🦠Microbiology Unit 20 – Laboratory Analysis of the Immune Response

Key Concepts and Terminology

• Innate immunity provides immediate, non-specific defense against pathogens and includes physical barriers (skin and mucous membranes), chemical barriers (enzymes and pH), and cellular components (neutrophils and macrophages)
• Adaptive immunity develops more slowly but provides specific, long-lasting protection against pathogens through the action of lymphocytes (B cells and T cells)
  • B cells produce antibodies that neutralize and eliminate pathogens
  • T cells directly attack infected cells (cytotoxic T cells) or help coordinate the immune response (helper T cells)
• Antigens are substances that trigger an immune response, typically proteins or polysaccharides on the surface of pathogens or foreign cells
• Antibodies (immunoglobulins) are proteins produced by B cells that specifically bind to antigens, neutralizing them or marking them for destruction by other immune cells
• Cytokines are signaling molecules secreted by immune cells that regulate the intensity and duration of the immune response (interleukins and interferons)
• Major histocompatibility complex (MHC) molecules are cell surface proteins that present antigens to T cells, enabling them to recognize infected or abnormal cells
• Complement system consists of plasma proteins that enhance the immune response by promoting inflammation, opsonization, and lysis of pathogens

Immune System Components

• Lymphoid organs are sites where lymphocytes develop, mature, and interact with antigens
  • Primary lymphoid organs include the bone marrow and thymus, where lymphocytes originate and mature
  • Secondary lymphoid organs include lymph nodes, spleen, and mucosa-associated lymphoid tissue (MALT), where lymphocytes encounter antigens and mount an immune response
• Lymphocytes are the key cellular components of the adaptive immune system
  • B lymphocytes (B cells) mature in the bone marrow and produce antibodies
  • T lymphocytes (T cells) mature in the thymus and include helper T cells (CD4+) and cytotoxic T cells (CD8+)
• Antigen-presenting cells (APCs) capture, process, and present antigens to T cells, initiating the adaptive immune response (dendritic cells, macrophages, and B cells)
• Granulocytes are innate immune cells characterized by the presence of granules in their cytoplasm (neutrophils, eosinophils, and basophils)
• Natural killer (NK) cells are innate lymphocytes that recognize and kill virus-infected or tumor cells without prior sensitization
• Mast cells are tissue-resident cells that release histamine and other mediators during allergic reactions and contribute to innate immunity

Common Lab Techniques

• Enzyme-linked immunosorbent assay (ELISA) detects and quantifies specific antibodies or antigens in a sample using enzyme-labeled antibodies and a colorimetric substrate
  • Indirect ELISA measures the concentration of specific antibodies in a sample
  • Sandwich ELISA measures the concentration of specific antigens in a sample
• Flow cytometry analyzes the physical and chemical characteristics of individual cells in a suspension as they pass through a laser beam
  • Immunophenotyping identifies and quantifies specific cell populations based on the expression of surface markers using fluorescently labeled antibodies
• Western blot detects specific proteins in a sample by separating them based on size using gel electrophoresis, transferring them to a membrane, and probing with specific antibodies
• Immunohistochemistry (IHC) detects the presence and location of specific antigens in tissue sections using labeled antibodies and microscopy
• Polymerase chain reaction (PCR) amplifies specific DNA sequences, enabling the detection of pathogens or genetic markers associated with immune function
• Cytokine assays measure the concentration of specific cytokines in a sample using ELISA, multiplex assays, or functional assays (cell proliferation or cytotoxicity)

Antigen-Antibody Interactions

• Antibodies bind to specific epitopes (antigenic determinants) on the surface of antigens through their variable regions (Fab fragments)
• Antibody-antigen binding is mediated by non-covalent interactions (hydrogen bonds, van der Waals forces, and electrostatic interactions) and is characterized by specificity and affinity
• Affinity is the strength of the interaction between a single antibody binding site and its corresponding epitope, while avidity refers to the overall strength of the antibody-antigen complex
• Antibodies can neutralize pathogens by blocking their entry into host cells, promoting their phagocytosis (opsonization), or activating the complement system
• Agglutination occurs when antibodies cross-link particulate antigens (bacteria or red blood cells), causing them to clump together
• Precipitation occurs when antibodies form insoluble complexes with soluble antigens, which can be visualized as a visible precipitate
• Monoclonal antibodies are produced by a single B cell clone and recognize a single epitope, while polyclonal antibodies are produced by multiple B cell clones and recognize multiple epitopes on an antigen

