L08 I&I Immunodiagnostics

Overview

This lecture covers how infection and immune status are measured in the lab: detecting the infectious agent itself (antigen) versus detecting the host’s response to it (antibody or T cell response). It works through the main assay formats (agglutination, immunofluorescence, neutralization, enzyme immunoassay, immunochromatographic tests), extends the same antibody-detection logic to autoimmunity (ANA testing) and allergy (Type I-IV hypersensitivity, skin prick/patch/tuberculin testing), covers a cell-mediated assay (IGRA/QuantiFERON-TB), and finishes on how serology is used to establish whether an infection is recent (IgM/IgG patterns, seroconversion, IgG avidity) with worked case studies (syphilis, toxoplasmosis in pregnancy, SLE).

Framework for immunodiagnostics

• Immunodiagnostics splits into detection of the infectious agent (antigen) and detection of the immune response (antibody or T cell response).
• Antigen detection is needed when the organism is difficult or impossible to culture (bacteria, viruses, fungi, parasites), including latent infections.
• Detecting the immune response answers three clinical questions: (1) is the patient infected (including when the organism cannot be cultured, or infection is latent); (2) did infection occur recently; (3) is the patient immune to infection.
• The same antibody-detection principle extends beyond infection to autoimmunity (immune response against self antigens), allergy (immune response against innocuous environmental antigens), and transfusion/transplantation (donor-recipient histocompatibility).
• Humoral immunity is measured via B lymphocyte proliferation to antibody-secreting cells; cellular immunity is measured via T lymphocyte proliferation (with antigen-presenting cells) to effector T cells.

Kinetics of infection markers

During an acute infection (illustrated over ~50 days, acute illness roughly days 0-6):

• Viraemia/antigen (detectable by RT-PCR) rises early and falls sharply by about day 6 - this is the window for antigen detection.
• NS1 antigen is detectable over a wider early window extending to around day 6.
• IgM rises as viraemia declines, peaks around day 14, then declines by day 50.
• IgG rises somewhat later than IgM and persists at a high level through day 50.
• Antigen detection therefore works best early in acute illness; antibody (IgM then IgG) detection works from mid-acute illness onward and IgG persists long-term.

Methods for detecting antigen and antibody

Each method can be configured to detect either antibody or antigen, depending on what is bound to the fixed/labelled reagent.

• Agglutination: particles are coated with antigen (to detect antibody) or with antibody (to detect antigen); when the target binds, the particles cross-link and visibly clump (reactive), versus remaining smooth/separate (non-reactive).
• Immunofluorescence:
  • Direct: a fluorescently-labelled antibody binds directly to antigen on a cell - detects antigen.
  • Indirect: an unlabelled primary antibody binds antigen on a cell, then a fluorescently-labelled secondary antibody binds the primary antibody - detects antibody (the secondary step amplifies signal).
• Neutralization: tests whether patient antibody can block infection. Without neutralizing antibody, virus particles freely infect the cell, causing damage/plaques. With neutralizing antibody, antibody coats virus particles and prevents them contacting the cell, keeping it healthy.
• Enzyme immunoassay (EIA/ELISA):
  • Detection of antibody: antigen-coated well → add serum (patient antibody binds if present) → add enzyme-conjugated anti-antibody → add substrate, which changes colour if antibody was present.
  • Detection of antigen: antibody-coated well → add serum (antigen binds if present) → add enzyme-conjugated antigen-specific antibody → add substrate, colour change indicates antigen present.
• Immunochromatographic (lateral flow) test (e.g. rapid HIV tests, pregnancy tests, rapid SARS-CoV-2 antigen tests): sample is added to the sample pad, where mobile antibodies to the analyte conjugated to colloidal gold are present; capillary flow carries the sample/antibody-gold complex along the strip; at the test line, analyte-antibody-gold complexes bind (positive result line); excess conjugate binds at the control line regardless (confirms the test ran correctly, i.e. valid test).

