A lateral-flow assay (LFA) for β-2-Transferrin (β2T) was developed (Figure 1). In the assay, the target protein binds to the capture antibodies at the test line producing a visible readout. All antibody deposition was completed by reagent applicator to maximize signal localization. We found a direct LFA β2T concentration of 0.16 mg/mL produced a visible positive line in an assay that involved the direct deposition of β-2-transferrin onto the nitrocellulose-based lateral flow device. This indicated the successful capture of antibody and colorimetric indicator. No control lines were utilized in preliminary devices. LFAs were initially designed for readout by antibody conjugation to streptavidin bound gold nanoparticles, a common technique used in many commercial lateral flow assays, including the at-home pregnancy test. However, in the limited time allotted with a capillary flow-driven device, the conjugation of the anti-β2T detection antibody to the streptavidin bound gold nanoparticles was not successful. After multiple tests confirmed the same result, the LFAs were transitioned to visualization by HRP tagged avidin. With HRP-avidin tagged LFAs, the readout produced a positive signal, but the readout was not well-localized to a single line as was initially intended due to the need for a colorimetric indicator (TMB). Direct β2T assay results demonstrated the ability to bind β-2-transferrin to HRP-conjugated antibodies and produce a qualitative readout (Figure 2). This result can be interpreted without the need for expensive in-lab equipment. Later experiments used in-vitro ELISAs to analyze the ability of β2T to bind in a sandwich with anti-β2T and more general anti-transferrin antibodies. These preliminary assays worked towards the eventual goal of using β2T as a capture analyte on a flow-through assay. Further work on this project will involve optimization of a β2T sandwich lateral or flow-through assay for clinical use.
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