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Chemistry

Principles

Understanding the analytical principles behind endocrinology laboratory tests is critical for ensuring accurate and reliable results. Fluorescence and immunoassay techniques are commonly used

Fluorescence Assays

  • Principle
    • Fluorescence: is a phenomenon in which a molecule (fluorophore) absorbs light at a specific wavelength (excitation wavelength) and emits light at a longer wavelength (emission wavelength)
    • The intensity of the emitted light is directly proportional to the concentration of the fluorophore
    • Fluorescence assays are highly sensitive and specific, making them useful for measuring low concentrations of hormones
  • Components
    • Light Source: Provides the excitation light (e.g., xenon lamp, laser)
    • Excitation Filter: Selects the excitation wavelength
    • Sample: Contains the hormone to be measured, labeled with a fluorophore
    • Emission Filter: Selects the emission wavelength
    • Detector: Measures the intensity of the emitted light (e.g., photomultiplier tube, photodiode)
  • Types of Fluorescence Assays
    • Direct Fluorescence Assay
      • The hormone itself is labeled with a fluorophore
      • The intensity of the emitted light is measured directly
    • Indirect Fluorescence Assay
      • A secondary reagent labeled with a fluorophore binds to the hormone
      • The intensity of the emitted light is measured indirectly
    • Fluorescence Polarization Immunoassay (FPIA)
      • Based on the change in polarization of emitted light
      • Small fluorescently labeled hormone molecules emit light with rapid depolarization
      • When the labeled hormone binds to antibodies, the resulting complex rotates more slowly, emitting light with increased polarization
      • The degree of polarization is inversely proportional to the concentration of the hormone
    • Time-Resolved Fluorescence Immunoassay (TR-FIA)
      • Uses lanthanide chelates as fluorophores
      • Lanthanide chelates have long emission lifetimes, allowing for time-resolved measurements
      • Background fluorescence is minimized by measuring the emission after a delay period
      • TR-FIA is highly sensitive and specific
  • Applications in Endocrinology
    • Thyroid Hormones: Measurement of T3 and T4
    • Steroid Hormones: Measurement of cortisol, estradiol, and testosterone
    • Peptide Hormones: Measurement of insulin and growth hormone
  • Advantages
    • High sensitivity
    • High specificity
    • Relatively simple instrumentation
    • Wide range of applications
  • Disadvantages
    • Susceptible to interference from fluorescent compounds in the sample (autofluorescence)
    • Photobleaching of the fluorophore
    • Quenching of fluorescence by other molecules

Immunoassays

  • Principle
    • Immunoassays: are based on the specific binding of antibodies to antigens (or haptens)
      • The hormone to be measured acts as the antigen
      • The amount of hormone is determined by measuring the amount of antibody-antigen complex formed
  • Components
    • Antibody: A protein produced by the immune system that binds to a specific antigen
    • Antigen: The hormone to be measured
    • Label: A molecule that is attached to the antibody or antigen to allow for detection
    • Detection System: Measures the amount of label bound to the antibody-antigen complex
  • Types of Immunoassays
    • Competitive Immunoassay
      • Unlabeled hormone in the sample competes with labeled hormone for binding to a limited number of antibodies
      • The amount of labeled hormone bound to the antibody is inversely proportional to the concentration of unlabeled hormone in the sample
      • Applications: Measurement of small hormones, such as steroid hormones and thyroid hormones
    • Non-Competitive Immunoassay (Sandwich Immunoassay)
      • Two antibodies are used: a capture antibody and a detection antibody
      • The capture antibody binds to the hormone in the sample
      • The detection antibody, which is labeled, binds to a different epitope on the hormone
      • The amount of labeled antibody bound to the hormone is directly proportional to the concentration of the hormone in the sample
      • Applications: Measurement of large hormones, such as peptide hormones and protein hormones
    • Enzyme-Linked Immunosorbent Assay (ELISA)
      • A type of immunoassay that uses an enzyme as the label
      • The enzyme converts a substrate to a detectable product, such as a colored compound
      • Applications: Measurement of a wide range of hormones
    • Chemiluminescent Immunoassay (CLIA)
      • A type of immunoassay that uses a chemiluminescent compound as the label
      • The chemiluminescent compound emits light when it undergoes a chemical reaction
      • The amount of light emitted is proportional to the concentration of the hormone
      • Applications: Measurement of hormones requiring high sensitivity
    • Radioimmunoassay (RIA)
      • A type of immunoassay that uses a radioactive isotope as the label
      • The amount of radioactivity is measured to determine the concentration of the hormone
      • Applications: Historically used for many hormones, but now less common due to safety concerns and regulatory requirements
  • Applications in Endocrinology
    • Thyroid Hormones: Measurement of T3, T4, and TSH
    • Steroid Hormones: Measurement of cortisol, estradiol, testosterone, and progesterone
    • Peptide Hormones: Measurement of insulin, growth hormone, prolactin, FSH, LH, ACTH, PTH, and hCG
  • Advantages
    • High sensitivity
    • High specificity
    • Wide range of applications
  • Disadvantages
    • Susceptible to interference from heterophile antibodies and other factors
    • Cross-reactivity with structurally similar compounds
    • Requirement for specific antibodies

Comparison of Fluorescence and Immunoassay Techniques

Characteristic Fluorescence Assay Immunoassay
Principle Measures emitted light from fluorophore Measures binding of antibodies to antigens
Sensitivity High High
Specificity High High
Instrumentation Relatively simple Can be complex, depending on the type of immunoassay
Interferences Autofluorescence, quenching Heterophile antibodies, cross-reactivity
Applications Measurement of hormones, drug monitoring Measurement of hormones, proteins, and other analytes

Key Terms

  • Fluorescence: Emission of light by a molecule after absorbing light at a specific wavelength
  • Fluorophore: A molecule that emits fluorescence
  • Immunoassay: An assay based on the specific binding of antibodies to antigens
  • Antibody: A protein produced by the immune system that binds to a specific antigen
  • Antigen: A molecule that binds to an antibody
  • Competitive Immunoassay: An immunoassay in which unlabeled antigen competes with labeled antigen for antibody binding
  • Non-Competitive Immunoassay: An immunoassay in which labeled antibody binds to antigen
  • Enzyme-Linked Immunosorbent Assay (ELISA): An immunoassay that uses an enzyme as the label
  • Chemiluminescent Immunoassay (CLIA): An immunoassay that uses a chemiluminescent compound as the label
  • Radioimmunoassay (RIA): An immunoassay that uses a radioactive isotope as the label
  • Heterophile Antibodies: Antibodies that bind to multiple antigens
  • Cross-Reactivity: The ability of an antibody to bind to multiple antigens
  • Epitope: The specific region of an antigen that is recognized by an antibody
  • Homogeneous Assay: An immunoassay where separation of bound and unbound reactants is not required
  • Heterogeneous Assay: An immunoassay where separation of bound and unbound reactants is required
  • Polarization: The alignment of the electric field vector of light waves
  • Quenching: A process that decreases the fluorescence intensity of a fluorophore
  • Photobleaching: The destruction of a fluorophore by light
  • Lanthanide: A rare earth element that exhibits long-lived fluorescence