Allergy and hypersensitivity testing

Hypersensitivity reactions and how each is tested:

Type	Mechanism	Examples	Testing
I - IgE-mediated	Histamine release/degranulation	Asthma, rhinitis, eczema, allergic conjunctivitis	Specific IgE testing, skin prick testing
II - IgG-mediated cytotoxic	Antibody-dependent cellular cytotoxicity, complement activation	-	-
III - Immune complex-mediated	Free-floating immune complex activates complement, recruits neutrophils	Food/drug allergy, anaphylaxis, urticaria	-
IV - Cell-mediated	Sensitized Th1 cell releases cytokines, activating macrophages and cytotoxic T cells	Contact dermatitis	Patch testing

• Skin prick testing (Type I): a lancet applies aqueous allergen to the skin; allergen crosses into skin and binds IgE already on mast cells, triggering mediator release. This produces a wheal (raised bump) and flare (surrounding redness) within 20-30 minutes; reaction size is measured against a ruler and compared to negative/positive controls.
• Patch testing (Type IV, delayed): allergen-containing patches are applied to the back and read at 48-96 hours; a positive reaction is red, inflamed, textured (eczematous/contact dermatitis) skin at the patch site.
• Tuberculin skin test (Type IV, delayed): intradermal (Mantoux) injection produces a small bleb; read at 72 hours as a raised, indurated wheal measured against a ruler.

Cell-mediated response testing: IGRAs

• Interferon-gamma release assays (IGRAs) measure T cell-mediated response: an antigen-presenting cell processes and presents antigen to an antigen-specific T cell, which then produces IFN-gamma.
• QuantiFERON-TB Gold In-Tube assay: blood is collected into three tubes - Nil (negative control), Mitogen (positive control), and TB antigen. Tubes are incubated overnight, then an ELISA for interferon-gamma is run (read as colour development on a 96-well plate).

Determining timing of infection

• Acute and convalescent serum sampling distinguishes recent from distant infection:
  • IgM negative, IgG positive = distant infection.
  • IgM positive, IgG positive or negative = recent infection.
  • Caveat: IgM can persist or reactivate, so IgM alone is not definitive.
• Practical approach: measure early (acute) sample and again 2-3 weeks later (convalescent). Seroconversion (negative to positive) or a four-fold increase in antibody titre (e.g. titre of 8 to 32) confirms recent infection. If the acute sample is taken too late, antibody titre may already be near plateau, masking the rise.
• In a typical primary antibody response: IgM rises first, peaks at 10-14 days, then declines toward baseline; IgG rises slightly later and plateaus/persists at a lower but sustained level. On re-exposure, a second, smaller IgM peak can occur.
• IgG avidity is an alternative way to time infection:
  • Avidity = accumulated strength of multiple antibody-antigen binding interactions (versus affinity, the strength of a single interaction).
  • Somatic hypermutation in the germinal centre increases antibody affinity over time, so early infection produces low-avidity antibody and distant infection produces high-avidity antibody.
  • Binding strength (Keq) increases from a single interaction (affinity, ~10^4) to avidity from a few interactions (~10^6) to avidity from a multivalent complex with many simultaneous binding sites (~10
  • Measured using a chaotropic agent (e.g. urea): serum is added to wells with or without urea, then enzyme-conjugated secondary antibody. Urea disrupts weaker (low-avidity) antibody-antigen binding, reducing signal relative to the untreated well. The ratio gives an avidity index (example: 0.33 = low avidity, consistent with recent infection).
• Germinal centre affinity maturation (why avidity rises over time): a naive B cell enters the germinal centre and undergoes clonal expansion and somatic hypermutation in the dark zone, generating diversity (some mutations are disadvantageous and those cells become apoptotic). Cells with improved affinity move to the light zone, where they undergo selection (interacting with follicular dendritic cells and T cells) and differentiation with class switching. This process has two outcomes: (1) affinity maturation and (2) class switching. Naive/dark zone B cells express IgM and IgD; output plasma cells secrete IgA, IgG, IgE or IgD, alongside memory B cells.

Clinical case applications

• Case 1 (secondary syphilis): 28-year-old man who has sex with men, diffuse rash for 1 week, prior painless penile lesion 9 months ago (healed). Results: HIV 1+2 negative; syphilis EIA reactive; Treponema pallidum latex agglutination (TPLA) reactive; rapid plasma reagin (RPR) positive at titre 128. Diagnosis: secondary syphilis. RPR is a non-treponemal test detecting antibodies against damaged cell wall components; it correlates with disease activity and decreases with successful treatment. Treponema pallidum cannot be cultured; PCR has now replaced direct immunofluorescence for direct detection of the organism.
• Case 2 (toxoplasmosis in pregnancy): 28-year-old woman, 12 weeks pregnant, 1 week of malaise, fever, cervical lymphadenopathy; primary school teacher; 3-month-old kitten at home. Results: CMV IgM negative, CMV IgG positive; Toxoplasma IgM positive, Toxoplasma IgG positive. This IgM+/IgG+ pattern indicates recent infection (applying the timing-of-infection framework above), raising concern for recently acquired toxoplasmosis in pregnancy.
• Case 3 (systemic lupus erythematosus): 33-year-old woman, 6-month history of painful joints in hands/wrists, fatigue, proteinuria, and a malar (butterfly-shaped) facial rash sparing the nasolabial folds. Anti-nuclear antibody (ANA) testing by indirect immunofluorescence on HEp-2 cells showed a homogeneous nuclear staining pattern (suggesting antibodies against dsDNA and/or histones/nucleosomes). ELISA for anti-dsDNA antibodies measured 14 IU/mL. Interpretation: 0-4 IU/mL within normal limits; 4-8 IU/mL may be seen in other connective tissue diseases; >8 IU/mL high titres largely confined to SLE. Diagnosis: systemic lupus erythematosus.

