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Comprehensive notes on Immunological Assays

  • Objectives (from Page 2):

    • To understand basic concepts and mechanisms of immunologic assays
    • To define and understand immunologic measures and methods
    • To understand clinical applications of immunologic assays

  • Key terms and definitions

    • Immunoassay: a test that uses antibody–antigen complexes to generate a measurable result. \text{Immunoassay} \equiv \text{Ab-Ag interactions producing a detectable signal}
    • Analyte: anything measured by a laboratory test; in immunoassay testing, the analyte may be either an antibody or an antigen.
    • Analyte categories:
    • Naturally present (e.g., thyroid hormone)
    • Produced by body but not typically present (e.g., a cancer-associated antigen)
    • Do not naturally occur in the body (e.g., an abused drug)

  • Precipitation concepts

    • Precipitation: combining soluble antigen (Ag) with soluble antibody (Ab) to form insoluble complexes that are visible.
    • Ab and Ag interactions: affinity vs avidity
    • AFFINITY: strength of binding between a single antigenic determinant and an antibody paratope; high affinity means strong binding to the target determinant.
    • AVIDITY: overall strength of multiple affinities in multivalent interactions (multivalent antigen with multivalent antibody is more robust by cumulative avidity).
    • Antigenic determinants: antigenic sites; cross-reacting antigens may share determinants but have lower affinity.

  • Prozone–Zone–Postzone (Precipitation curve)

    • Prozone: antibody excess; limited lattice formation leading to weak precipitation.
    • Zone of equivalence: optimal ratio of Ag to Ab resulting in maximal lattice formation and visible precipitate.
    • Postzone: antigen excess; reduced lattice formation due to insufficient cross-linking.
    • Conceptually: as Ag concentration increases with constant Ab, lattice formation increases until the zone of equivalence, then decreases if Ag becomes limiting.

  • Measurement of precipitation via light scattering

    • Turbidimetry: measures turbidity (light transmission) caused by suspended particles; reports on relative concentration based on transmitted light.
    • Nephelometry: measures scattered light at various angles (e.g., 70°, 90°, forward scatter) to quantify particle concentration more sensitively than turbidimetry.
    • Instrument arrangements (conceptual): light source → sample cell → detectors at defined angles (e.g., 90°, forward, back scattered).

  • Passive immunodiffusion techniques

    • Principle: diffusion of soluble antigen and antibody in a support medium with no electric current; forms a visible precipitin line where they meet in optimal proportions.
    • Radial (single) immunodiffusion: antibody fixed in a gel (agar) on a slide; test antigen diffuses radially; precipitin ring diameter relates to antigen concentration.
    • Ouchterlony double diffusion: both antibody and antigen diffuse in a gel; patterns (A, B, C) used to assess identity, non-identity, and partial identity.

  • Radial immunodiffusion (RID)

    • Setup: glass slide coated with agar containing specific antibody.
    • Measurement: diameter of precipitin ring (mm)² is proportional to the amount of antigen present.
    • Typical relationship: D^2 \propto C where D is ring diameter and C is antigen concentration.
    • Example visualization (from slide): multiple standards and unknowns can be compared by ring diameter; standard curves are generated to quantify antigen levels.

  • Ouchterlony double diffusion (pattern interpretations)

    • Identity: Ab and Ag form smooth, continuous arcs that fuse (Ag ≡ Ag1, Ab ≡ anti-1).
    • Nonidentity: crossing lines indicate differing specificities or multiple antigens (Ag ≠ Ag1, Ag2; Ab ≠ anti-1, anti-2).
    • Partial identity: line segments indicate related antigens sharing some, but not all, determinants; indicated by spur formations (Ag1a is a part of Ag1 but a simpler antigen).
    • Practical layout: center well contains antibody/antiserum; outer wells contain antigens; the patterns indicate relatedness of antigens.

  • Immunoelectrophoresis and related electrophoretic immunodiffusion techniques

    • Concept: diffusion of antigens/antibodies followed by electrophoresis to separate proteins by charge/mobility, then immunodiffusion to visualize antigen–antibody complexes.
    • Techniques (Page 17):
    • Rocket immunoelectrophoresis (one-dimensional electroimmunodiffusion): rapid, quantitative; shows a rocket-shaped precipitation peak.
    • Immunoelectrophoresis: diffusion plus electrophoresis to differentiate proteins.
    • Immunofixation electrophoresis: immunoblot-like separation with subsequent fixation of bands; often used for HIV, Lyme disease, syphilis.
    • Double diffusion: combination of diffusion with electrophoresis to resolve multiple components.

  • Immunoblotting and HIV testing (Pages 19–20)

    • Immunoblot concept: transfer of proteins separated by SDS-PAGE to a membrane (e.g., nitrocellulose) followed by probing with patient serum; detected with enzyme-labeled secondary antibodies.
    • HIV testing workflow (illustrated):
    • SDS-PAGE separation of viral proteins; transfer to nitrocellulose; overlay with HIV-specific antiserum (patient sera).
    • Detect bound antibody using enzyme-linked anti-IgG.
    • Immunofixation electrophoresis: similar idea, but uses immunofixation to identify specific proteins with antiserum.

