The Bright Side of Diagnostics: Understanding Chemiluminescence

1.  Introduction

Chemiluminescence immunoassay (CLIA) is a highly sensitive and specific in-vitro diagnostic (IVD) technique that detects and quantifies analytes in biological samples through light emission from chemical reactions [1]. This method measures emitted light to determine the concentration of target substances. CLIA includes direct assays with luminescent labels attached to antibodies, enzyme-linked assays that use enzyme-catalyzed light production, and electrochemical assays combining electrochemical detection with chemiluminescence. These approaches make CLIA versatile and valuable for rapid, accurate clinical diagnostics across various diseases.

2.  Comparison of Common Chemiluminescence Immunoassay Types

2.1   Direct Chemiluminescence Immunoassay

In direct chemiluminescence, the target antigen or antibody is directly labeled with luminophore markers such as acridinium and ruthenium esters. Upon oxidation in an alkaline solution, the labeled molecule emits light, producing a signal proportional to the amount of antigen-antibody complex formed [6]. This approach simplifies the assay by eliminating the need for secondary antibodies, reducing potential cross-reactivity, and streamlining the workflow with a single labeling step. While direct chemiluminescence achieves reliable sensitivity, it generally provides lower signal amplification compared to indirect methods, making it less suitable for detecting low-abundance targets.

2.2   Enzyme-Linked Chemiluminescence Immunoassay,

Enzymatic chemiluminescence immunoassay (CLEIA) employs enzyme-labeled antibodies to detect specific antigens in biological samples through emitted light, enabling precise quantification. There are 2 common types of substrates utilized in CLEIA horseradish peroxidase (e.g. luminol, acridan-based reagents) and alkaline phosphatase (e.g. AMPPD, APS-5). As a newly developed alkaline phosphatases substrate for CLEIA, APS-5 distinguishes itself with several notable advantages. It enables rapid signal development, reaching peak luminescence within two minutes, and demonstrates exceptional stability, ensuring reliable performance during prolonged experiments or extended storage at 2–8°C. Furthermore, APS-5 maintains robust functionality across a wide temperature range (25–35°C) and offers a broad dynamic range, making it well-suited for both qualitative and quantitative analyses [1]. Its low background noise further enhances analytical precision and reliability, contributing to its widespread application in CLEIA.

2.3  Electrochemical Luminescence Immunoassay

Electrogenerated chemiluminescence (ECL) is a luminescence phenomenon produced during electrochemical reactions, where light is emitted by excited luminophore species generated at the electrode surface through electron-transfer reactions. This process begins with an electrochemical reaction that creates reactive intermediates, which then undergo exergonic chemical reactions to form an electronically excited state of the luminophore. Upon returning to its ground state, the luminophore emits a photon [4]. A widely used ECL luminophore is [Ru(bpy)₃]²⁺, which has been instrumental in various applications since its introduction in the 1970s. ECL is noted for its high sensitivity and selectivity, making it valuable for analytical and clinical applications.

3.  Advantages of Enzymatic Chemiluminescence Immunoassay vs. Traditional Methods

CLEIA combine superior sensitivity, rapid signal acquisition, and an extended dynamic range, outperforming traditional immunoassays like ELISA. With detection capabilities down to zeptomole, picogram, or femtogram levels, CLEIA is particularly effective for identifying low-abundance biomarkers critical in early disease diagnosis[2]. Automated platforms allow for simultaneous multi-analyte detection and quantitative results in 30–60 minutes, making CLEIA highly suitable for high-throughput and time-sensitive applications. Additionally, CLEIA reduces reagent use and incubation times, supports standardization through full automation, minimizes human error, and provides enhanced specificity, reducing false positives. Its ability to process smaller sample volumes and accommodate diverse sample types further highlights its versatility and efficiency in clinical diagnostics. As results of employing APS-5 as substrates by Wondfo’s Accre and SmarLumi CLIA families, Wondfo’s reagents are recognized as a dependable choice for clinical diagnostics and research applications, supporting a comprehensive spectrum of analytical parameters.

4.  Conclusion

In conclusion, CLIA provide exceptional sensitivity and specificity, enabling rapid and accurate quantification of analytes through light emission. Direct CLIA streamlines workflows by eliminating the need for secondary antibodies, though it may offer less signal amplification for low-abundance targets. Enzyme-linked CLIA improves signal amplification, while electrochemical luminescence CLIA offers superior sensitivity and selectivity. Compared to traditional methods, CLIA provides a broader dynamic range, faster results, and reduced reagent consumption, positioning it as a critical tool in disease diagnosis and management.

Reference:

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