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ELISA Kits are fully validated and ready-to-use, containing 96-well strip plates pre-coated with capture antibody to detect sample antigen.

Sandwich Kit Principle

In a competitive ELISA assay, sample antigen and labeled antigen compete for capture antibody binding. The more target protein there is in the sample, the less labeled antigen will be captured and the weaker the signal.

Competitive Kit Principle

 

Sandwich ELISA Kit Assay Procedure

Prepare all reagents, standard solutions and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature.

Prepare duplicate standard points by serially diluting the standard stock solution.

Determine the number of strips required for the assay. Insert the strips in the frames for use. The unused strips should be stored at 2-8°C.

Add 50μlstandard to standard well

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Add 40μl sample to sample wells and then add 10μl anti-TNF alpha antibody to sample wells, then add 50μl streptavidin-HRP to sample wells and standard wells (Not blank control well ). Mix well. Cover the plate with a sealer. Incubate 60 minutes at 37°C.

 

Remove the sealer and wash the plate 5 times with wash buffer. Soak wells with at least 0.35 ml wash buffer for 30 seconds to 1 minute for each wash. For automated washing, aspirate all wells and wash 5 times with wash buffer, overfilling wells with wash Blot the plate onto paper towels or other absorbent material.

 

Add 50μl substratesolution A to each well and then add 50μl substrate solution B to each well. Incubate plate covered with a new sealer for 10 minutes at 37°C in the dark.

 

Add 50μl Stop Solution to each well, the blue color will change into yellow immediately.

 

Determine the optical density (OD value) of each well immediately using a microplate reader set to 450 nmwithin 30 min after adding the stop solution.