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Discover Creative Proteomics’ ECL assay service for high-sensitivity, multiplex electrochemiluminescence immunoassays in biomarker research.

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Electrochemiluminescence (ECL) Assay Service

What is Electrochemiluminescence (ECL)?

Electrochemiluminescence (ECL) is a luminescence phenomenon in which light is generated through an electrochemical reaction. In ECL-based immunoassays, chemical labels attached to detection antibodies are activated by a controlled electrical stimulus, causing them to emit light. Unlike traditional fluorescence or colorimetric detection, the excitation (electricity) is separate from the signal (light), which minimizes background noise and enables highly sensitive detection of biomolecules. ECL combines high sensitivity, broad dynamic range, and compatibility with multiplexed assays while requiring minimal sample volumes.

How Does ECL Work?

ECL is the detection principle used in plate-based ECL immunoassays to deliver high sensitivity and low background. Non-specific background signals are largely eliminated because only labels close to the electrode surface emit light upon electrical stimulation. The result is a clean, highly sensitive readout suitable for quantifying proteins at very low concentrations. The process proceeds as follows:

Schematic illustration of EC-sensing systems classified.

Figure 1. Schematic illustration of EC-sensing systems classified according to the reactions that drive the ECL signal emission (Yoo S M, et al., 2022).

Advantages of ECL Assays

Differences between ECL, ELISA and Olink

Feature / Parameter Plate-based ECL platform ELISA Olink Proteomics
Detection Principle Electrochemiluminescence; ECL labels on detection antibodies produce light upon electrode stimulation Colorimetric (HRP/TMB), fluorescent, or luminescent enzymatic reactions Proximity Extension Assay (PEA); dual oligonucleotide-labelled antibodies generate qPCR signal upon target binding
Multiplexing Capacity Multiple analytes per well (typical commercial implementations: up to ~8–10 spots per well) Single-analyte per well; multi-analyte requires multiple plates Extremely high; 92–3072 proteins per panel
Sensitivity / LLOD High; ultra-sensitive formats can reach low fg/mL range Moderate; pg/mL range Very high; low fg/mL range depending on panel
Sample Volume Required Low; 10–25 μL per well for multiplex assays Moderate; 50–100 μL per analyte Very low; 1–5 μL per sample per panel
Throughput High; 96-well plate, rapid read times, automated options Low to moderate; multiple wash and incubation steps slow workflow Moderate to high; 96- or 384-well plate, mostly automated
Assay Flexibility High; validated kits, custom multiplex, or user-developed assays Limited; minor customization only Moderate; panel selection fixed, limited customizability
Best Use Cases Biomarker panels, PK/PD studies, immunogenicity testing, cell/gene therapy studies Single-protein quantification, low-throughput studies, validation of exploratory hits Large-scale biomarker discovery, high-throughput profiling, cohort studies
Advantages Multiplexing with low sample volumes, broad dynamic range, ultra-sensitivity, robust across matrices Simplicity, established method, widely understood Extremely high multiplexing, minimal sample consumption, consistent data across large panels
Limitations Higher instrument cost, limited maximum multiplex (up to 10 per well), requires specialized plates Single-analyte throughput, higher sample consumption, limited sensitivity Panel dependence and proprietary workflows

Workflow of Our ECL Assay Service

Our ECL assay service follows a streamlined workflow to deliver reproducible, publication-ready results:

Applications of ECL in Biomedical Research

Simple Requirements for ECL Assay

Sample Type Serum, plasma, cell culture supernatants, whole blood, CSF, urine, tissue extracts
Sample Volume Typically 10–50 µL per analyte (multiplexing reduces total volume needed)
Sample Quality Fresh or properly stored (e.g., -80°C for plasma/serum), free of hemolysis or debris
Multiplexing Capability Up to 10 analytes per well for standard panels; customizable for more analytes with special designs
Assay Format Sandwich, direct, indirect, or bridging assay depending on project requirements

Why Choose Creative Proteomics for ECL Assay

FAQ

Q1: Can ECL multiplex assays reliably measure multiple analytes in one sample without cross‐reactivity or bias?

