Diagnostic Testing 2

Lecture Overview

  • Laboratory Diagnostics_2 Topics:

    • Immunologic methods

    • Serology

    • Precipitation Assays

    • Agglutination Assays

    • Neutralization Assays

    • Complement fixation Assays

    • EIA/ELISAs

    • ICAs

    • Western Blotting

  • Source Material: Openstax Microbiology textbook, Chapter 20: Laboratory analysis of the immune response

  • Key Abbreviations:

    • Antibody = Ab or Ig

    • Antigen = Ag

Immunologic Methods in Laboratory Diagnostics

  • Definition:

    • Immunologic-based diagnostic tests involve the interactions of antibodies with specific antigens.

    • Known antibodies can identify unknown pathogens (antigens).

    • Known antigens can determine the presence of unknown antibodies.

    • Tests can be performed on various fluids such as blood, cerebrospinal fluid, and urine.

  • Serology:

    • Defined as the study of the serum components in a patient’s blood.

    • Goal: To determine the presence of specific antigens or antibodies in a patient's blood.

    • Serum is the liquid part of blood devoid of clotting factors, obtained after blood clots have formed.

Advantages and Limitations of Immunological Tests

  • Advantages:

    • Rapid results, typically returning in minutes to hours.

    • Low technological requirements, making them easy to use.

    • Ability to detect antibodies or antigens even when cultures are difficult.

    • Capable of detecting organisms that cannot be cultured.

  • Disadvantages:

    • May not differentiate between current and past infections.

    • Require high-quality antibodies for accuracy.

    • Potential for false positives and negatives due to cross-reactivity or timing issues.

    • Overall lower sensitivity and specificity compared to some other methods.

Functions of Antibodies (Immunoglobulins, Ig)

  • Key Functions:

    • Neutralization: Blocks pathogen binding to cells.

    • Agglutination: Clumps antigens, making them easier for phagocytes to identify.

    • Opsonization: Enhances phagocytosis of pathogens.

    • Complement Activation: Engages the classical complement pathway leading to pathogen lysis.

    • ADCC: Mediates antibody-dependent cell-mediated cytotoxicity, where antibodies mark infected cells for destruction.

Types of Assays Detecting Antigen–Antibody Complexes

  • The binding between antigens and antibodies underlies many diagnostic tests. Common assay types include:

    • Precipitation Tests:

    • Formation of visible precipitate from the reaction of soluble antigens with antibodies.

    • Agglutination Tests:

    • Clumping of particles or cells due to antibody binding, visible as agglutinates.

    • Immunoassays (e.g., ELISA, EIA, FEIA):

    • Identification of bound antibody or antigen using labels for detection.

Precipitation Tests

  • Principle:

    • Occurs when soluble antigens and antibodies interact to form insoluble complexes that precipitate.

    • A visible precipitate appears when antigen and antibody concentrations are optimal, known as the zone of equivalence.

  • Key Steps:

    • Mix antigen with corresponding antibody in a liquid or gel medium.

    • Allow time for antigen-antibody complexes to form.

    • Observe for the formation of a precipitate line or cloudiness, indicating a positive reaction.

  • Diagnostic Applications:

    • Radial Immunodiffusion: Quantifies serum proteins, such as IgG and complement.

    • Ouchterlony Double Diffusion: Identifies specific antibodies or antigens (e.g., for fungal infections or autoantibodies).

  • Advantages:

    • Simple and inexpensive.

    • Provides visual results, useful for measuring antibody levels or antigen presence.

    • High specificity with purified antigens.

Agglutination Tests

  • Principle:

    • Occurs when particulate antigens bind to antibodies, creating visible clumps (agglutinates).

    • Used to detect both antigens and antibodies in patient samples.

  • Key Steps:

    • Mix patient sample (antigen or antibody) with known antigen or antibody suspensions.

    • Allow time for binding, where clumping indicates a positive reaction.

    • Can be direct (natural antigen on cell surface) or indirect (antigen/antibody attached to carrier particles).

  • Diagnostic Applications:

    • Blood typing (ABO, Rh).

    • Latex agglutination tests (for C-reactive protein, rheumatoid factor, meningitis pathogens).

    • Widal test for detecting Salmonella antibodies.

  • Advantages:

    • Rapid results are easy to interpret visually.

    • Capable of detecting small amounts of antigen or antibody.

    • Can be automated or performed as slide tests in clinical labs.

Direct vs. Indirect Agglutination

  • Direct Agglutination:

    • Detects antigens directly present on the surface of cells or particles.

    • Examples include blood typing and bacterial identification.

  • Indirect Agglutination:

    • Involves latex beads or carrier particles that are coated with either antigens or antibodies.

    • Example: Coagulase test for identifying Staphylococcus aureus.

Neutralization Reactions

  • Principle:

    • Detects antibodies that neutralize the biological activity of antigens, such as viruses or toxins.

