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FAQ: ELISA Kits

What is a Sandwich ELISA?
An antigen can be detected or measured by a sandwich ELISA. In this technique, the monoclonal or polyclonal antibody (rather than the antigen) is immobilized on a microtiter well. A sample containing the antigen is added and allowed to react with the immobilized antibody. After the well is washed, a second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen. After any free secondary antibody is removed by washing, substrate is added, and the colored reaction product is measured. Sandwich ELISAs are used to measure soluble molecules/antigens that are present in biological fluids (such as plasma, serum, urine, etc).

What is a Competitive ELISA?
The amounts of antigen‘s (Ag) can also be detected by Competitive ELISA. In this technique, an antibody (Ab) is first incubated in solution with a sample containing the antigen. The antigen-antibody (Ag-Ab) mixture is then added to an antigen-coated microtiter well. The more antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. Addition of an enzyme-conjugated secondary antibody specific for the isotype of the primary antibody or addition of an enzyme-conjugated streptavidin, can be used to determine the amount of the primary antibody bound to the well. In the competitive assay, however, the higher the concentration of an antigen in the original sample, the lower is the absorbance.

What are intracellular ELISA Kits from AdipoGen?
Intracellular ELISA Kits are Sandwich ELISA’s. However, while normal Sandwich ELISAs are used to measure soluble molecules/antigens that are present in biological fluids (such as plasma, serum, urine, etc), intracellular ELISAs detect molecules present intracellularly and therefore need an additional step of cell lysis.

Can I extend the standard curve (in either direction)?
AdipoGen cannot support kit results outside of the stated range. AdipoGen has chosen a specific range because of confidence in the reproducibility of the assay within that range.

Can AdipoGen ELISA kits be used with all type of samples?
All positively tested sample types are indicated on the product datasheet and in the manual. Please contact our Technical Support for advice, before using kits in non-stated and non-tested sample types.

Why is there a sample dilution necessary in this ELISA Kit?
There are several reasons for dilutions. In some assays most samples read above the standard curve, thus requiring a dilution to be read within the range of the assay. Another common reason, is to limit interference or matrix effects in a sample.

What are TwinPlex and PentaPlex kits?
TwinPlex and PentaPlex are convenient and low priced multiple kit packages from the same production lot. The TwinPlex Kit provides enough reagents and plates to perform 2 x 96 wells and PentaPlex Kit provides enough reagents and plates to peform 5 x 96 wells.