An ELISA (Enzyme-Linked Immunosorbent Assay) is a multi-well plate-based immunoassay within which one of the assay components, typically an antibody or sample, is adsorbed onto a solid surface, in this case, a plate. Offering rapid, quantitative, and sensitive analyte detection at relatively low cost, ELISAs represent one of the simplest assay formats to perform. Furthermore, the ease of adapting an ELISA to a higher throughput screening method empowers researchers to test large sample numbers in a single run.
Our ELISAs are a convenient and user-friendly method for studying soluble protein biomarkers in a variety of matrices including serum, plasma, cell culture supernatant, or lysate. Our ELISA offering consists of >1,000 Research Use Only assays, against a wide variety of species including human, mouse, rat, and various other animal models (Agricultural and Companion). Each assay comes with a specific protocol and the required reagents to run a 96-well plate. ELISAs primarily exist in a 96-well format with the vendor providing the reagents necessary to detect and quantitate the target. There are a variety of ELISA methods including Sandwich, Direct, and Competitive.
The sandwich assay uses a pair of monoclonal antibodies (mAbs) against different epitopes against the same target. The primary mAb, bound to the plate, pulls the protein out of the solution while the second mAb is used to complete the “sandwich” and provide the signal which will indicate the presence of the target. A known amount of the recombinant version of the protein is provided with the kit, allowing the user to create a standard curve against which the signal from one of the samples will be interpreted. The majority of our portfolio is in this sandwich ELISA format.
The direct ELISA and competitive assays are rarer. The direct assay uses a single mAb to detect the sample which is bound to the plate. The mAb is then bound by a reporter secondary antibody to provide the signal.
The competitive assay has a known amount of the biomarker pre-bound to the plate. A labeled antibody is co-incubated with the sample which “mops up” the antibody depending on the concentration of the target in the sample. The free antibody is then able to bind to the antigen on the plate and provide a signal after the sample is washed away. In this case, the signal is inversely proportional to the concentration of the target biomarker.
The Conferma® ELISA brand consists of sandwich assays designed to minimize lot-to-lot variability at the critical reagent level, a key concern of assay users. Evaluating the individual lots of critical reagents with a range of analytical techniques aligned with a detailed quality control (QC) protocol allows us to control and limit variability over time.
In order to allow scientists to evaluate the reproducibility of their method we further designed the Belysa® Immunoassay Curve Fitting Software. With this tool, a user can examine their sandwich immunoassay to ensure parameters such as %CV (coefficient of variation) and %Recovery were as expected. Then they can move on to compare the standard curves of multiple plates to confirm that their method was consistent.
This article offers 4 popular ELISA protocols: Sandwich ELISA protocol, Phosphorylation Assay Procedure, EIA Assay Procedure, & Cell-based Assay Procedure.
Troubleshoot and optimize ELISAs using this guide that includes solutions to some of the most common sources of problems for assay development.
There are several ELISA configurations, with the most common formats being sandwich, competitive and signaling assays.
Learn about the Conferma® ELISA development and manufacturing methods that provide strong sample detection and long-term assay and lot consistency, giving you confidence in your research.
Explore how to effortlessly monitor immunoassay method reproducibility using Belysa® analysis software.
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