ELISA Assay Theory

Neogen’s direct competitive ELISAs operate on the basis of competition between the horseradish peroxidase (HRP) enzyme conjugate and the analyte in the sample for a limited number of specific binding sites on the precoated microplate.

ELISA Assay Procedure

Samples, standards or calibrators are first added to the precoated antibody microplate. Next, the enzyme conjugate is added and the mixture is incubated at room temperature. During incubation, competition for binding sites on the microplate is taking place. The plate is then washed removing all unbound material. The bound enzyme conjugate is detected by the addition of a TMB based substrate.

ELISA test results may be obtained by measuring and comparing the absorbance reading of the wells of the samples against the standards with a microplate reader at 650 nm or 450 nm if acid stop is used. The extent of color development is inversely proportional to the amount of analyte in the sample or standard. For example, the absence of the analyte in the sample will result in a dark blue color, whereas the presence of the analyte will result in a light blue color or no color as the concentration of the analyte increases. If acid stop is used to halt the assay then the dark blue color will change to a dark yellow color and the light blue color to no color will change to light yellow to no color.

ELISA Assay Test Principle Procedure

Plates are precoated with the antibody. The plate is ready for use.
DO NOT WASH

A sample or control is added to each well. Next the drug-enzyme conjugate is added and the mixture is incubated at room temperature.

Wash the plate to remove all unbound materials

The bound materials now remain in the microplate

Add TMB substrate to each well and allow the color to develop.

Qualitative results are obtained by measuring the absorbance reading at 650 nm or 450 nm if acid stop is used.