研究目的
To develop and optimize a fluorescence-based lateral flow immunoassay for detecting antibodies in plasma, specifically targeting EBNA-1 and HPV biomarkers, with a focus on improving sensitivity, reducing costs, and ensuring stability under storage conditions for use in low and middle-income countries.
研究成果
The optimized LFA using PBST (0.5% Tween-20) as washing buffer improved signal-to-noise ratio without the need for blocking, reducing costs and production time. Strips stored at room temperature maintained detectable signals for up to three weeks, challenging the necessity of cold storage. These findings support the development of cost-effective, portable diagnostics for low and middle-income countries, with future work needed on long-term stability and adaptation to HPV biomarkers.
研究不足
The study is limited to fluorescence-based serological LFAs; results may not generalize to other assay types. The sample size for HPV testing was small due to limited access to HPV plasma samples. Long-term storage effects beyond three weeks were not investigated, and temperature variations in real-world settings were not fully explored. The assay's performance might be affected by batch-to-batch variability in materials.
1：Experimental Design and Method Selection:
The study designed a fluorescence-based lateral flow immunoassay (LFA) to detect antibodies in plasma samples. It involved optimizing washing and blocking conditions to enhance sensitivity and reproducibility. Theoretical models included immunoassay principles and capillary flow dynamics. Methods included covalent conjugation of fluorescent microspheres to antibodies, assembly of LFA strips, and performance of assays with various buffers and conditions.
2：Sample Selection and Data Sources:
Plasma samples were purchased from Blood Centers of the Pacific, validated as seropositive for EBNA-1 antibodies using ELISA. Samples were de-identified and approved by the institutional review board.
3：List of Experimental Equipment and Materials:
Materials included EBNA-1 protein, PBS, BlockAid, TBS, glass slides, Tween-20, EDC, Sulfo-NHS, F1Y050 fluorescent microspheres, goat anti-human IgG, nitrocellulose membrane (Hi-Flow Plus HF07502XSS), and various buffers. Equipment included a laser cutter for cutting nitrocellulose, a Nordson EFD Ultimus V Multipurpose fluid dispenser for protein dispensing, a hot plate for drying, and a custom-built fluorescent reader for detection.
4：Experimental Procedures and Operational Workflow:
Steps included conjugating fluorescent microspheres to antibodies using EDC/Sulfo-NHS, assembling LFA strips by laser-cutting nitrocellulose and mounting on glass slides, dispensing proteins for test and control lines, applying plasma samples and washing buffers, adding conjugated microspheres, washing again, drying strips, and reading results with the fluorescent reader. Assays were performed with different washing buffers (PBST and TBST), Tween-20 concentrations, and blocking conditions using BSA. Storage tests involved sealing strips with desiccants and storing at room temperature or 4°C, with weekly assays.
5：Data Analysis Methods:
The fluorescent reader detected signals as photocurrent, converted to voltage output. Signal intensity was measured by the time to reach an 8 V threshold, with smaller times indicating higher fluorescence. Signals were normalized to negative control (BSA) sites, and signal-to-noise ratios were calculated.
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