Transcript flag

Slide 23’s clinical photo (papulosquamous rash on the trunk, Case 1) was noted only for visual severity and distribution; no further detail could be inferred from the transcript.

Self-test

1. Distinguish detection of an infectious agent from detection of the immune response, and explain why antigen detection is sometimes the only option.
2. Describe the typical timeline of viraemia/antigen, IgM, and IgG during an acute infection, and explain what this means for choosing when to test for antigen versus antibody.
3. Describe the steps of an agglutination assay set up to detect antigen.
4. Distinguish direct from indirect immunofluorescence, including which one is used to detect antigen versus antibody, and why the indirect method typically gives a stronger signal.
5. Describe the steps of a neutralization assay and how you would tell from the cell monolayer whether neutralizing antibody was present.
6. Describe the steps of an enzyme immunoassay (ELISA) designed to detect antibody in patient serum.
7. Describe the steps of a lateral flow (immunochromatographic) test, explaining the purpose of both the test line and the control line.
8. List the four types of hypersensitivity reaction with the underlying mechanism of each, and state which test is used for Type I and which for Type IV.
9. Describe what happens physiologically to produce a wheal and flare in skin prick testing, and the timeframe in which it is read.
10. Distinguish skin prick testing from patch testing in terms of hypersensitivity type, mechanism, and read time.
11. Describe the steps of an interferon-gamma release assay (IGRA), and explain the purpose of the Nil and Mitogen tubes in the QuantiFERON-TB Gold In-Tube assay.
12. A patient’s IgM is negative and IgG is positive for a given pathogen. What does this indicate about timing of infection, and what caveat limits this interpretation?
13. Explain how acute and convalescent serum sampling is used to confirm recent infection, including what result would confirm seroconversion.
14. Distinguish antibody affinity from antibody avidity, and explain how each changes over the course of an immune response.
15. Describe how IgG avidity is measured in the lab, and explain why treatment with a chaotropic agent like urea lowers the signal more for a recent infection than a distant one.
16. Describe the steps of affinity maturation in the germinal centre, from naive B cell entry to the two possible mature outputs.
17. A pregnant patient has Toxoplasma IgM positive and IgG positive. Using the timing-of-infection framework, what does this suggest, and why is this clinically significant in pregnancy?
18. A patient has a reactive syphilis EIA, reactive TPLA, and RPR positive at titre 128. Explain what type of test RPR is, what it correlates with, and how it could be used to monitor treatment response.

