Fluorescent Immunoassay

Dr. Arvind Kumar

Introduction

  • Immunoassays are analytical techniques that use the specific binding between an antigen and an antibody to detect and quantify molecules of biological or clinical importance.
  • Since the 1960s, when radioimmunoassay (RIA) was first developed, immunoassay technology has advanced tremendously, introducing alternatives that avoid the drawbacks of radioactive methods.
  • Among these, the fluorescent immunoassay (FIA) has emerged as one of the most powerful tools, combining immunological specificity with the high sensitivity of fluorescence detection.

 

  • FIA uses fluorophores as labels instead of radioisotopes or enzymes.
  • These fluorophores absorb light at a certain wavelength and emit light at a longer wavelength, allowing sensitive measurement of antigen–antibody interactions.
  • Due to its unique properties, FIA has become indispensable in clinical diagnostics, biomedical research, pharmacology, food safety, and environmental monitoring.

Definition

A fluorescent immunoassay (FIA) is a laboratory method used to detect and quantify specific biological molecules (antigens or antibodies) by employing fluorophore-labeled immunoreagents.

  • When a fluorophore-labeled antibody binds to its antigen, or vice versa, the complex can be excited with light of a particular wavelength.

  • The emitted fluorescence is detected, and its intensity is proportional to the concentration of the target molecule.

Thus, FIA is defined as:

“An immunoassay that utilizes fluorescent labels to detect and measure antigen–antibody binding reactions with high sensitivity and specificity.”

Principle

The principle of FIA rests on three key foundations: immunological specificity, fluorescence, and quantitative signal measurement.

1 Immunological Specificity

  • Antigens are molecules capable of stimulating an immune response.

  • Antibodies are Y-shaped proteins that bind to antigens with high specificity at their antigen-binding sites.

  • This lock-and-key mechanism ensures that FIA provides highly selective results.

2 Fluorescence

  • Fluorescence is the ability of a molecule (fluorophore) to absorb photons at an excitation wavelength and emit photons at a longer emission wavelength.

  • Example: Fluorescein absorbs blue light (~490 nm) and emits green light (~520 nm).

  • By tagging antibodies or antigens with fluorophores, binding events can be visualized or quantified.

3 Quantitative Detection

  • The intensity of emitted fluorescence is proportional to the concentration of antigen–antibody complexes.

  • Specialised instruments such as fluorescence microscopes, plate readers, flow cytometers, and spectrofluorometers are used for measurement.

 

Components 

  1. Antigen – The target analyte (e.g., protein, hormone, pathogen, toxin).

  2. Antibody – Specific immunoglobulin designed to bind antigen.

  3. Fluorophore – The fluorescent label attached to antibody or antigen. Common fluorophores include:

    • Fluorescein isothiocyanate (FITC)

    • Rhodamine

    • Alexa Fluor dyes

    • Cyanine dyes (Cy3, Cy5)

    • Quantum dots (nanoparticle fluorophores)

  4. Detection System – Device to measure fluorescence (e.g., fluorometer, microscope, microplate reader).

  5. Solid Support (optional) – Nitrocellulose, polystyrene beads, or microplates to immobilize immunoreagents.

 

Types 

FIA can be classified based on assay design and detection strategy:

1 Direct Fluorescent Immunoassay (DFA)

  • Antibody specific to the antigen is directly labeled with a fluorophore.

  • When antigen is present, the fluorescent antibody binds and emits signal.

  • Advantages: Fast, fewer steps.

  • Limitations: Lower sensitivity because no signal amplification.

  • Example: DFA test for Rabies virus in brain tissue.

2 Indirect Fluorescent Immunoassay (IFA)

  • Antigen is first bound by an unlabeled primary antibody.

  • A fluorophore-labeled secondary antibody binds the primary antibody.

  • Advantages: Increased sensitivity (multiple secondary antibodies can bind each primary).

  • Example: Detection of antinuclear antibodies (ANA) in autoimmune disease diagnosis.

3 Competitive Fluorescent Immunoassay

  • Labeled antigen and unlabeled antigen (sample) compete for antibody binding sites.

  • Higher concentration of unlabeled antigen → lower fluorescence intensity.

  • Useful for small molecules like drugs, hormones, and toxins.

  • Example: Measurement of cortisol levels in serum.

4 Sandwich Fluorescent Immunoassay

  • Antigen is captured by an immobilized antibody.

  • A second, fluorophore-labeled antibody binds a different epitope.

  • Advantages: Very high sensitivity and specificity.

  • Example: Detection of viral proteins, cytokines, and biomarkers.

5 Fluorescence Polarization Immunoassay (FPIA)

  • Based on differences in rotational motion of molecules.

