Infection Science – Serological Diagnostics Vocabulary

Learning Outcomes

• By the end of the session (plus targeted reading) you should be able to:

  • Discuss the principles behind a range of diagnostic (serological) methods.

  • Compare and contrast advantages, disadvantages, and real-world applications of each method.

  • Correctly interpret serological test data in a clinical context.

Key Definitions

• Serology: study of serum components, primarily antibodies.

  • In vitro immune reactions exploited include precipitation, agglutination, complement fixation, etc.

• Serological test: any diagnostic procedure based on an antigen–antibody reaction.

  • Measures serum antibody titres or detects specific antigens (e.g., \text{HLA}, HIV p24).

• Immunoassay: laboratory method that uses an antibody (or antigen) as a reagent to detect or quantify its binding partner.

  • Unlabelled immunoassays: visibility of reaction is intrinsic (precipitate / clump).

  • Labelled immunoassays: reaction revealed by a tag (radio‐isotope, enzyme, fluorochrome, etc.).

Blood Components Overview

• Whole blood: cellular elements (erythrocytes, leucocytes, platelets) + plasma.

• Plasma (clear yellow fluid): water, electrolytes, glucose, proteins (coagulation factors, albumin, antibodies), etc.

• Serum (Latin whey): plasma minus fibrinogen & other clotting factors.

  • In diagnostics, serum is preferred because clotting proteins can interfere with tests.

Serology — Principal Applications

• Clinical diagnostics:

  • Detect antibodies generated against pathogens, allergens, tumours, self-antigens (autoimmunity), or altered gene products.

  • Detect circulating antigens (e.g., microbial, viral, HLA typing, blood-group antigens).

• Research & biotechnology:

  • Antibody-based purification (affinity chromatography), protein quantification, vaccine development, etc.

• Blood-grouping and transfusion compatibility.

Immunoassay Techniques — Bird’s-Eye View

• Unlabelled (visual):

  • Precipitation reactions (e.g., radial immunodiffusion).

  • Agglutination reactions (haemagglutination, latex, bacterial slide tests).

• Labelled (signal amplification):

  • Radioimmunoassay (RIA).

  • Enzyme-Linked Immunosorbent Assay (ELISA) – multiple formats.

  • Western Blot.

  • Flow Cytometry.

Unlabelled Immunoassays

Precipitation Reactions

• Occur in aqueous or semi-solid media (agar).

• Lattice formation: bivalent antibodies + bi/poly-valent soluble antigens → visible precipitin lines.

  • Monovalent antigens do not precipitate.

• Suitable for qualitative pattern analysis (identity, partial identity) and semi-quantification.

Radial Immunodiffusion (RID)

• Agar contains antibody; antigen diffuses radially ➞ ring diameter \propto concentration.

• Quantifies serum immunoglobulins (IgG, IgM, IgA), complement proteins (C3, C4), microbial antigens.

• Clinical uses: diagnose primary immunodeficiencies (e.g., X-linked agammaglobulinaemia), confirm reduced complement.

• Limitations: slow (24–72 h), relatively expensive, poor sensitivity for low-abundance analytes (IgE, IgD).

Agglutination Reactions

• Antibodies bind particulate (cellular) antigens ➞ visible clumping.

Haemagglutination (HA)

• ABO blood typing: anti-A or anti-B sera added to erythrocytes; agglutination pattern reveals group.

• Viral quantitation: e.g., influenza, rubella titres measured by ability to agglutinate red cells.

Bacterial Agglutination

• Direct slide test: identify bacteria in culture by reacting with specific antisera.

• Indirect (serum) test: patient serum + known bacterial antigen ➞ presence of antibodies indicates infection; titre reflects disease stage.

Latex Agglutination

• Pathogen antigens coated onto latex beads; patient serum supplies antibody.

• Rapid, point-of-care diagnosis (minutes). Examples: Group B strep, H. influenzae type b.

Labelled Immunoassays

Radioimmunoassay (RIA)

• Invented by Yalow & Berson (1950s); Yalow received 1977 Nobel Prize.

• Competitive binding: trace radiolabelled antigen competes with sample antigen for limited antibody sites.

• Highly sensitive (pg/mL range); applications include hormones, drugs, vitamins, viral antigens.

• Drawback: radioactive waste & specialised instrumentation.

Enzyme-Linked Immunosorbent Assay (ELISA)

• Concept by Engvall & Perlmann (1971); enzyme replaces radio-isotope.

• Core steps:

  1. Coating/Capture – immobilise antigen or antibody on microplate.

  2. Blocking – saturate unbound surface with irrelevant protein (e.g., BSA) to prevent non-specific binding.

  3. Probing/Detection – add sample + enzyme-linked antibody (direct) or primary + enzyme-linked secondary (indirect).

  4. Signal – add substrate; measure colour/fluorescence \propto analyte amount.

• Amenable to automation (96–1536-well plates); rapid and quantitative.

