Introduction
Enzyme-linked immunosorbent assay (ELISA) analysis is a plate-based bioanalytical technique used to detect and quantify biomolecules such as proteins, peptides, antibodies, and hormones. ELISA assays have multiple advantages. These include its high-throughput, rapid, versatility, sensitivity, accuracy, quantitative, and multiplexing capabilities.
ELISA Assay Method
In the ELISA Lab, the target antigen is immobilized on a microplate. It then undergoes complexation with an antibody linked to a reporter enzyme. In the ELISA assay method, detection involves measuring the activity of the reporter enzyme.
The key step of ELISA analysis involves the highly specific antigen-antibody interaction. ELISA Assays can be of different types. These include direct, indirect, sandwich, and competitive ELISA assays. Sandwich and competitive are the most frequently used ELISA assay methods. Different types of ELISA assays have advantages and limitations. For example, while sandwich ELISA is more sensitive, competitive ELISA is faster and easier to perform. The type of ELISA selected largely depends on the availability of antibodies and the complexity of the sample.
Despite the different variants, the basic steps of ELISA remain the same. These include coating or capture, which involves the immobilization of antigens on the microplate wells. Thereafter, the plate is blocked. In this step, the unblocked binding sites in the microplate get blocked using other irrelevant molecules. The next step is probing or detection. In this step, the antigen-specific antibodies are allowed to bind to the antigens/proteins adhered to the surface of the blocked microplate. The final step is the signal measurement, where the signal generated by the reporter molecule is detected. Different detection methods may be employed based on the type of reporter molecule used for ELISA analysis. Detection methods include colorimetry, chemiluminescence, and fluorescence, and the appropriate protocol needs to be adapted accordingly.
ELISA Guide
Having gained an understanding of the different ELISA assay methods available and the working principle, this section will provide a detailed insight into the ELISA protocol. The first step involves coating the microwell plate. It is important to note that binding proteins to polystyrene plates depends on the type of protein. Hence, one should optimize the protein binding step before the experiment. The binding step can be done at 4-37 ⁰C, based on the stability of the target protein. Using three buffers of different pH values is highly recommended. The protein to be immobilized is first diluted in the coating buffer, followed by adding the protein solution to the microplate wells and overnight incubation. Next, the plate is washed four times with PBS buffer (pH 7.2). Each time, the plate is soaked for about a minute in PBS, followed by tapping the wells to dry. The wells are then blocked using a blocking buffer for around 2 hours at room temperature. The microwell plate is washed with PBS buffer to remove unbound molecules. The wash step is similar to that described above. The microwell plate is now coated with the target protein.
Must Read: HPLC Lab Basics: What You Should Know
A dilute solution of the anti-target protein antibody is added to the microwells, and the plate is covered and incubated for 1-2 hours at room temperature. The incubation may be performed overnight at 4 ⁰C to obtain higher sensitivity if the target protein concentration is low or the epitope is hidden partially. Post incubation, the plate is washed about 4 times with PBS-Tween buffer, carefully following the wash step described before. Gradually, a dilute solution of the secondary antibody in the buffer is added to the microwells. The plate is covered and incubated at room temperature for about 45 minutes. The plate is then washed with PBS-Tween buffer, followed by adding the substrate solution to the microwell plate. Commonly used substrates for protein detection include p-nitrophenyl phosphate (pNPP), 2,2′-azino-bis[3-ethylbenz-thiazoline-6-sulfonic acid] (ABTS), o-phenylenediamine (OPD), and 3,3′,5,5′-Tetramethylbenzidine (TMB).
Notably, the substrate should be prepared immediately before its use. Post-substrate addition, the color development process should be monitored for 45 minutes. Finally, the stopping reagent is added at a specific time, and the product is measured.
Tips for Successful ELISA Lab
ELISA kits such as PK ELISA assay kits are commercially available and contain the manufacturer’s instructions for conducting the test. However, remembering a few points can be helpful while performing an ELISA analysis. Detection antibodies should be allowed to bind at least for one hour at room temperature. If the concentration of the target protein is very low, increasing incubation time to a couple of hours or even overnight can help enhance sensitivity. Performing the ELISA steps on a shaker can help improve the test outcome. Antibodies should be diluted based on their types. Hence, it is better to follow the manufacturer’s suggestion or use an initial concentration similar to that used in Western blot analysis. Including suitable negative controls (without any protein, such as dilution buffer, blank reagent, etc.) is recommended. These negative controls should be treated similarly to the samples. Finally, when developing a new ELISA assay method, the different parameters of the assay (such as incubation time and protein concentration) should be optimized, along with the validation of the procedure.
