ELISA stands for Enzyme Linked Immunosorbent Assay. The immunoassay utilizes the specific lock-and-key recognition between antibodies and antigens. This recognition occurs naturally in the adaptive immune system; antibodies are created by the immune system when an antigen such as a virus or bacteria invades the body. The immune system recognizes the foreign invader and creates antibodies that specifically recognize surface proteins on the virus or bacteria. The antibodies are either attached to the surface of an immune cell or move freely through the body to tag the invader and begin a cascade of destruction and elimination. The most useful part of this process from a biotechnology and engineering perspective is the specificity of the antibody-antigen recognition.
The ELISA is performed in a 96-well polystyrene plate in one of two formats: either antigen is attached to the bottom of each well, or the capture antibody is attached to the bottom of each well. In the case where the goal of the assay is to detect the presence of an antibody in a clinical sample, the antigen is attached to the bottom of each well. If the goal is to detect the antigen in a clinical sample, then the capture antibody is attached to the bottom of each well (also known as a sandwich ELISA). The ELISA is such a common and standardized assay that it is possible to purchase pre-coated plates that are pre-blocked and ready to receive the clinical or experimental sample immediately upon opening the package.
The ELISA process doesn’t end after the introduction and incubation with the target sample. The next step is to add a labeled detection antibody in order to detect whether or not binding occurred between the capture and target antibody and antigen. The detection antibody is labeled with a fluorescent, chemiluminiscent, or chromogenic molecule that can be quantified by an ELISA reader.
An ELISA reader is an instrument that is used to read the fluorescent, chemiluminiscent, or chromogenic response of the ELISA in a 96-well plate. The instrument is designed to fit standard 96-well plates, and will provide quantitative information about the extent of the response for each individual well of the plate. This enables up to 96 different conditions to be examined at once. The ELISA reader is both a fluorimeter (able to input light at an excitation wavelength for the flurorophore being used as the label, and to record the intensity of the emitted light at the emission wavelength), and a spectrophotometer (able to record the intensity of light emitted from chemiluminescent reactions and chromogenic reactions across a range of wavelengths. Most ELISA readers also come with software that converts the raw intensity values into quantitative curves including dilution information and error. The software is equipped with calibration curves to ensure that the highest standard of quality and reproducibility is met every time the instrument is used.