MEVE 018: Unit 10 - Immunoassays

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UNIT 10: IMMUNOASSAYS

10.0 Introduction

Immunoassays are analytical methods based on the specific binding between an antigen and its corresponding antibody. These assays are widely used in medical diagnostics, biotechnology, pharmaceutical research, and increasingly in environmental monitoring. The ability of immunoassays to detect trace levels of analytes with high specificity makes them invaluable for detecting pollutants, toxins, and biological contaminants.

10.1 Objectives

After studying this unit, the learner will be able to:

  • Understand the principles of immunoassay techniques.
  • Differentiate between various types of immunoassays.
  • Describe how immunoassays are applied in environmental analysis.
  • Comprehend advanced labeling and detection methods such as DELFIA and SFLIA.

10.2 Radio Immuno-Assays (RIA)

Radioimmunoassay (RIA) is one of the earliest immunoassay techniques. It involves labeling an antigen or antibody with a radioactive isotope (commonly iodine-125).

Principle:

  • A known amount of radio-labeled antigen competes with an unlabeled sample antigen for binding to a specific antibody.
  • The amount of radioactivity bound to the antibody is inversely proportional to the concentration of the analyte in the sample.

Applications:

  • Detection of hormones, drugs, and environmental toxins.
  • Sensitive analysis of trace contaminants such as dioxins and polychlorinated biphenyls (PCBs).

Advantages: High sensitivity
Disadvantages: Use of radioactive materials poses safety and disposal concerns.

10.3 Enzymatic-Immunoassay (EIA)

Also known as Enzyme-Linked Immunosorbent Assay (ELISA), this method uses enzymes as labels instead of radioisotopes.

Principle:

  • An enzyme-linked antigen or antibody reacts with a substrate to produce a measurable color change.
  • Color intensity is proportional to the amount of analyte in the sample.

Types:

  • Direct ELISA
  • Indirect ELISA
  • Sandwich ELISA
  • Competitive ELISA

Applications in Environment:

  • Monitoring pesticide residues.
  • Detection of microbial pathogens in water.

Advantages:

  • Safer than RIA, no radioactivity.
  • Easily automated and scalable.

10.4 Immuno-Fluorescence Analysis (IFA)

Immunofluorescence uses fluorescent dyes (fluorochromes) conjugated to antibodies to detect target antigens.

Principle:

  • The antibody-fluorophore conjugate binds to the antigen, and the fluorescence is measured using a fluorescence microscope or reader.

Types:

  • Direct IFA (labeled primary antibody)
  • Indirect IFA (labeled secondary antibody)

Applications:

  • Detecting microbial contamination in soil and water.
  • Identification of toxins and allergens.

Advantages:

  • Visual and quantitative analysis.
  • High sensitivity and specificity.

10.5 Stable Isotope Labeling

In this method, non-radioactive isotopes (e.g., 13C, 15N) are used to label antibodies or antigens.

Principle:

  • Isotopically labeled compounds are tracked using mass spectrometry or nuclear magnetic resonance (NMR).

Applications:

  • Tracking of labeled contaminants through ecosystems.
  • Understanding environmental fate and transport of pollutants.

Advantages:

  • Safe compared to radioisotopes.
  • Excellent for long-term studies.

10.6 Neutron Activation Analysis (NAA)

Although not a classic immunoassay, NAA can be used for elemental analysis in biological systems.

Principle:

  • Samples are bombarded with neutrons, causing elements to form radioactive isotopes.
  • The emitted gamma rays are measured to identify and quantify the elements.

Applications:

  • Tracing elements in soil and water samples.
  • Detecting heavy metal accumulation in organisms.

Advantages:

  • Extremely precise.
  • Non-destructive to samples.

10.7 Substrate-Labeled Fluorescence Immunoassay (SFLIA)

SFLIA is an advanced method where the substrate, upon enzyme interaction, produces fluorescence.

Principle:

  • The antigen-antibody reaction leads to enzyme activity, which cleaves a fluorogenic substrate.
  • The resulting fluorescence is measured quantitatively.

Applications:

  • Detection of pesticide residues in crops and water.
  • Monitoring foodborne pathogens.

Advantages:

  • Very sensitive and specific.
  • Can detect multiple analytes simultaneously.

10.8 Delayed Enhanced Lanthanide Fluorescence Immunoassay (DELFIA)

DELFIA uses lanthanide chelates (e.g., europium) as labels which emit delayed fluorescence.

Principle:

  • Lanthanide ions are chelated with specific molecules and attached to antibodies.
  • Upon excitation, they emit long-lived fluorescence, allowing time-resolved measurements and low background noise.

Applications:

  • Environmental biosensing of toxins.
  • Multiplex detection of environmental biomarkers.

Advantages:

  • Very low detection limits.
  • Ideal for complex environmental samples.

10.9 Application of Immunoassay in Environmental Monitoring

Immunoassays have revolutionized the detection of pollutants in environmental matrices due to their specificity, sensitivity, and adaptability.

Key Applications:

  • Pesticide detection in food, soil, and water (e.g., atrazine, glyphosate).
  • Detection of PAHs, PCBs, and dioxins in sediments and water.
  • Monitoring microbial contamination in drinking water.
  • Biosensors based on immunoassay principles for field testing.

Benefits:

  • Rapid, on-site detection.
  • Cost-effective for large-scale monitoring.
  • Minimally invasive sample preparation.

10.10 Let Us Sum Up

  • Immunoassays are antibody-based techniques widely used in both medical and environmental sciences.
  • Techniques include RIA, ELISA, IFA, SFLIA, and DELFIA, each with unique strengths.
  • These methods are highly effective for detecting trace levels of pollutants and toxicants in complex environmental samples.
  • Recent advances include stable isotope labeling and lanthanide fluorescence, improving accuracy and sensitivity.

Keywords

  • Immunoassay-Analytical method using antibodies to detect specific antigens.
  • Radioimmunoassay (RIA)-Uses radioactive isotopes to quantify analytes via antibody binding.
  • ELISA (EIA)-Enzyme-based immunoassay that produces a colorimetric signal.
  • Immunofluorescence (IFA)-Uses fluorescent-labeled antibodies to detect antigens visually.
  • Stable Isotope Labeling-Use of non-radioactive isotopes for tracking molecules.
  • Neutron Activation Analysis (NAA)-Technique to detect elements by neutron-induced radioactivity.
  • SFLIA-A fluorescence immunoassay where the substrate emits light after enzymatic conversion.
  • DELFIA-Time-resolved immunoassay using lanthanide-based delayed fluorescence.
  • Lanthanides-Elements like europium and terbium used in time-resolved fluorescence detection.
  • Environmental Monitoring-The process of analyzing environmental samples to detect pollutants or biohazards.

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