CARDIAC HIGH-THROUGHPUT ASSAY TECHNOLOGIES
Comparison of fluorescent and chemiluminescent immunoassay technologies used in high-throughput cardiac biomarker testing
FEATURE
FIA | Fluorescence Immunoassay
CLIA | Chemiluminescent Immunoassay Enzyme-labeled antibodies catalyze light-emitting reactions measured without external excitation
Fluorophore-labeled antibodies emit light upon optical excitation; signal intensity proportional to analyte concentration
DETECTION PRINCIPLE
Fluorescence emission
Chemiluminescence emission
SIGNAL TYPE
Typically picogram per milliliter (pg/mL) range; may be limited by background fluorescence or optical noise Determined by antibody affinity & assay design; generally comparable to CLIA Used in certain high-sensitivity & electro- chemiluminescent (ECL) platforms; including Roche Cobas analyzers
Often femtogram per milliliter (fg/ mL) range; high signal amplification & broader dynamic range
DETECTION SENSITIVITY
Also antibody-dependent; comparable specificity to FIA
SPECIFICITY
Standard in automated high-throughput systems such as Abbott Architect, Siemens ADVIA Centaur, Roche Cobas e-Series, Beckman Coulter Access Very high sensitivity, wide dynamic range, optimized for continuous, high-volume testing
COMMON APPLICATIONS
High sensitivity, rapid detection, suitable for compact analyzers
PERFORMANCE SUMMARY
EVALUATION METHOD
Antibody pairs specific to cTnI , CRP , NT-proBNP , and CK-MB were evaluated in a two-step sandwich format using either a fluorescence immunoassay (FIA) or a chemiluminescent immunoassay (CLIA). In FIA, capture antibodies were coated on a nitrocellulose (NC) membrane and detection antibodies were conjugated to Europium. In CLIA, capture antibodies were coated on magnetic beads and detection antibodies were conjugated to alkaline phosphatase (ALP). The limit of detection (LOD) was determined for each antibody pair.
TWO-STEP SANDWICH ASSAY
DETECTION ANTIBODY CONJUGATE FIA | Ab-Europium Conjugate CLIA | Ab-AKP Conjugate
ADD SAMPLE
ADD DETECTOR
ANTIGEN
FIA | Coated on NC Membrane CLIA | Coated on Magnetic Bead CAPTURE ANTIBODY
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