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Today, multiple immunoassay platforms exist, providing scientists and clinicians with the means to measure a variety of biomarkers^1,2,3. However, research indicates some immunoassay platforms have limitations that can inhibit progress in the laboratory^4,5,6. In an effort to provide researchers with a tool to overcome some of these limitations, ALPCO developed its STELLUX^® chemiluminescence immunoassay platform. Be sure to download our Guide to STELLUX^® Chemiluminescence ELISAs infographic to learn more about how chemiluminescence ELISAs work. Download Infographic STELLUX^® chemiluminescence ELISAs offer robust features such as higher analytical sensitivity, broader dynamic range, simplified sample preparation and assay protocols, enabling scientists to advance research and physicians to improve clinical outcomes.

What is a Chemiluminescence ELISA?

A chemiluminescence ELISA is a type of immunoassay that measures the amount of light generated from a chemical reaction to quantify the concentration of an analyte in a sample^6,7. [caption id="attachment_5298" align="alignnone" width="778"] Figure 1: Defining a chemiluminescence ELISA[/caption]

What Happens in a Plate Well During a Chemiluminescence Reaction?

In a microtiter plate well, the chemical reaction between the enzyme label and luminescent substrate generates excited state intermediates which decay back to ground state, emitting light^6,7. The light emitted from the chemiluminescent reaction is detected by a plate reader capable of reading luminescence (luminometer) and recorded as relative light units (RLU). The intensity of light in the well is directly proportional to the concentration of the analyte in the sample^6,7,8. [caption id="attachment_5299" align="alignnone" width="565"] Figure 2: A chemiluminescent reaction to a microtiter plate well[/caption]

Chemiluminescence Plate Readers

There are many commercially available chemiluminescence plate readers. Single mode chemiluminescence plate readers read only the luminescence given off by a chemiluminescent ELISA. However, multimode plate readers are capable of reading multiple ELISA outputs such as absorbance from colorimetric ELISAs and luminescence. The table below provides a list of chemiluminescent plate reader manufacturers. [caption id="attachment_5300" align="alignnone" width="423"] Table 1: Chemiluminescence plate reader manufacturers[/caption]

Features of the STELLUX^® Chemiluminescence Immunoassay Platform

ALPCO developed the STELLUX^® chemiluminescence immunoassay platform to provide better tools for researchers and clinicians. STELLUX^® chemiluminescence ELISAs are designed to meet the analytical sensitivity and specificity benefits of RIAs without the use of radioactive materials. Furthermore, the STELLUX^® platform was designed to maintain the simplicity of colorimetric ELISAs, thus providing easy-to-use and robust chemiluminescence immunoassays for laboratories. ALPCO’s STELLUX^® ELISAs offer many robust features for users compared to RIAs and colorimetric assays including:

• Broader dynamic range
• Higher sensitivity and/or specificity
• Simplified sample preparation

Broader Dynamic Range

STELLUX^® assays exhibit a broad dynamic range, which eliminates the need to dilute samples to fit within a standard curve, as well as the need to purchase multiple assays with differing analytical measurement ranges. The broad dynamic range of STELLUX^® assays is achieved using chemiluminescence. When read using a luminometer, the photo multiplier tube (PMT) within the plate reader allows for multiple logs of measurement, affording the ability to quantitate biomarkers to levels limited by the antibody pairs as opposed to limitations by the measurement capacity of traditional spectrophotometers⁸. [caption id="attachment_5301" align="alignnone" width="635"] Figure 3: The Broad Dynamic Range of the STELLUX^® Chemi Rodent Insulin ELISA[/caption]

Higher Sensitivity and/or Specificity

The STELLUX^® chemiluminescence immunoassay platform can provide superior sensitivity compared to colorimetric ELISAs. The high level of sensitivity is achieved through the use of luminometers, which overcome challenges faced when using typical spectrophotometers. As a result, STELLUX^® assays afford the opportunity for users to confidently measure normal versus disease state levels of biomarkers in the samples, while also minimizing the number of non-detects that they may have observed using standard colorimetric immunoassays. Additionally, STELLUX^® chemiluminescence ELISAs afford researchers the ability to specifically measure the desired analytes at physiologically relevant levels without concern for cross-reactivity with similar proteins.

Simplified Sample Preparation

STELLUX^® assays are designed with simplified sample preparation, allowing for a better user experience in the lab. The broad dynamic range of STELLUX^® assays eliminates any upfront dilution steps required by many colorimetric ELISAs. In turn, this saves users sample volume, time and money, as they would normally have to repeat tests that reveal samples above the measurable assay range.