Answers

Reveal answers

1. Detection of the infectious agent (antigen) identifies the organism directly, while detection of the immune response identifies antibody or T cell reactivity to it. Antigen detection is sometimes the only option because many bacteria, viruses, fungi and parasites are difficult or impossible to culture, or the infection is latent.
2. Viraemia/antigen rises early in acute illness and falls sharply by about day 6 (the acute illness window), so RT-PCR/antigen tests are most useful early. IgM rises as viraemia falls, peaking around day 14, then declining by day 50. IgG rises slightly later than IgM and persists at a high level long-term. So antigen tests suit very early infection, IgM suits sub-acute/early convalescent infection, and IgG indicates past exposure/persists long-term.
3. Particles coated with antibody are mixed with patient serum/sample; if antigen is present it cross-links the antibody-coated particles, causing visible clumping (reactive); if absent the suspension stays smooth (non-reactive).
4. Direct immunofluorescence uses a fluorescently-labelled antibody that binds directly to antigen on a cell, so it detects antigen. Indirect immunofluorescence uses an unlabelled primary antibody to bind antigen, followed by a fluorescently-labelled secondary antibody that binds the primary antibody, so it detects antibody; the added secondary-antibody step amplifies the fluorescent signal.
5. Cells are exposed to virus with or without patient serum. Without neutralizing antibody, virus infects the cell freely, causing damage and plaques (cell death) in a monolayer. With neutralizing antibody, antibody coats the virus and blocks it contacting the cell, so the monolayer stays healthy with no plaques - fewer/no plaques indicates neutralizing antibody was present.
6. An antigen-coated well has serum added (patient antibody binds the antigen if present); an enzyme-conjugated anti-antibody is added, which binds the patient antibody; substrate is then added, and a colour change indicates antibody was present.
7. Sample containing analyte is added to the sample pad, where mobile gold-conjugated antibodies to the analyte are present; capillary flow carries the sample/antibody-gold complex along the strip. At the test line, analyte-antibody-gold complexes bind, producing a coloured line if analyte is present (positive result). At the control line, excess conjugate binds regardless of analyte presence, confirming the test ran correctly (valid test).
8. Type I: IgE-mediated, histamine release/degranulation (e.g. asthma, rhinitis, eczema); tested by specific IgE testing or skin prick testing. Type II: IgG-mediated cytotoxic, via antibody-dependent cellular cytotoxicity and complement activation. Type III: immune complex-mediated, free immune complexes activate complement and recruit neutrophils (e.g. food/drug allergy, anaphylaxis, urticaria). Type IV: cell-mediated, sensitized Th1 cells release cytokines activating macrophages and cytotoxic T cells (e.g. contact dermatitis); tested by patch testing.
9. A lancet introduces aqueous allergen into the skin; the allergen binds IgE already bound to mast cells, triggering mediator (e.g. histamine) release, which produces a raised wheal and surrounding red flare. It is read within 20-30 minutes.
10. Skin prick testing detects Type I (IgE-mediated) hypersensitivity via mast cell degranulation and is read within 20-30 minutes as a wheal and flare. Patch testing detects Type IV (cell-mediated, delayed) hypersensitivity via sensitized Th1 cells/macrophages and is read at 48-96 hours as localized skin inflammation.
11. An antigen-presenting cell processes and presents antigen to an antigen-specific T cell, which produces IFN-gamma if it recognises the antigen. In QuantiFERON-TB, the Nil tube is a negative control (no stimulation, establishes background), and the Mitogen tube is a positive control (non-specific stimulation, confirms the immune cells can respond); the TB antigen tube tests for a specific T cell response to TB antigens.
12. IgM negative, IgG positive indicates distant (past) infection rather than recent infection. The caveat is that IgM can persist or reactivate in some cases, so a negative IgM/positive IgG result alone is not always definitive proof of distant infection.
13. An acute serum sample is taken early, and a convalescent sample 2-3 weeks later. Seroconversion (antibody status changing from negative to positive) or a four-fold rise in antibody titre (e.g. 8 to 32) between the two samples confirms recent infection. If the acute sample is taken too late, titre may already be near plateau, masking a rise.
14. Affinity is the strength of a single interaction between one antibody-binding site and its epitope; avidity is the accumulated strength of multiple such interactions occurring simultaneously (e.g. from a multivalent antibody). Affinity/avidity is low early in infection and increases over the course of the response via somatic hypermutation, so distant infection is associated with high avidity.
15. Serum is added to wells with or without a chaotropic agent (e.g. urea), followed by enzyme-conjugated secondary antibody; signal in the urea-treated well is compared to the untreated well to calculate an avidity index. Urea disrupts weaker (low-avidity) antibody-antigen bonds more readily than strong (high-avidity) bonds, so a recent (low-avidity) infection shows a bigger drop in signal with urea, giving a lower avidity index, than a distant (high-avidity) infection.
16. A naive B cell enters the germinal centre and, in the dark zone, undergoes clonal expansion and somatic hypermutation, generating diversity (cells with disadvantageous mutations become apoptotic). Cells with improved affinity move to the light zone, where they undergo selection (via interaction with follicular dendritic cells and T cells) and differentiation with class switching. The two outcomes are affinity maturation and class switching, producing plasma cells secreting IgA, IgG, IgE or IgD, and memory B cells.
17. Toxoplasma IgM positive with IgG positive fits the “recent infection” pattern from the timing-of-infection framework, suggesting the infection may have been recently acquired. This is clinically significant in pregnancy because recently acquired toxoplasmosis carries risk of transmission to the fetus, unlike a distant (IgM negative, IgG positive) infection.
18. RPR is a non-treponemal test detecting antibody against damaged cell wall components (not against Treponema pallidum itself). Its titre correlates with disease activity and decreases with successful treatment, so serial RPR titres can be used to monitor treatment response, alongside treponemal-specific tests (EIA, TPLA) which confirm the diagnosis but do not track activity in the same way.