Chemiluminescence immunoassay (CLIA)

Principle

• An immunoassay in which the label produces light through a chemical reaction; no excitation light source is needed, so background is low and sensitivity high.
• Common labels: acridinium esters (flash of light when alkaline hydrogen peroxide is added); luminol or isoluminol derivatives oxidised with a catalyst such as horseradish peroxidase (enhanced chemiluminescence); alkaline phosphatase acting on dioxetane substrates (glow signal).
• Electrochemiluminescence (ECL) uses a ruthenium complex that emits light at an electrode surface with tripropylamine.
• Light is measured by a photomultiplier tube as relative light units (RLU).

Formats

• Sandwich (immunometric) for large molecules (TSH, hCG, troponin, ferritin, tumour markers): signal is directly proportional to analyte.
• Competitive for small molecules (free T4, cortisol, drugs, steroids): signal is inversely proportional to analyte.
• Paramagnetic microparticles are usually the solid phase; unbound material is removed by magnetic washing.

Specimen

• Serum or plasma per assay; avoid gross haemolysis, lipaemia and fibrin clots.

Quality control and pitfalls

• Biotin supplements interfere with streptavidin–biotin assays: falsely low in sandwich assays and falsely high in competitive assays.
• Heterophile antibodies and human anti-mouse antibodies (HAMA) can bridge capture and detection antibodies, usually giving falsely high sandwich results.
• High-dose hook effect in sandwich assays (e.g. very high hCG or prolactin) gives falsely low values; repeat on dilution.
• Macro-forms (e.g. macroprolactin) may cause misleading results.
• Run multilevel controls; verify calibration after reagent lot changes.

Clinical use

• Thyroid function, fertility hormones, cardiac markers, tumour markers, vitamin D, infectious disease serology (HBsAg, anti-HIV, anti-HCV).