Chemiluminescence Immunoassay: Innovative Approach Unveiled for Analyte Detection A New Robust Technique

Chemiluminescence Immunoassay

Chemiluminescence immunoassay (CLIA) is a technique used for detecting and quantifying analytes such as antibiotics, hormones, vitamins, and proteins. It combines the specificity of immunoassay with the sensitivity and versatility of chemiluminescence detection. In a CLIA, the analyte of interest is labeled with an enzyme that generates a luminescent product during the enzymatic reaction. This light signal is then detected by a photomultiplier tube or a charge-coupled device (CCD) camera, allowing accurate quantification of the analyte present in the sample.

Principle of Chemiluminescence Immunoassay

The basic principle of CLIA involves an enzymatic reaction that produces light emission from a substrate. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) are two commonly used labeling enzymes. In an HRP-based CLIA, the analyte is tagged with HRP either directly or through an antigen-antibody complex. When HRP-catalyzed hydrogen peroxide oxidizes Chemiluminescence Immunoassay  or other luminophore, blue-green light is emitted. This light is directly proportional to the amount of analyte present in the sample. Similarly, in an ALP-based CLIA, the enzyme catalyzes the dephosphorylation of appropriate substrates like adamantyl 1,2-dioxetane phosphate (AMPPD) to produce a light flash. The light produced is measured via a photomultiplier tube, allowing highly sensitive detection of analytes.

Immunoassay Procedure

CLIA can be performed in direct, indirect, or competitive format depending on the analyte to be detected. A direct CLIA involves direct conjugation of the analyte to an enzyme label. An indirect CLIA uses a primary antibody that binds the analyte and a secondary antibody tagged with an enzyme like HRP or ALP. In a competitive CLIA, the analyte competes with an enzyme-labeled analyte for binding to a limited number of antibody sites. The steps involved in a typical CLIA are:

1. Immobilization of capture antibody onto a solid surface like microtiter plate wells.

2. Blocking of non-specific binding sites.

3. Addition of calibrators/controls/samples and an enzyme-labeled antibody or analyte.

4. Incubation to allow formation of antigen-antibody complexes.

5. Washing to remove unbound complexes.

6. Addition of substrate for the labeling enzyme.

7. Measurement of light signal produced in a luminometer or chemiluminometer.

8. Quantification of analyte concentration based on a standard curve.

Applications and Advantages of CLIA

CLIA has emerged as an important technique for clinical diagnostics, pharmaceutical analysis, food safety testing, and environmental monitoring due to several advantages:

- High sensitivity: CLIA is more sensitive than enzymatic colorimetric methods, allowing detection of analytes in femtomolar to picomolar range. This makes it suitable for low-abundance analytes.

- Wide dynamic range: A dynamic range of 5-6 orders of magnitude allows accurate quantification of analytes over a broad concentration range.

- Rapid results: Due to no separation step, CLIA gives results within 1-2 hours, facilitating high-throughput testing.

- Versatility: CLIA can be adapted to both microtiter plate and automated analyzer formats for detecting various analytes. Labels like HRP, ALP, and acridinium ester allow flexibility.

- Robust technology: Luminometer-based detection provides resistance to contaminants, making CLIA suitable for complex sample matrices like serum, plasma, food, and environmental samples.

- Low interference: Luminescence readout reduces background noise, giving cleaner signals compared to colorimetric detection. Interference from sample pigments is negligible.

CLIA finds use in quantitative detection of hormones (hCG, TSH), tumor markers (PSA), cardiac markers (myoglobin, troponin), drugs of abuse, therapeutic drugs, vitamins, and infectious disease biomarkers like HIV and hepatitis antigens in clinical analysis. CLIAs are also applied to residue analysis in food industry and environmental testing. Automated CLIA systems capable of processing 100-200 samples per hour are commercially available from multiple vendors.

Standardization and Validation

To obtain accurate and reliable results from CLIA, strict quality assurance measures must be followed during assay development, validation, and routine use. Parameters like specificity, precision, accuracy, sensitivity, reportable range, and reference interval need to be established as per appropriate guidelines like CLSI. Cross-reactivity studies check interference from related analytes. Precision is determined by repeat testing of samples, and accuracy by recovery studies using spiked samples or method comparison. Proper storage of reagents, calibrators, controls and calibrations are necessary for consistent long-term performance. Periodic equipment calibration and participation in external quality assessment programs help standardize CLIA testing across laboratories. With rigorous validation and control, CLIA emerges as a robust immunoassay technology suited for a variety of clinical, industrial and research applications.

Chemiluminescence immunoassay offers high sensitivity, wide dynamic range and versatility for detecting different analytes. Using enzymatic chemiluminescence as the detection principle, CLIA adds advantages of luminescence measurements like low background, resistance to contaminants and interference-free results. Its microtiter plate as well as automated analyzer formats facilitate quantitative measurement of clinically important markers, therapeutic drugs, food residues, hormones and environmental toxins. With standardization and validation practices followed, CLIA establishes itself as a powerful technique in research, clinical diagnostics and industrial analysis.

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Money Singh is a seasoned content writer with over four years of experience in the market research sector. Her expertise spans various industries, including food and beverages, biotechnology, chemical and materials, defense and aerospace, consumer goods, etc. (https://www.linkedin.com/in/money-singh-590844163)_