Direct ELISA

• Single enzyme-conjugated antibody detects antigen.

• Simple & quick but lower sensitivity (signal not amplified) and higher background.

Indirect ELISA

• Primary antibody unlabelled; enzyme-linked secondary antibody binds Fc region.

• Increased sensitivity & flexible (same secondary for many primaries) but extra incubation/wash.

Sandwich (Capture) ELISA

• Two antibodies recognise different epitopes on same antigen ("antigen captured between 2 arms").

• High specificity; widely used for detecting viral, bacterial, fungal antigens (e.g., HIV p24).

  • Versions: Direct (tag on capture Ab) and Indirect (tag on detection Ab).

Competitive ELISA

• Useful when antigen has single epitope or steric hindrance.

• Sample antigen competes with immobilised antigen for binding antibody OR vice-versa.

  • Inverse signal relationship: more antigen in sample → less antibody available to bind plate → lower optical density.

ELISA Formats Summary

• Direct capture, direct sandwich, indirect sandwich, indirect, and competitive; choice depends on antigen size, epitope number, matrix complexity, desired sensitivity.

Western Blot

• Proteins separated by SDS-PAGE (size) in semi-solid polyacrylamide → transferred to membrane.

• Membrane probed with specific antibody; signal (enzyme/fluor) indicates presence & molecular weight of antigen.

• Confirmatory test in virology (e.g., HIV), autoimmune profiling, research.

Flow Cytometry

• Suspension of labelled cells passed single-file through laser beam; detectors measure:

  • Forward scatter (FSC) – approx. cell size.

  • Side scatter (SSC) – internal complexity/granularity.

  • Fluorescence – bound Ab-fluor conjugates reveal antigen expression (phenotyping).

• Example immunophenotyping panel:

  • CD3-FITC (\lambda{ex}=492\,\text{nm},\;\lambda{em}=518\,\text{nm}) – pan-T cells.

  • CD4-APC (\lambda_{em}=660\,\text{nm}) – helper T cells.

  • CD8-PE (\lambda_{em}=578\,\text{nm}) – cytotoxic T cells.

• Generates multiparametric data per cell (10⁴–10⁵ cells/s).

Interpretation & Clinical Use of Serology

1. Acute infection diagnosis – measure IgM vs IgG by ELISA:

   • (+) IgM, (–) IgG → recent infection.

   • (–) IgM, (+) IgG → past infection (> 6 months).

2. Immunological memory – verify protective antibodies after infection or vaccination (e.g., MMR).

3. Screening of blood/organs – mandatory tests for HIV, HBV, HCV, syphilis.

4. Congenital infection – IgM in neonate or persisting IgG > 6 months indicates maternal–foetal transmission.

5. Primary immunodeficiency – abnormal immunoglobulin pattern (quantified by RID or nephelometry).

• Rationale for serology over culture:

  • Antigens/antibodies abundant and stable in fluids.

  • Pathogen culture may be slow, insensitive, or impossible (anaerobes, intracellular microbes).

Automation of Serological Tests

• Emerged with enzyme immunoassay technology (1970s).

• Advantages:

  • High throughput (96/384/1536 wells).

  • Reduced human error; consistent incubation & wash cycles.

  • Cost-effective on volume; integration with LIMS.

• Robotic ELISA workstations illustrate modern laboratory workflows.

Exam Revision — Guidance

• Be able to define serology and justify its use in infectious-disease diagnostics.

• Provide at least two unlabelled immunoassay examples (RID, HA, latex) and outline principles.

• Provide at least two labelled immunoassay examples (RIA, ELISA, Western, Flow Cytometry) and outline principles.

• Discuss advantages/limitations (sensitivity, speed, cost, hazards).

Recommended Reading

• Rittenhouse-Olson K., De Nardin E. (2013) Contemporary Clinical Immunology & Serology – Chapters 6 & 7.

• Crocker J. & Burnett D. (2005) The Science of Laboratory Diagnosis – Chapters 20 & 21.

• Kuby Immunology 7ᵗʰ Ed – Chapter 20.

• Murphy K. Janeway’s Immunobiology – Appendix I.

Extra Notes & Anecdotes

• Humorous intro slide: “SIPILESS: A simple stress test – I do your blood work, send it to the lab, and never get back to you with the results.”

  • Highlights the critical need for proper result communication.

• Ethical considerations:

  • Handling of patient samples & data confidentiality.

  • Radiation safety for RIA.

  • Proper disposal of biohazardous & chemical waste.

• Practical caveats:

  • Cross-reactivity can cause false positives (e.g., rheumatoid factor → false IgM detection).

  • Window periods (time between infection & detectable antibodies) require antigen tests or nucleic acid assays.