  • Free fluorescent antigen rotates rapidly → depolarized emission.

  • Antibody-bound antigen rotates slowly → polarized emission.

  • Application: Therapeutic drug monitoring (e.g., theophylline, cyclosporine).

6 Time-Resolved Fluorescent Immunoassay (TR-FIA)

  • Uses special fluorophores (lanthanides like Europium, Terbium) with long-lived fluorescence.

  • Allows measurement after background autofluorescence has decayed.

  • Advantages: Very high sensitivity, reduced noise.

  • Applications: Detection of very low-abundance biomarkers in clinical samples.

7 Multiplex Fluorescent Immunoassay

  • Uses beads, arrays, or microchips coated with different antibodies, each tagged with distinct fluorophores.

  • Detects multiple analytes simultaneously in a single sample.

  • Example: Luminex xMAP technology – simultaneous detection of 50+ cytokines.

8 Flow Cytometry-Based FIA

  • Fluorescently labeled antibodies bind to cell-surface or intracellular markers.

  • Laser excitation detects fluorescence from individual cells.

  • Application: Immunophenotyping of blood cells, cancer diagnostics, HIV monitoring (CD4 counts).

 

Applications

FIA has wide-ranging applications across many fields:

1 Clinical Diagnostics

  • Infectious Diseases:

    • DFA for Rabies virus, Chlamydia trachomatis, Respiratory syncytial virus.

  • Endocrinology:

    • Measurement of insulin, thyroid hormones, cortisol.

  • Oncology:

    • Tumor markers (PSA, CA-125, CEA).

  • Autoimmune Disorders:

    • Indirect immunofluorescence for ANA in lupus and other autoimmune diseases.

  • Hematology:

    • Blood group antigen typing.

2 Biomedical Research

  • Localization of proteins in cells using immunofluorescence microscopy.

  • Tracking cellular signaling pathways.

  • Analysis of protein–protein interactions.

  • High-throughput screening of drug candidates.

3 Pharmaceutical Industry

  • Therapeutic drug monitoring (FPIA assays for antibiotics, immunosuppressants).

  • Pharmacokinetics and pharmacodynamics studies.

  • Biomarker discovery and validation in clinical trials.

4 Food and Agriculture

  • Detection of foodborne pathogens (Salmonella, E. coli O157:H7).

  • Identification of allergens (gluten, peanut proteins).

  • Monitoring of pesticide residues.

5 Environmental Monitoring

  • Detection of toxins in water (cyanobacterial microcystins).

  • Monitoring microbial contamination in soil and water.

  • Identification of endocrine-disrupting chemicals.

6 Point-of-Care Testing

  • Portable FIA devices for bedside testing.

  • Examples:

    • COVID-19 antigen fluorescent assays.

    • Troponin tests for myocardial infarction.

    • Rapid flu and dengue antigen detection kits.

 

Advantages 

  1. High Sensitivity – Detects femtomolar–picomolar concentrations.

  2. High Specificity – Due to antigen–antibody recognition.

  3. Multiplexing Capability – Multiple analytes can be measured simultaneously.

  4. Wide Dynamic Range – Works over broad concentration ranges.

  5. Non-radioactive – Safer than radioimmunoassays.

  6. Versatility – Can be adapted to microscopy, flow cytometry, microarrays, or plate assays.

  7. Quantitative and Qualitative – Provides both presence/absence and precise concentration.

 

Limitations 

  1. Autofluorescence – Some biological samples (blood, tissues) emit natural fluorescence that interferes with signal.

  2. Photobleaching – Fluorophores lose fluorescence when exposed to light for prolonged periods.

  3. Quenching – Fluorescence can be reduced by chemical interactions or environmental conditions.

  4. Cost – High-quality fluorophores and instruments are expensive.

  5. Technical Expertise – Requires trained personnel for accurate execution.

  6. Cross-Reactivity – Non-specific binding can give false positives.

  7. Hook Effect – Extremely high antigen concentration may give falsely low results.

  8. Stability Issues – Some fluorophores degrade under temperature or pH changes.

 

Recent Advances in FIA

  • Quantum Dots (QDs): Nanoparticles with exceptional brightness and photostability.

  • Near-Infrared (NIR) Fluorophores: Reduce background noise and allow deeper tissue penetration.

  • Microfluidic FIA Systems: Lab-on-a-chip devices for rapid, portable analysis.

  • Smartphone-Based FIA: Mobile phone cameras used for fluorescence detection in low-resource settings.

  • AI and Image Analysis: Automated interpretation of immunofluorescence microscopy.

  • Dual-Label and Ratiometric FIAs: Improve accuracy by reducing background fluctuations.