Sandwich kits are fully validated and ready-to-use, containing 96- well strip plates pre-coated with capture antibody to detect sample antigen.
The plate is coated with a capture antibody, then antigen-containing sample is applied to the plate and any antigen present binds to capture antibody; During a washing step the plate is washed to remove unbound antigen; then detecting antibody is added and binds to antigen; Then enzyme-linked secondary antibody is added and binds to detecting antibody; substrate is added and is converted by enzyme to detectable form. The absorbance of the plate wells is measured to determine the presence and quantity of antigen. The color develops in proportion to the amount of the quantity of antigen.
Competitive Kit In a competitive ELISA assay, the sample antigen competes with a reference antigen for binding to a specific amount of labeled antibody. The plates are pre-coated with detecting antibody. The sample and biotinylated antibody are added to the wells. During a washing step the plate is washed to remove unbound antigen. substrate is added and is converted by enzyme to detectable form. The absorbance of the plate wells is measured to determine the presence and quantity of antigen. The color develops inversely in proportion to the amount of the quantity of antigen. This means the more antigen there is in the sample, the less reference antigen will be detected and the weaker the signal.
Some competitive ELISA kits use labeled antigen instead of a labeled antibody. The labeled antigen and the sample antigen (unlabeled) compete for binding to the primary antibody. The lower the amount of antigen in the sample, the stronger the signal due to more labeled antigen in the well. It is suitable for detecting small antigens.
Qualitative Kit Qualitative results provide a simple positive or negative result for a sample. In quantitative ELISA, the optical density (OD) of the sample is compared to a standard curve, which is typically a serial dilution of a known-concentration solution of the target molecule.
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
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.