(J-381) Lateral Flow Depletion ELISA for Antibody Titer Measurement

Introduction

Serological tests measure antibody expressions in bodily fluids induced against pathogenic agents [1, 2]. The serological tests are relevant for a variety of applications including serosurveys, screening of potential convalescent donors, vaccine efficacy evaluation, and immunity assessment from reinfection [1, 2]. ELISA is the gold standard method for reliably quantifying antibody expressions [2, 3]. In ELISA, the antibody titer is measured on a sample serially diluted across a well plate. The titer is determined by the well where the signal falls below a reference threshold, i.e., the amount of dilution required on the original sample to drop to an undetectable level. However, ELISA is a labor-intensive assay involving multiple steps conventionally performed by trained staff in laboratories [4, 5].

In this work, we report a paper-imprinted device that automatically performs a multi-well ELISA assay for titer measurements. Instead of serial dilution and manual dispensing of the sample, our approach relies on gradual dilution of the target analyte as it is depleted from the sample by immobilized capture elements during flow to create a concentration gradient. To automate the sequence and timing of applying different reagents used to perform ELISA, we equipped the device with a paper-based flow [6, 7] that eliminated the need for manual supervision of the assay. To demonstrate practical applications, we created the devices to measure severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody titers with the device only requiring the sample and ELISA reagents to be loaded for operation.

Materials and Methods

Our device design focused on two main tasks: (1) serially diluting the target analyte and distributing it to isolated wells and (2) delivering reagents in a programmed manner with preset incubation times for an optimal ELISA. We aimed to decrease the concentration of target analyte in the sample by gradually depleting it through immunocapture. This in-situ process is referred to as depletion ELISA. To create an immunodepletion path for the analyte, our device uses vertically-stacked paper layers, each functionalized with a relevant antigen (Figure 1a). In operation, the sample applied on the top layer is wicked towards the bottom layer and the analyte is gradually depleted by immunocapture leaving the analyte at a lower concentration for the subsequent layer. The presence of immunocaptured antibodies on layers is transduced into an optical signal by ELISA (Figure 1b). The antibody titer can then be determined by counting the number of colored.

In line with our depletion ELISA strategy, we created a paper-based 96-well sheet by simply patterning cellulose paper with water-insoluble ink (Figure 1c). To enable automated ELISA operations on the device, we employed a paper-based flow controller that delivers reagents to the wells in a programmed sequence (Figure 1d). The flow controller utilized timer that rely on the delamination of wetted paper from a laminating tape [6, 7]. To integrate the 96-well sheet with the flow controller, the sheet was folded and sandwiched between the flow controller and a blotting paper, and then plugged into the device case (Figure 1e).

Results and Discussion

We used recombinant SARS-CoV-2 nucleocapsid (N) protein as the antigen and recombinant SARS-CoV-2 nucleocapsid IgG antibody (IgG antibody) as the target antibody as a model pair. To associate the output of our assay, i.e., the number of colored wells with specific antibody titers, we compared our results with those from conventional ELISA assay performed on matched samples.

In testing our device, a 1:19683 titer sample (810 μg/ml in PBS) was first serially diluted 3-fold from 1:19683 to 1:27 titer, and 50 μl aliquots were deposited on different wells along with negative control (NC). The number of colored wells in each row on the unfolded well sheet was determined in independent experiments (n=3) by measuring color intensities with digital image processing. The number of the colored wells, 10 for the 1:19683 titer sample, was found to decrease by one in each row of 3x diluted antibody titer with the sample of the lowest titer tested (1:27) producing only 4 colored wells (Figure 2a). All of these results from our depletion ELISA were consistent with the conventional ELISA performed on a plastic 96-well plate by manually 3-fold diluting the identical samples (Figure 2b). Next, we attempted to verify the observed antibody dilution factor in our assay by estimating the dilution factor directly from the measured intensity of optical signals. Based on the optical signal amplitude from a well, we calculated the effective concentration of IgG in that well for the ELISA reaction by employing the Hill equation as [8]. We found the computed well-to-well dilution factors were on average ~ 3.07-fold (STD = 0.21) (Table S1).

Conclusions

ELISA is widely accepted as the gold standard assay for reliable measurement of antibody titers. We developed the depletion ELISA device to effortlessly perform this well-established yet intrinsically-complex assay as a dipstick test. Importantly, our assay integrates serial sample dilution and automated manipulation of different reagents, all on a disposable paper substrate, and provides results in a well plate format. Given ELISA’s current popularity in its laborious form, the ability to self-test using dipstick ELISA tests can be transformative in the decentralized delivery of healthcare.  

References

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Acknowledgments

This work was supported by the start-up funds provided to A.F.S. by Georgia Institute of Technology, School of Electrical and Computer Engineering.