Current methods for antibody quantification present limitations. ELISA, while highly accurate and multiplexable, is slow, cumbersome, and requires specialized equipment and personnel. Lateral flow immunoassays are faster and simpler but are qualitative and have limited multiplexing capabilities. This research aims to develop a new technology that combines the advantages of both methods. E-DNA scaffold sensors offer a reagentless, single-step electrochemical approach for antibody quantification. They are based on a DNA scaffold attached to an electrode, presenting an antibody-binding epitope. Antibody binding hinders electron transfer, providing a quantifiable signal. Prior work demonstrated the analytical performance using monoclonal antibodies, but this study extends the research to authentic human serum samples to assess clinical potential.

The introduction cites ELISA as the gold standard for antibody quantification, highlighting its drawbacks. Lateral flow immunoassays are presented as the point-of-care alternative, acknowledging their limitations in terms of quantitative results and multiplexing. The paper reviews existing literature on the immunogenicity of different gp41 epitopes, informing the selection of epitopes for the E-DNA sensors. The authors cite previous research characterizing the analytical performance of E-DNA scaffold sensors using purified monoclonal antibodies and exploring the size limitations of antigens presented on the platform. However, the clinical performance with real-world samples had not been previously explored.

The study used 15 anonymized human serum samples (10 HIV-positive, 5 HIV-negative) to compare E-DNA sensors with commercial ELISA and lateral flow immunoassays. Three gp41 epitopes (4B3, 2F5, 4E10) with varying immunogenicity were selected. A novel ELISA mimicking the E-DNA sensor architecture was used to validate the epitopes' immunogenicity. E-DNA sensors were developed for each epitope, and their performance was assessed using the patient samples. The ELISA results were quantified using absorbance at 450 nm, and lateral flow assays were analyzed using ImageJ. The E-DNA sensor signal was measured via square wave voltammetry. Analytical performance was compared by titrating E-DNA sensors with varying concentrations of HIV-positive serum, while maintaining constant overall serum concentration. Receiving operator characteristic (ROC) curves were generated to compare the clinical performance of the different methods.

Both commercial ELISA and lateral flow immunoassays achieved 90% sensitivity and 100% specificity in the test set, with one HIV-positive sample incorrectly identified as negative by both methods. The novel ELISA confirmed the expected immunogenicity of the three gp41 epitopes: 4B3 (high), 2F5 (low), 4E10 (near zero). E-DNA sensors using the 4B3 epitope showed results matching the commercial tests and the novel ELISA, correctly identifying nine of ten HIV-positive samples. The E-DNA sensor with the 4E10 epitope showed no significant signal change, as expected. The E-DNA sensor with the 2F5 epitope showed reduced sensitivity compared to ELISA at the tested serum dilution (1:80), failing to detect anti-2F5 antibodies in two samples positive by ELISA. Analytical sensitivity comparison showed that ELISA had better detection limits due to enzymatic amplification and wash steps, but the E-DNA sensors still provided clinically relevant results. ROC analysis showed that the E-DNA sensor's performance fell between ELISA and lateral flow immunoassays (ROC area: ELISA 0.96, E-DNA scaffold sensor 0.92, lateral flow immunoassay 0.90).

The study demonstrates that E-DNA scaffold sensors offer comparable clinical sensitivity and specificity to established methods for HIV antibody detection. While ELISA exhibits superior analytical sensitivity, its time and resource requirements make it unsuitable for point-of-care applications. E-DNA sensors offer a more practical alternative, combining quantitative results with speed and ease of use. The improved speed of diagnosis provided by E-DNA sensors could significantly impact the timely initiation of treatment for sexually transmitted diseases, particularly given the challenges in ensuring follow-up visits. The lower sensitivity of E-DNA sensor for the 2F5 epitope at the tested serum dilution may be improved by optimizing the assay conditions or increasing the sample concentration.

E-DNA scaffold sensors provide a promising point-of-care diagnostic tool for HIV antibodies, achieving comparable clinical performance to established methods while offering significant advantages in speed and simplicity. Future research could focus on optimizing sensor design to improve sensitivity for low-immunogenicity epitopes and explore its application for other disease biomarkers.

The study's sample size was relatively small, limiting the generalizability of the findings. The discrepancy in sensitivity between ELISA and E-DNA sensor for the 2F5 epitope requires further investigation. The study focused solely on HIV antibodies, and further research is needed to evaluate the platform's performance with other biomarkers.