The STELLUX^® Chemiluminescence ELISA Suite

STELLUX^® Chemiluminescence ELISAs enable laboratories to accurately measure key metabolic biomarkers such as C-peptide, insulin, proinsulin, and glucagon-like peptide-1. STELLUX^® ELISAs allow for the measurement of these biomarkers in biological fluids and islet supernatants from human, mouse, and rat samples. STELLUX^® assays are designed to support various model systems including:

• Fasted vs. fed
• Perifusion
• Glucose-stimulated insulin secretion
• Diet-induced obesity

Setting Up a STELLUX^® Chemiluminescence ELISA

Setting up a STELLUX^® chemiluminescence ELISA is similar to that of a colorimetric ELISA. First, select and order the right STELLUX^® assay to fit your research needs. Before running the ELISA, review the components provided in the kit box and gather the materials necessary to run the kit which are not provided with the kit. Be sure to set up a plate map before beginning the assay in order to keep track of what goes into each well on the microtiter plate. Prepare samples and reagents according to the instructions for use (IFU) provided with the kit. It is recommended to run all standards, controls, and samples in duplicate. [caption id="attachment_5306" align="alignnone" width="500"] Figure 4: STELLUX^® Chemi Human Insulin ELISA Components[/caption]

Running a STELLUX^® Chemiluminescence ELISA

All of ALPCO’s STELLUX^® chemiluminescence ELISAs are sandwich ELISAs. When samples are added to wells of a STELLUX^® microtiter plate, the analyte in the sample binds to the antibody coated plate during a specified incubation period. After a wash step is performed, detector antibody is added to create the antibody-analyte sandwich in the wells. Next, an enzyme label is added to the wells which binds to the detector antibody. The final step requires the addition of luminol, a chemiluminescent substrate, into the wells. Luminol reacts with the enzyme label triggering a reaction that produces light. The plate is then inserted into a luminometer which reads the intensity of the chemiluminescent reaction (luminosity) as relative light units (RLU). Next, using a software program capable of 4-PL and 5-PL curve fitting, a standard curve is drawn and the concentration of the analyte in each well can be determined. The assay procedure for each STELLUX^® ELISA may vary slightly, therefore it is important to always read and follow the instructions for use (IFU) provided with the kit.

A Guide to STELLUX^® Chemiluminscence ELISAs

Researchers and clinicians now have many different types of immunoassay platforms to choose from to measure biomarkers^1,2,3. However, research shows some platforms have limitations that create challenges and hinder progress in the laboratory^4,5,6. ALPCO’s STELLUX^® chemiluminescence immunoassay platform provides a broad dynamic range, superior sensitivity and specificity, and simplified sample preparation procedures. Laboratories can use STELLUX^® chemiluminescence ELISAs to confidently measure key biomarkers, such as insulin and GLP-1, in a wide range of model systems. You can download our Guide to STELLUX^® Chemiluminescence ELISAs infographic to further explore chemiluminescence ELISAs and discover how easy it is to run a STELLUX^® assay in your lab.

References

1. LeQuin (2005). Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA). Clinical Chemistry. 51:12 2415-2418. DOI: 10.1373/clinchem.2005.051532.
2. Darwish (2006). Immunoassay Methods and their Applications in Pharmaceutical Analysis: Basic Methodology and Recent Advances. Int J Biomed Sci. 2006 Sep; 2(3): 217–235. PMID: 23674985.
3. Gibbs (2012). Selecting the Detection System - Colorimetric, Fluorescent, Luminescent Methods. ELISA Technical Bulletin - No. 5. Corning incorporated Life Sciences.
4. Jandreski (1998). Chemiluminescence Technology in Immunoassays. Laboratory Medicine, Volume 29, Issue 9, 1 September 1998, Pages 555–560. DOI: https://doi.org/10.1093/labmed/29.9.555
5. Zhao, et al (2009). Chemiluminescence Immunoassay. TrAC Trends in Analytical Chemistry 28(4):404-415. DOI: 10.1016/j.trac.2008.12.006.
6. Azim, et al (2015). Chemiluminescence Immunoassays: Basic Mechanism and Applications. Bangladesh Journal of Nuclear Medicine, [S.l.], v. 18, n. 2, p. 171-178, Jan. 2018. ISSN 1562-3831. doi:http://dx.doi.org/10.3329/bjnm.v18i2.35240.
7. Baeyens, et al (1998). Chemiluminescence-based detection: principles and analytical applications in flowing streams and in immunoassays. J Pharm Biomed Anal. 1998 Sep 1;17(6-7):941-53. PMID: 9884186.
8. Wild (2001). The Immunoassay Handbook Second Edition. Nature Publishing Group. ISBN 1-56159-270-6.