What is ELISA Assay Development?

Understanding ELISA AssayEnzyme-linked immunosorbent assay or ELISA assay is a strong tool for quantifying and detecting the specific proteins in a given sample matrix.

In the ELISA assay development, an antigen is captured on the solid surface, which is then complexed to an enzyme-linked antibody. Detection is achieved with the help of complexed enzyme’s activity by incubating it with a substrate.

However, ELISA assay development requires selection of the right type of ELISA method, construction of a working protocol, and then strategically optimizing the ELISA assay for accurate and robust results.

Types of ELISA AssayThe first step is to decide the type of ELISA assay to be used: direct, sandwiched, or indirect.

1. The direct method uses a direct primary antibody for detection.
2. The indirect method uses a primary antibody and an indirect secondary antibody for detection.
3. The sandwiched method sandwiches antigen between two direct (primary) antibody. Then, detection is achieved using a secondary antibody.

All of these ELISA assay types offer different sensitivity, specificity, speed, and simplicity. The components and features are also varied. Take sandwiched ELISA assay, for instance. It requires antibody-matched pair. When the assay is being developed from scratch, it is possible that the antibodies may not be accessible. Hence, in this case, antibody production is necessary.

ELISA OptimizationAfter ELISA assay development, scientists optimize the antibody concentration. This is achieved in the following manner.

• Use different coating buffer concentrations to prepare capture antibody in varied concentrations.
• Place equal volume corresponding to every antibody concentration on the plate and start the ELISA procedure or protocol.
• Observe low background and strong signal.

How Does It Work? The human body produces antibodies when it encounters a foreign element or infection. The antibody ELISA assay identifies the same by allowing scientists to know whether the body has come in contact with a particular virus. When we are developing an antibody ELISA assay, the antigen is captured on the plastic surface and then the sample is poured on the plate. The antibodies related to the particular disease conjugate with the antigen. The secondary antibody helps in detection by changing the color when the substrate is added. If the disease antibodies are not present in the given sample, the second antibody won’t show any results. It will not conjugate or complex to the surface. Here are the steps of the process:

• The test sample or serum is complexed to an antigen which is captured on a 96-well place.
• The secondary antibodies are added to the sample.
• The solution is washed and the substrate is added to observe the color change marker.

ConclusionELISA assays are imperative for drug discovery and development. The assay, when correctly developed and executed help scientists in evaluating the robustness, repeatability, tolerance, and specificity of the drug compound. Usually, sandwiched ELISA assay is considered as the best method for drug discovery and development due to efficiency and sandwiched antigen. But, different methods may be utilized for different drug testing procedures. Hence, consider all the factors discussed above before selecting any type of ELISA assay.

Maria Brown is a medical expert who blog on patient safety and drug efficacy. She’s working for a top medical equipment company. Maria Brown has granted a large online following for herself as an expert medical thought leader.