Clinical Immunology and Serology: Assays and Autoantibody Patterns
Serology questions test both method principles and the interpretation of antibody patterns. You need to know the difference between a sensitive screening assay and a specific confirmatory one, and how interference produces false positives.
Core rules
- Sandwich ELISA: capture antibody binds antigen, an enzyme-labelled second antibody detects it; high-dose hook effect occurs with very high antigen concentrations and produces a falsely low or negative result, requiring dilution.
- Indirect immunofluorescence for ANA: report titre (for example 1:320) and pattern. Homogeneous pattern suggests anti-dsDNA or anti-histone; speckled suggests anti-ENA such as Sm, RNP, SSA/SSB; nucleolar suggests scleroderma; centromere suggests limited cutaneous scleroderma.
- Nephelometry and turbidimetry measure immunoglobulin and complement concentrations by light scatter or absorption in antigen excess-controlled conditions; rheumatoid factor interferes with nephelometric assays.
- Complement: C3 and C4 are consumed in active SLE and cryoglobulinaemia; they are useful for monitoring activity.
- Syphilis testing uses the reverse algorithm: a treponemal EIA is the screen, and a reactive result is followed by RPR (a marker of activity) and a second treponemal test such as TPPA for confirmation.
Worked example
A 55-year-old woman with dry eyes and dry mouth has ANA 1:320 speckled, anti-dsDNA negative, anti-Sm negative, anti-SSA strongly positive and anti-SSB positive. Reasoning: the speckled pattern reflects antibodies to extractable nuclear antigens; the specific identity comes from the individual assays, not the pattern. SSA and SSB with sicca symptoms fit Sjogren syndrome. Anti-SSA also crosses the placenta and is associated with congenital heart block, so the finding has an obstetrical implication, and a high-titre ANA without specific antibodies should not be used alone to diagnose lupus.
Traps that cost marks
- Prozone phenomenon in agglutination tests: antibody excess blocks lattice formation and gives a false negative; dilute and repeat.
- Heterophile (Monospot) tests are false positive in lymphoma, hepatitis and with rheumatoid factor.
- A positive screen does not equal disease; predictive value depends on prevalence, and the examination frequently asks you to apply sensitivity and specificity logic.
- IgM antibody may persist for months to years, so serology cannot reliably date an infection; paired sera showing a fourfold rise or a documented seroconversion is required.
- Serum separator tubes are acceptable for most serology, but complement assays require EDTA or heparin plasma shipped on ice.