  • Comparative table: precipitating techniques (Table 8-1 summary)

    • Nephelometry
    • Applications: immunoglobulins, complement, C-reactive protein, other serum proteins.
    • Sensitivity: approximately 1–10 μg/mL.
    • Notes: automated, sensitive, expensive; requires specialized equipment.
    • Radial immunodiffusion (RID)
    • Applications: immunoglobulins, complement.
    • Sensitivity: about 10–50 μg/mL.
    • Notes: quantitative but slower and less sensitive than nephelometry.
    • Ouchterlony double diffusion
    • Applications: complex antigens such as fungal antigens.
    • Sensitivity: 20–200 μg/mL; semi-quantitative; interpretation can be difficult.
    • Rocket immunoelectrophoresis
    • Applications: immunoglobulins, complement; e.g., alpha-fetoprotein.
    • Sensitivity: around 2 μg/mL; fast, quantitative but technically demanding.
    • Immunoelectrophoresis
    • Applications: serum protein differentiation.
    • Sensitivity: 20–200 μg/mL; slow, semi-quantitative, interpretation can be difficult.
    • Immunofixation electrophoresis
    • Applications: HIV, Lyme disease, syphilis; variable sensitivity.
    • Notes: fairly rapid, semi-quantitative, sensitive.
    • Immunofixation (summary): emphasizes ability to identify specific antibody/antigen bands with higher specificity.

  • Agglutination concepts and methods

    • Agglutination: visible aggregation of particles caused by antibody binding; agglutinins are antibodies that induce agglutination.
    • Two key steps:
    • Sensitization: antibody binds to particulate antigen without visible aggregation yet.
    • Lattice formation: cross-linking of particles leads to visible clumping.
    • Antigen with multiple determinants on particulate surface improves agglutination.

  • Agglutination formats and variants

    • Antibody–antigen interactions with particulate Ags (lattice formation) vs soluble Ags.
    • Reverse passive agglutination: carrier particles coated with antigen or antibody to promote visible agglutination in the presence of the complementary antibody/antigen.
    • Coated-particle agglutination: solubilized antigen or antibody is attached to beads; binding of target leads to visible clumping.

  • Hemagglutination inhibition test

    • Principle: mixture of patient sample (potential antibody) with viral antigen forms immune complexes; if antibodies are present, they inhibit hemagglutination of red blood cells (RBCs).
    • Test interpretation:
    • If antibody present: immune complexes form; no agglutination occurs (negative in terms of agglutination).
    • If antibody absent: agglutination occurs (positive for lack of antibody).
    • Practical implication: used to detect antibodies to certain viruses via interference with RBC agglutination.

  • Red cell agglutination scoring (grading)

    • Grading scales described for hemagglutination tests (examples):
    • 4+ : One solid clump
    • 3+ : One or more large clumps
    • 2+ : Moderate clumping
    • 1+ : Barely discernible clumps; cloudiness present
    • 0 (Negative): Smooth suspension; no clumps
    • Scoring is based on the size and number of clumps after the assay and, in some cases, following centrifugation.

  • Agglutination examples and modern contexts

    • Demonstrations using specific reagents (e.g., SARS-CoV-2 RBD) show agglutination with polyclonal anti-RBD antibodies but not with sera lacking anti-RBD antibodies; illustrates antigen–antibody specificity in solid-phase formats.
    • Example components shown include: VHH anti-Glycophorin A on RBCs and SARS-CoV-2 RBD antigen constructs; detection relies on labeled secondary antibodies that produce a visible signal upon binding.

  • Labeled immunoassays: overview

    • Two broad categories:
    • Competitive (1): labeled antigen competes with patient antigen for limited antibody; the signal inversely correlates with amount of patient antigen.
    • Non-competitive (2): sandwich or indirect formats where a labeled detection antibody binds to target.

  • Radioimmunoassay (RIA) and Enzyme immunoassays (EIA)

    • Radioimmunoassay (RIA): competitive format using radiolabeled antigen/antibody; quantitative based on radioactivity measurement.
    • Enzyme immunoassay (EIA): competitive format using enzyme-labeled reagents; read via colorimetric change.
    • Other labeled methods: Immunofluorescence (IF), Chemiluminescence (CL), and Flow cytometry-based immunoassays.

  • Indirect ELISA (Indirect Enzyme-Linked Immunosorbent Assay)

    • Principle: solid-phase antigen captures patient antibody; enzyme-labeled secondary antibody detects bound patient antibody.
    • Steps (as depicted):
    • Coat plate with antigen (solid-phase antigen)
    • Block non-specific sites (washing steps implied)
    • Add patient serum (primary antibody binding to antigen)
    • Add enzyme-labeled anti-immunoglobulin (secondary antibody)
    • Wash to remove unbound components
    • Add substrate; enzyme catalyzes color change indicating binding
    • Key concepts: Specific binding; washing to reduce background; colorimetric readout.

