Lateral Flow Assay Enhanced for Multiplex Detection

Lateral flow assays (LFAs) are popular point-of-care diagnostic tools because they are rapid and easy to use. However, LFAs often lack analytical sensitivity and quantitative output and may be difficult to multiplex, limiting their usefulness in biomarker measurement.

Scientists at SRI International (Menlo Park, CA, USA) designed a quantitative, multiplex LFA with readily available near-infrared (NIR) detection to improve analytical sensitivity. They constructed and optimized a multiplex LFA that simultaneously and accurately measured interleukin (IL)-6 and C- reactive protein (CRP) in 10% human plasma on one test strip.


Commercially available antibodies were conjugated with the fluorescent dye IRDye 800CW (Li-Cor; Lincoln, NE, USA). The investigators used singleplex, optimized NIR-LFAs to measure IL-6 from 0 to 200 pg/mL and developed duplex assays to simultaneously measure IL-6 from 0 to 100 pg/mL (0 to 4.5 pmol/L) and CRP from 50 to 2,500 ng/mL (0.4 to 20 nmol/L) on a single test strip. Assays were tested on 60 different spiked samples and compared to enzyme-linked immunosorbent assay (ELISA) results. The intensity ratios for unknown samples were compared to the calibration curve to determine analyte concentrations and samples were run in quadruplicate.

The choice of an inhibition-style assay rather than a sandwich-style assay avoided the complications due to a potential hook effect at high CRP concentrations. With this approach, free CRP present in the sample inhibited binding of the dye-conjugated antibody to CRP striped on the membrane. As the concentration of CRP in the sample increases, the signal directly decreases. The NIR-LFAs detected IL-6 in a 10% plasma matrix with a limit of detection of 4 pg/mL (182 fmol/L). The duplex NIR-LFAs quantitatively measured IL-6 and CRP concentrations simultaneously. The values strongly correlated to ELISA measurements, with linear regression R2 values of 0.9825 and 0.9711 for IL-6 and CRP, respectively.

The authors concluded that NIR-LFAs exhibit quantitative measurement at pg/mL concentrations owing to a high signal-to-background ratio and robust detection antibody clearance through the test strip. Moreover, NIR-LFAs are able to detect molecules present at vastly different concentrations in multiplex format and compare favorably to ELISAs. LFAs with direct NIR detection may be a valuable tool for biomarker evaluation in the point-of-care setting. The study was published on January 30, 2013, in the journal Clinical Chemistry.

Related Links:
SRI International
Li-Cor