Rapid Antibody Selection Using Surface Plasmon Resonance for High-Speed and Sensitive Hazelnut Lateral Flow Prototypes

Abstract

Lateral Flow Immunoassays (LFIAs) allow for rapid, low-cost, screening of many biomolecules such as food allergens. Despite being classified as rapid tests, many LFIAs take 10–20 min to complete. For a really high-speed LFIA, it is necessary to assess antibody association kinetics. By using a label-free optical technique such as Surface Plasmon Resonance (SPR), it is possible to screen crude monoclonal antibody (mAb) preparations for their association rates against a target. Herein, we describe an SPR-based method for screening and selecting crude anti-hazelnut antibodies based on their relative association rates, cross reactivity and sandwich pairing capabilities, for subsequent application in a rapid ligand binding assay. Thanks to the SPR selection process, only the fast mAb (F-50-6B12) and the slow (S-50-5H9) mAb needed purification for labelling with carbon nanoparticles to exploit high-speed LFIA prototypes. The kinetics observed in SPR were reflected in LFIA, with the test line appearing within 30 s, almost two times faster when F-50-6B12 was used, compared with S-50-5H9. Additionally, the LFIAs have demonstrated their future applicability to real life samples by detecting hazelnut in the sub-ppm range in a cookie matrix. Finally, these LFIAs not only provide a qualitative result when read visually, but also generate semi-quantitative data when exploiting freely downloadable smartphone apps.

A Critical Comparison between Flow-through and Lateral Flow Immunoassay Formats for Visual and Smartphone-Based Multiplex Allergen Detection

“…However, these visual readings are performed by a trained person, and the distinction between signal and no signal at the lowest concentrations is not trivial. In comparison, when the same anti-hazelnut antibody was applied in a single-plex LFIA, an LOD of 0.1 ppm in spiked buffer was reached, which suggests that having an additional test line on the LFIA can compromise the overall sensitivity [30]. Still, the multiplex LODs are in accordance with commercially available allergen single-plex LFIAs, which report LODs within this range.…”

“…Following the addition of the CNP-mAbs to the passive flow-through assay, the positive spots appeared within 5 s, a detection speed which is unparalleled by LFIA. Even when using the high-speed LFIA described in [30] the appearance of the positive result took 30 s, due to MTL limitations of the solution that needs to wick through the membrane before reaching test lines. Three drops of RB were added to the flow-through assay to wash the unbound CNPs from the membrane.…”

“…But for HPC, the hook-effect was greater in 25:75 compared with PHC with concentrations of 100 and 50 ppm experiencing reduced intensity on both the control and the peanut lines, complicating quantitative analysis. The larger hook-effect in this configuration could be because the upstream (hazelnut) test line comes into contact with the sample first, and this mAb has a rapid association rate and high affinity for THP, and so it becomes quickly saturated [30].…”

“…Most often, smartphone analysis is based on specific apps which relate a particular color intensity to a certain concentration of analyte. In the absence of a specific app, it has been shown by Ross et al [30] that it is possible to use freely downloadable apps from the Google Play Store to analyze endpoint, smartphone image color intensity values. By converting RGB values to LAB values, luminosity or intensity can be plotted as a function of concentration in a calibration curve.…”

“…Antibodies can be selected for their binding kinetics by in depth surface plasmon resonance (SPR)-based antibody screening and characterization. In this way a carbon nanoparticle-based hazelnut allergen LFIA has been developed, with a 30 s assay time, which as far as we know is a world record for allergen assay speed [30].…”