ELISA Assay: Accurate and Flexible Analysis in 96 well plates
Enzyme Linked Immunosorbent Assay (ELISA) assay is a widely utilized immunoassay for quantitating and detecting proteins, hormones, peptides, cytokines, antibodies, and other drugs along with their metabolites. ELISA assays are effective in quantitatively detecting any molecule, or antigen, that can be ascertained by an antibody. For example, ELISA assays are used in pregnancy testing, infectious disease identification, and detection of cytokines, and soluble receptor proteins, etc. Due to the precision, sensitivity, assay speed, and ease of quantitation, ELISA assay development is a common choice for several diagnostic and research applications.
While there are various formats of ELISA assay, the most common is a sandwich ELISA assay. In this format, the analyte whose concentration is to be measured is sandwiched in-between two antibodies that bind to a different region (epitopes) on the antigen. These antibodies are referred to as detection antibody and capture antibody. In this assay, the capture antibody is coated to a microtiter plate in a 96-well format. The antigen binds to the capture antibody, and a detection antibody is used to measure the analyte. The antibody is conjugated to an enzyme, typically horseradish peroxidase (HRP), and detected via catalysis of a substrate which yields a colored product. Spectrophotometry is used to monitor the colored product, and a standard curve is utilized for calculating the antigen concentration in the sample. When the antigen is small, or two antibodies that can simultaneously bind to the antigen are unavailable, then another variation of ELISA, termed competitive ELISA assay is utilized using a single epitope.
It is notable that the ELISA assay typically uses 96 microwell plates for parallel analysis of many samples, standards, and controls in a single experiment. The surface of these plates is treated with special absorbents such that the antigen or antibody can adhere properly. ELISA assay offers increased sensitivity and specificity compared to other standard antibody-based assays. In ELISA assay, the interactions with analyte or antibody occur through serial binding that is done to some solid surface such as polystyrene in the 96 microwell plates discussed above.
Step-by-step ELISA Assay Method Development
Enzyme Linked Immunosorbent Assay or ELISA Assay development is a complex task involving multiple sequential steps for the measurement of analyte concentration in a sample.
Plate Coating
Whenever an ELISA assay is developed for any analyte, a surface attachment strategy is established first. Once the analyte is affixed to the surface of the plate, it is immobilized and helps with the sequential addition of other reagents and washing cycles in the next steps, without mixing. ELISA Assay is thus a solid-phase assay.
The process of immobilizing an antibody or antigen to the plate is essential, as the method of immobilization utilized is what determines the efficacy of the ELISA assay. Many times, the immobilization may cause a conformational change in the analyte or antibody, preventing the detection antibody from binding. Thus, it is critical to immobilize the antibody or antigen while preserving its conformational structure.
Antibody and Antigen Pairs
The specificity and sensitivity of the assay are determined using a range of concentrations of antibody and/or antigen. The affinity of the detection antibody and sensitivity of the detection reagents corresponding to a specific antigen are principally responsible for defining the performance of an optimized ELISA assay. Selection of the best capture and detection antibody is critical for effective assay development.
Detection
The antibodies which are labeled with an enzyme (detection antibodies) contribute to the signal output of this assay. Different types of enzymes can be utilized, such as alkaline phosphatase, horseradish peroxidase, etc., each of which produces a color upon reacting with their substrates. These colored products are detected with spectrophotometry, where the signal intensity is proportional to the amount of antigen in the microwell. Afterward, a standard curve along with positive and negative controls is used to quantify the amount of analyte within a given sample.