  • Immunofluorescent immunoassays

    • Principle: use labeled antibodies to generate fluorescence when bound to target antigen; detection via fluorescence signals under appropriate illumination.
    • Formats: Solid-phase antigen with labeled antibody; or labeled secondary antibodies; visualization of antigen–antibody complexes via fluorescence.
    • Representative note: pictorial demonstration shows two-step binding and detection by fluorescent signal.

  • Chemiluminescent immunoassays

    • Principle: light emitted from a chemical reaction (e.g., luminol-based reactions) is measured to quantify target Ab/Ag interactions.
    • Key advantage: high sensitivity and wide dynamic range; often used in clinical laboratories.

  • Luminex multiplex immunoassays

    • Principle: bead-based (microsphere) suspension assay capable of detecting multiple analytes simultaneously.
    • Features: 100 color-codes allow 100 simultaneous tests; cytokine-specific beads; streptavidin–phycoerythrin (PE) reporter system for readout.
    • Workflow highlights: each bead set is coupled to a specific capture antibody; samples are run in a single well with multiplex detection; precision fluidics align beads in single file for laser-based readouts.

  • Flow cytometry and immunophenotyping (FACS)

    • Definition: Flow cytometry measures physical/chemical characteristics of cells/particles as they flow in a fluid stream through a focused laser beam.
    • FACS (Fluorescence Activated Cell Sorting): a flow cytometer configured to physically sort cells based on fluorescence and other optical properties; BD Immunocytometry Systems is a common provider reference; not all flow cytometers are FACS.
    • Key components: focused laser, detectors, fluidics, and optical filters; single-file passage is preferred for accurate measurements.

  • Safety, ethics, and practical lab notes (Pages 43–45)

    • Lab safety note: handling of human specimens requires fixation of cells and adheres to biosafety guidelines; instructions emphasize fixed samples when handling potentially infectious material.
    • Protocol reminders: ensure sheath fluid is not run dry; check waste management; follow facility-specific biosafety and instrument-use policies.
    • Flow cytometer operation ethics: many labs require fixed human cells or non-pathogenic samples; a few slides mention prohibitions on sorting unfixed human cells or cells capable of human infection due to safety concerns.

  • Nucleic acid analyses and diagnostic workflows (Page 29)

    • RT-PCR: time-consuming, lab-based (3–4 hours), labor-intensive; detects viral RNA; considered a standard for active infection.
    • RT-LAMP: lab-based or point-of-care testing; relatively fast (<1 hour); detects active infection.
    • Antigen tests (rapid antigen tests): detect viral proteins; useful for active infection; faster and easier but generally less sensitive than RT-PCR.
    • ELISA: lab-based; time-consuming (1–3 hours); detects antibodies or antigens depending on setup.
    • Lateral flow assays (POCT): rapid, 15–20 minutes; easy to perform; can detect active infection (antigen tests) or past infection (antibody tests).
    • Summary: multiple modalities exist for COVID-19 diagnosis with differing aims (active infection vs past exposure), speeds, and required infrastructure.

  • Summary of how to choose an assay in practice

    • Consider the analyte type (antigen vs antibody vs nucleic acid).
    • Consider the clinical question (active infection vs past exposure).
    • Consider the required turnaround time and available laboratory infrastructure.
    • Consider sensitivity/specificity requirements and potential costs.
    • Consider safety and biosafety requirements for specimen handling.

  • Notation of key relationships and formulas

    • Precipitation assays often rely on lattice formation; quantitative interpretation may use ring diameters or light-scattering intensities.
    • RID quantitative relationship: D^2 \propto C where D is the precipitin ring diameter and C is the antigen concentration.
    • In immunoassays, signal readouts may be colorimetric, fluorescent, luminescent, or light-scatter-based depending on the detection method.

  • Practical exam-ready takeaways

    • Precipitation vs agglutination: Precipitation involves soluble Ag/Ab forming insoluble complexes in a gel or solution; agglutination involves particulate Ag or Ab forming visible aggregates with lattice formation.
    • Prozone and Postzone effects can lead to false negatives if antibody or antigen is in extreme excess; optimal detection requires balancing concentrations.
    • Agglutination inhibition formats can be used to infer the presence of specific antibodies by the absence of agglutination.
    • Labeled immunoassays offer a spectrum from competitive to non-competitive formats and include methods such as RIA, ELISA, IF, CL, and flow-based systems.
    • Modern multiplex and flow-based platforms (e.g., Luminex, flow cytometry) enable high-throughput, multi-parameter analyses with high sensitivity and specificity.

  • Connections to foundational principles and real-world relevance

    • Immunoassays rely on specific antigen–antibody interactions, a cornerstone of adaptive immunity.
    • The choice of assay is guided by kinetics (affinity/avidity), antigen/antibody valency, and the physical state of the analyte (soluble vs particulate).
    • Clinical utility spans infectious disease diagnostics (HIV, SARS-CoV-2), autoimmune panels, cancer biomarkers, and monitoring of immune status (immunoglobulins, complement).
    • Ethical and practical considerations include biosafety, quality control, and interpretation of semi-quantitative results in heterogeneous patient populations.