Combining sandwich and competitive immunoassay formats in a duplex lateral flow test for rapid iron-status assessment.

BACKGROUND: Reliable assessment of iron status benefits from the combined measurement of complementary biomarkers, but implementation in rapid lateral flow formats is complicated by large differences in biomarker concentration ranges and assay behavior. Soluble transferrin receptor (sTfR) occurs at substantially higher physiological concentrations than ferritin, creating a risk of high-dose hook effects in conventional sandwich lateral flow assays (LFAs).

RESULTS: Here, we report a mixed-format duplex LFA for the simultaneous quantitative measurement of ferritin and sTfR in human plasma. The assay integrates a sandwich immunoassay for ferritin with an indirect competitive immunoassay for sTfR on a single test strip. Quantitative readout was achieved using spectrally distinct upconverting nanoparticle reporters and spatially separated test lines. The finalized duplex assay showed limits of detection of 0.25 ng/mL for ferritin and 0.1 μg/mL for sTfR and enabled quantitative measurement across broad functional dynamic ranges. The assay was completed in approximately 12 min and was evaluated using lithium heparin plasma samples from healthy adult donors. Results showed analytical compatibility with routine central laboratory reference methods for both biomarkers.

SIGNIFICANCE: These findings demonstrate that combining competitive and non-competitive immunoassay formats within one lateral flow device can overcome dynamic-range and hook-effect limitations in multiplexed point-of-care testing. The mixed-format duplex assay provides a rapid quantitative approach for iron status assessment, with potential applications in blood donor screening and testing in settings with limited laboratory infrastructure.