A lateral flow protein microarray for rapid and sensitive antibody assays

Abstract

Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (<30 ng/mL) determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.

Keywords: lateral flow; point of care; protein microarray.

Figure 1

(A) Three cardboard-backed nitrocellulose strips attached to a glass array slide. Two arrays were printed per strip, but only the data from the upstream array was used in this study; (B) Here, an anti-HisABP antibody was used to reveal the pattern of the printed antigens which all had been recombinantly fused with a His₆ABP-tag.

Figure 2

(A) Lateral flow and glass microarray results from the analysis of 26 antibody samples are compared. Green dots (in the top right quadrant) indicate concordant positive signals, black (bottom left quadrant) are concordant negative signals and blue dots (top left and bottom right quadrants) represent positive signals that only arose on one platform. All expected antibodies were detected by the correct antigen spot on both platforms. The Pearson correlation coefficient for the dataset was 0.74. Only signals higher than the mean for each array are shown; (B) A receiver-operator-characteristics analysis was performed, employing the vertical threshold for the glass array signals indicated in (A). The area under the curve (AUC) was found to be 98%, suggesting a very good binary classification concordance between the methods.

Figure 3

Dilution series of two antibodies, HPA Ab 9 and HPA Ab 22. The antibodies were diluted 1:100, 1:1000, 1:10.000 and 1:100.000 and the signal from the expected positive antigen spot was measured. It was found that a linear relation between the intensity and the log of the concentration was found between dilutions 1:100 and 1:10.000. As no significant signals were found at dilutions 1:100.000, it is suggested that the sensitivity may reside at or lower than around 30 ng/mL for the lateral flow analysis of the two antibodies.

Figure 4

Four antibody mixes were prepared, two containing two antibodies, one containing four antibodies, and one containing eight antibodies. In all cases, the signal/noise on the expected antigen (Ag) spots were distinctly higher than the highest unexpected antigen signal on the same array, although the S/N decreased with increased number of antibodies in the mix.