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.


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.

Extensive ELISA Assay Development by NorthEast BioLab

NorthEast BioLab offers various client-centric ELISA assay development services.

Bioanalytical Assay Development and Validation

Proper ELISA assay development and validation are critical for the accurate quantification of an analyte. NorthEast BioLab helps you select the right ELISA assay format and develop a robust protocol for analysis. Our specialists optimize the assay by titrating various components to ensure accurate results.

Pharmacokinetics (PK) Studies

Our pharmacokinetic studies for quantitation of drug-responsive levels of a specific serum protein using ELISA assay development allow accurate and reliable pharmacokinetic data to help regulate dosage regimens.

Toxicokinetic (TK) Studies

As in pharmacokinetic studies, an ELISA assay can be used in toxicokinetic studies to quantitate a drug or compound when administered at high dosage. TK studies allow estimation of the level of toxicity that can be produced by these drug compounds.

Immunogenicity Testing Assay

An ELISA assay can be used for immunogenicity testing. These studies are carried out to assess how drug exposure induces an immune response in the body of humans or other animals. It further evaluates how anti-drug antibodies (ADA) can affect immunogenicity.

Cell-Based Assay

Our scientists help clients with the successful execution of cell-based assays to assess the toxicity of the compounds and ensure reliable drug manufacturing. For example, we perform cytotoxicity testing and mechanism of action (MOA) assay to understand the biochemical reactions triggered by drug compounds.

Biomarker Assay List

Biomarker research, discovery, and qualification are pivotal in modern drug development from demonstrating proof-of-concept (POC) to establishing drug safety and efficacy in the clinic…

Frictionless ELISA Assay Method from scratch or validated Research Use Only (RUO) kits

At NorthEast BioLab, we ensure robust ELISA assays to quantify large molecule analytes such as biologics, proteins, and antibodies in your samples. As often as it takes, our veteran scientists with 30+ years experience discuss and study your ELISA assay development and validation in depth. Our team continues without yielding until we have customized these assays per your requirement and satisfaction.

We encourage intensive collaboration between the client and our scientists for complete transparency throughout your study with us. NorthEast BioLab offers quick turnaround times, scalability, and flexibility, given our extensive experience with ELISA assay development and a thorough understanding of the science behind it. We enable our clients to expedite drug approval through efficient assay development, validation, and optimization while maintaining 100% regulatory compliance.

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