• Distribute60ulofstreptavidin-HRPsolutiontoallwells,includingblankwells.
• Coverandincubate30minat37℃.
• Removethecoverandemptywells,Washmicrowellstripsaccordingtostep,Proceedimmediatelytothenextstep.
• Add50ulSubstrateAandSubstrateBtoeachwell。Incubatefor10minat37℃。
• Theenzyme-substratereactionisstoppedbyquicklypipetting50ulofH2SO4.stopreagentintoeachwell,includingtheblankwells,tocompletelyanduniformlyinactivatetheenzyme.ResultsmustberedimmediatelyaftertheadditionofH2SO4.
• Readabsorbanceofeachwellonaspectrophotometerusing450nmastheprimarywavelengthandoptionally620nm(610nmto650nmisacceptable)asthereferencewavelength.

 
 
SUGGESTEDPLATESCHEME

Standard concentrations（pg/ml）

A

400

400

sample

sample

sample

sample

sample

sample

sample

sample

sample

sample

B

200

200

sample

sample

sample

sample

sample

sample

sample

sample

sample

sample

C

100

100

sample

sample

sample

sample

sample

sample

sample

sample

sample

sample

D

50

50

sample

sample

sample

sample

sample

sample

sample

sample

sample

sample

E

25

25

sample

sample

sample

sample

sample

sample

sample

sample

sample

sample

F

12.5

12.5

sample

sample

sample

sample

sample

sample

sample

sample

sample

sample

G

0

0

sample

sample

sample

sample

sample

sample

sample

sample

sample

sample

H

sample

sample

sample

sample

sample

sample

sample

sample

sample

sample

sample

sample

 
LIMITATIONSOFTHEPROCEDURE
Donotextrapolatethestandardcurvebeyondthemax standardcurvepoint.Thedose-responseisnon-linearinthisregionandgoodaccruacyisdifficulttoobtain.
 
CALCULATIONOFRESULTS
Theminimumdetectableconcentrationinthisassayisestimatedtobe1.0pg/ml