Introduction to ELISA Technology

The evolution of immunoassays from radioimmunoassays (RIAs) to enzyme linked immunosorbent assays (ELISAs) has revolutionized our understanding of the dynamics of the chemical communicating signals that maintain homeostasis. Insights gained, have had a direct impact on our understanding of metabolic diseases.

As with any technology that becomes an integral part of day-to-day laboratory activities, end-users often times loose sight of the critical factors that are necessary to insure that the analysis, particularly an indirect measure such as the immunoassay, is accurate. Herein lies the challenge for scientists: how do I know that what I am measuring is what I think I am measuring? And a corollary to this question: how do I know that I am measuring essentially all the analyte in a particular sample?

To answer these questions one must first review the priniciple of the immunoassay, the enzyme immunoassay.

There are two current strategies for immunoassays. The earliest method, the competitive immunoassay (EIA), relies upon the competition of the analyte with a labeled analyte for antibody binding. The second method is the sandwich immunoassay (ELISA), which is based on the trapping or capture of the analyte by one antibody and the detection by another antibody. The most commonly applied sandwich assay method is supposed to be superior because the two recognition sites on the analyte necessary for detection suggest greater specificity. Second, the sandwich assay system would appear to be more sensitive. Third, the assays are easy to use.

To meet this need, PromoKine offers highly sensitive and specific sandwich enzyme immunoassay kits (ELISA) for cell biology analysis i.e. for the measurement of cytokines, chemokines, growth factors, soluble receptors and CD markers as well as other target molecules that are playing important roles in other cellular processes and cell biology disciplines (e.g. apoptosis, cell metabolism, oncology, inflammation, immunity, infections and diseases).  Besides the complete, ready-to-use ELISA kits, economical ELISA Development Kits are available as well.

Principle of PromoKine's ELISAs

PromoKine's ELISAs are enzyme linked immunosorbent assays which measure the "free" forms of cytokines in cell culture supernatants, plasma, human serum and other body fluids. The kits are convenient coming with a user-friendly, comprehensible protocol. They include all necessary reagents to perform a highly sensitive, reliable and reproducible ELISA.

Typically, a microtiter plate pre-coated with a highly specific (mono- or polyclonal) antibody generated against the target antigen is used to capture the respective cytokine in the sample. Subsequently, a second antigen-specific (mono- or polyclonal) antibody labeled with biotin binds to the target cytokine in the sample, too. With the addition of a strepavidin-HRP-conjugate, followed by the addition of a color reagent solution containing the substrat for the horseradish peroxidase enzymatic reaction, the amount of cytokine is detected. The standard curve demonstrates a direct relationship between Optical Density (OD) and cytokine concentration: i.e. the higher the OD the higher the cytokine concentration in the sample.

PromoKine ELISA is designed to measure the amount of "free" cytokine in tissue cell culture supernatants and "free" but not "bound" cytokine in biological fluids (serum and plasma) and serum-free samples. There are enough reagents included in the kits for one 96-well immunoassay plate. We recommend running duplicate wells for samples and standards. Standards are included in the kit. 

ELISA Trouble Shooting Guide

The successful performance of immunoassays relies on a number of factors. Many of the more common errors may be avoided if the protocol is read and fully understood before sitting down to run the assay. For all kits and kit reagents, the proper storage temperature is 4°C; do NOT freeze them. Please store all kit components as recommended to ensure optimal test  performance. Spin down vials briefly before opening to to make sure to collect all the contents. Always use clean, disposable plastic pipette tips and containers for reagent preparation and storage. Avoid cross contamination of kit reagents by changing pipette tips with each addition of the standards, samples and reagents. Be sure the incubation times and temperatures prescribed in the assay procedure have been followed and no substitute kit reagents have been used. A standard curve should be run at each session, and standards and samples should be run in duplicate to improve the accuracy of the assay. Washing of the plates before usage is not necessary but recommended as precision may be lower in case of omission.


Note: ELISA technology is also topic of two training courses of our PromoCell Academy! >More Information