SANDWICH ELISA- BRIEF DESCRIPTION AND PROTOCOL

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Annuja Anandaradje

A BRIEF DESCRIPTION AND OUTLINE ON SANDWICH ELISA REF: R&D

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SANDWICHELISA- MIP1α
AIM: To performsandwichELISA forMIP-1α
PRINCIPLE:
MATERIALS ANDREAGENTS
PROTOCOL
PLATE PREPARATION
 Dilute the capture antibodytothe givenworkingconcentrationinPBSwithoutcarrier
protein.
 Immediatelycoatthe 96 well microplate with100ul /well of dilutedcapture antibody.
 Seal the plate andincubate O.N.at roomtemperature.
 Aspirate eachwell andwashwithwashbufferandrepeatthe processthrice
 Blockthe platesbyadding~300ul of reagentdiluenttoeachwell.
 Incubate at roomtemperature for1 hour
 Aspirate withwashbufferthrice
ASSAYPROCEDURE
 Add100ul of standardsand samplesinassaydiluent
 Coverwithan adhesive plate sealerfor2 hours
 Aspirate thrice withwashbuffer
 Add100ul of detectionantibodythatisdilutedinreagentdiluent
 Incubate for2 hoursin room temperature
 Aspirate thrice withwashbuffer
 Add100ul of working dilutionof streptavidinHRPtoeach well.
 Coverthe plate and incubate for20 minutesatroomtemperature
 NOTE: Performexperimentindark
 Aspirate the wellsthrice withwashbuffer
 Add100ul of substrate to eachwell
 Incubate 20 minindark at room temperature
 Soonaftercolour developmentaddstopsolutiontoeachwell.
 Determine the ODusingmicroplate readeratat 450nm.

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