Cell-Mediated Immunity Assays

• Lymphocyte proliferation assays measure the ability of T cells to proliferate in response to specific antigens or mitogens (phytohemagglutinin or concanavalin A) using radioactive or fluorescent labels
• Cytotoxicity assays assess the ability of cytotoxic T cells or NK cells to kill target cells
  • Chromium-51 release assay measures the release of radioactive chromium from lysed target cells
  • Flow cytometry-based assays use fluorescent dyes (propidium iodide or annexin V) to detect cell death
• ELISPOT (enzyme-linked immunospot) assay detects the secretion of cytokines (interferon-gamma or interleukin-2) by individual T cells in response to specific antigens
• Intracellular cytokine staining (ICS) measures the production of cytokines by T cells at the single-cell level using flow cytometry and fluorescently labeled antibodies
• Delayed-type hypersensitivity (DTH) skin test assesses the presence of antigen-specific T cells in vivo by measuring the local inflammatory response to an intradermal antigen injection (tuberculin skin test)

Immunological Disorders and Diagnostics

• Autoimmune disorders occur when the immune system mistakenly attacks the body's own tissues (rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis)
  • Diagnostic tests include autoantibody detection (anti-nuclear antibodies or rheumatoid factor), inflammatory marker measurement (C-reactive protein or erythrocyte sedimentation rate), and imaging studies
• Immunodeficiencies are characterized by a weakened immune response, leading to increased susceptibility to infections (severe combined immunodeficiency or AIDS)
  • Diagnostic tests include lymphocyte subset enumeration, antibody levels, and genetic testing
• Allergies are hypersensitivity reactions mediated by IgE antibodies in response to typically harmless antigens (pollen, food, or drugs)
  • Diagnostic tests include skin prick tests, allergen-specific IgE measurement, and oral food challenges
• Vaccination induces protective immunity against specific pathogens by exposing the immune system to inactivated or attenuated pathogens or their components
  • Vaccine efficacy can be assessed by measuring antibody titers or T cell responses post-vaccination
• Immunotherapy harnesses the immune system to treat cancer or other diseases
  • Monoclonal antibodies target specific antigens on cancer cells or immune checkpoints (rituximab or ipilimumab)
  • Chimeric antigen receptor (CAR) T cell therapy genetically modifies a patient's T cells to recognize and kill cancer cells

Data Analysis and Interpretation

• Sensitivity and specificity are key metrics for evaluating the performance of diagnostic tests
  • Sensitivity is the ability of a test to correctly identify individuals with the disease (true positive rate)
  • Specificity is the ability of a test to correctly identify individuals without the disease (true negative rate)
• Positive and negative predictive values depend on the prevalence of the disease in the population
  • Positive predictive value (PPV) is the probability that an individual with a positive test result actually has the disease
  • Negative predictive value (NPV) is the probability that an individual with a negative test result does not have the disease
• Receiver operating characteristic (ROC) curves plot the true positive rate against the false positive rate for different cut-off values of a diagnostic test, allowing the determination of the optimal cut-off value
• Reference ranges are established by testing a large number of healthy individuals and determining the central 95% of the values, which serve as a basis for interpreting patient results
• Quality control measures ensure the accuracy and reliability of laboratory results
  • Positive and negative controls validate the performance of the assay
  • Calibration curves and standards ensure the quantitative accuracy of the results
  • Proficiency testing and inter-laboratory comparisons assess the performance of the laboratory in relation to its peers

Safety and Ethical Considerations

• Biosafety guidelines protect laboratory personnel and the environment from exposure to infectious agents
  • Biosafety levels (BSL) 1-4 define the containment measures and practices required for handling different risk groups of microorganisms
  • Personal protective equipment (PPE) includes gloves, lab coats, and eye protection to minimize the risk of exposure
• Proper handling and disposal of biohazardous materials prevent the release of infectious agents into the environment
  • Autoclaving, chemical disinfection, and incineration are common methods for decontaminating biohazardous waste
• Informed consent must be obtained from patients or research participants before collecting samples or performing tests, ensuring that they understand the purpose, risks, and benefits of the procedure
• Confidentiality of patient information must be maintained in accordance with HIPAA (Health Insurance Portability and Accountability Act) regulations in the United States
  • Access to patient data should be limited to authorized personnel
  • Electronic health records and laboratory information systems must have appropriate security measures in place
• Ethical considerations in research involve the protection of human subjects and the responsible conduct of research
  • Institutional Review Boards (IRBs) review and approve research protocols to ensure that they meet ethical standards
  • Animal research must follow the principles of the 3 Rs (replacement, reduction, and refinement) to minimize animal use and suffering