    • Presence of antibodies blocks the pathogen or toxin from exerting its normal effects.

  • Key Steps:

    • Mix patient serum (which may contain neutralizing antibodies) with a known virus or toxin.

    • Incubate to allow antibody binding, followed by addition to susceptible cells or test animals.

    • A lack of cytopathic effect or no toxicity indicates a positive neutralization reaction.

  • Diagnostic Applications:

    • Virus neutralization tests (e.g., for measles, influenza, SARS-CoV-2).

    • Toxin neutralization tests (e.g., for Clostridium botulinum or diphtheria toxin).

  • Advantages:

    • High specificity—confirms the presence of functional, protective antibodies.

    • Indicates immunity or past infection instead of merely the presence of antibodies.

    • Valuable for studying vaccine efficacy.

Complement Fixation Tests

  • Principle:

    • Detects antigen-antibody complexes by measuring complement protein consumption.

    • Binding of antibodies in patient serum to their respective antigens results in fixed complement, unavailability for lysing indicator cells.

  • Key Steps:

    • Heat patient serum to inactivate native complement.

    • Mix it with a known antigen and a source of complement proteins.

    • Add indicator cells (e.g., sheep red blood cells coated with anti-sheep red blood cell antibodies) to observe hemolysis.

  • Interpretation:

    • No hemolysis indicates a positive reaction due to complement fixation.

    • Hemolysis indicates a negative result (complement is free, no antigen-antibody complex forms).

  • Diagnostic Applications:

    • Useful when direct pathogen detection is challenging (e.g., for influenza or rubella).

  • Advantages:

    • Effective for detecting small amounts of antibody.

    • Applicable when other tests like agglutination are not suitable.

Enzyme Immunoassay (EIA) or Enzyme-linked Immunosorbent Assays (ELISAs)

  • Principle:

    • Detects antigens or antibodies through an enzyme-labeled antibody.

    • The binding of an enzyme-linked antibody to its target produces a color change when a substrate is added.

  • Key Steps:

    • Antigens (or antibodies) are immobilized on a microplate well surface.

    • Patient sample is added, and binding occurs if the matching antibody or antigen is present.

    • An enzyme-linked antibody is added to detect binding.

    • Substrate is added, converting into a colored product—intensity proportional to the target presence.

  • Applications:

    • Used to detect HIV, hepatitis, hormones, drugs, allergens.

    • Basis for many rapid diagnostic kits.

  • Advantages:

    • High throughput capability, can provide quantitative or semi-quantitative results.

Immunochromatography Assay (ICA) or Lateral Flow Assay (LFA)

  • Principle:

    • Relies on capillary flow of a liquid sample across a porous strip coated with antibodies, facilitating Ab-Ag binding.

  • Key Components:

    • Nitrocellulose membrane with immobilized capture antibodies forming test and control lines.

    • Labeled antibodies (with colored particles) provide visible results.

  • Results Interpretation:

    • Test Line: Appears if the target antigen or antibody is present.

    • Control Line: Confirms proper flow and reagent function.

  • Examples of Use:

    • Pregnancy tests (hCG detection), rapid COVID-19 antigen tests, rapid strep and influenza tests.

  • Advantages:

    • Fast results within minutes.

    • Simple to use, requires no additional equipment.

    • Effective for point-of-care diagnostics.

Western Blotting (Immunoblotting)

  • Principle:

    • Detects specific proteins (antigens) or antibodies in a sample following gel electrophoresis and transfer to a membrane.

  • Key Steps:

    1. Protein Separation: Conduct SDS-PAGE based on protein size.

    2. Transfer: Proteins are transferred to a nitrocellulose or PVDF membrane.

    3. Blocking: Non-specific sites on the membrane are blocked to prevent background binding.

    4. Antibody Detection: Add primary antibody specific to the target protein, followed by an enzyme-linked secondary antibody that binds to the primary.

    5. Visualization: Add enzyme substrate to produce a signal (color or light) where the antibody is bound.

  • Diagnostic Applications:

    • Used primarily as a confirmatory test for conditions like HIV and Lyme disease.

    • Detects specific viral or bacterial proteins indicating infection.

  • Advantages:

    • High specificity for confirming the presence of true target proteins.

    • Useful as a follow-up for more sensitive initial screening assays (like ELISA).

Learning Objectives

  • After attending the lecture and completing any assigned readings, students should be able to:

    • Explain how antibodies in patient serum are utilized to diagnose diseases.

    • Define key terms such as serology, serum, and plasma.

    • Compare and distinguish between direct and indirect agglutination.

    • Describe the principles, key techniques, applications, and advantages of various assays including precipitation tests, agglutination tests, neutralization tests, complement-fixation tests, EIA/ELISA, ICAs, and Western Blotting.

    • Discuss the pros and cons of immunological diagnostics in comparison to other identification methods (i.e., culture or nucleic acid methods).