A1: Yes. Validated multiplex ECL assays (e.g., 8-plex for ETEC vaccine antigens, or SARS-CoV-2 antigen panels) have shown strong performance. These studies demonstrate good correlation with each analyte's ELISA, acceptable spike/recovery metrics, and reliable quantitation across sample types. Proper panel design and validation are essential to minimize cross‐reactivity.

Q2: How do you control for matrix effects and interference?

A2: We evaluate matrix effects via spike/recovery, dilution linearity, and parallelism testing across representative matrices; where required, we optimize diluents, blocking reagents, or alternate sample preparation to mitigate interference. Acceptance criteria are established during method qualification.

Q3: How is cross-reactivity evaluated in multiplex panels?

A3: Cross-reactivity is assessed by single-plex vs multiplex comparison, spiking individual analytes into pooled matrices, and testing potential interfering substances. Acceptable cross-reactivity thresholds and mitigation strategies are defined during panel design.

Demo

Demo: Sensitive electrochemiluminescence (ECL) immunoassays for detecting lipoarabinomannan (LAM) and ESAT-6 in urine and serum from tuberculosis patients

Results of LAM and ESAT-6 concentrations.

Figure 2. LAM and ESAT-6 concentrations in urine and serum samples (Broger T, et al., 2019).

Case Study

Case: An electrochemiluminescence based assay for quantitative detection of endogenous and exogenously applied MeCP2 protein variants.

Abstract:

Methyl-CpG-binding protein 2 (MeCP2) is a chromosomal protein crucial for gene regulation in the central nervous system. Both deficiency and overexpression of MeCP2 cause severe neurological disorders, including Rett syndrome (RTT) and MECP2 duplication syndrome. Current RTT treatments are limited, and protein replacement therapy using TAT-MeCP2 fusion proteins has emerged as a potential therapeutic approach. Accurate, sensitive quantification of MeCP2 is essential for developing such therapies. To express and purify recombinant TAT-MeCP2 fusion proteins and develop a sensitive, high-throughput electrochemiluminescence immunoassay (ECLIA) capable of measuring endogenous and exogenously applied MeCP2 protein levels in cells and brain tissue, supporting therapeutic and uptake studies.

Methods

  • TAT-MeCP2 and TAT-MeCP2-eGFP fusion proteins were expressed in E. coli and purified using biotin-binding affinity purification resin.
  • Human fibroblasts (healthy and MeCP2-deficient), HEK-293, NSC-34 cells, and mouse brain tissues were prepared for protein extraction.
  • A 96-well ECLIA was established using specific capture and detection antibodies, with electrochemiluminescence for quantitative readout.
  • In vitro BBB permeation studies were performed using cerebEND endothelial cells to evaluate TAT-MeCP2 transport.

Results

  • Milligram quantities of stable, purified TAT-MeCP2 and TAT-MeCP2-eGFP were obtained.
  • The ECLIA showed high sensitivity (LLOD 1.002 ng/mL), broad dynamic range (1–1800 ng/mL), low intra- and inter-assay variability, and superior performance compared to Western blot and commercial ELISA kits.
  • Endogenous MeCP2 levels were accurately measured in human and mouse samples, including detecting differences between wild-type and heterozygous mice.
  • TAT-MeCP2 uptake studies demonstrated time- and concentration-dependent nuclear accumulation in MeCP2-deficient fibroblasts.
  • In vitro BBB studies confirmed the ability of TAT-MeCP2 and TAT-MeCP2-eGFP to cross endothelial cell layers.
Determination of Mecp2 protein levels via ECLIA assay.

Figure 3. Determination of Mecp2 protein levels in mouse brains and human fibroblasts via MeCP2 ECLIA assay.

Transport of TAT-MeCP2 and TAT-MeCP2-eGFP across a BBB model.

Figure 4. Transport of TAT-MeCP2 and TAT-MeCP2-eGFP across a blood-brain barrier in vitro model.

Conclusion

The study successfully produced recombinant TAT-MeCP2 fusion proteins and established a quantitative, sensitive ECLIA for measuring MeCP2 levels. This platform enables precise dosage assessment for protein replacement therapy and facilitates studies on MeCP2 dynamics, including BBB transport, providing a valuable tool for RTT therapeutic development.

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References

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