基于化学交联法固定抗体的新型纸基酶联免疫吸附法的研究

Establishment of a novel paper-based enzyme-linked immunosorbent assay using chemical crosslinking of antibody

目的建立高灵敏度的新型纸基酶联免疫吸附测定法(paper-based ELISA,p-ELISA)。方法采用聚二甲基硅氧烷(polydimethylsiloxane,PDMS)印章法制备纸基微孔板,并对纸基微孔表面结构进行表征电镜扫描。用高碘酸钠氧化剂将纸基微孔表面羟基氧化为醛基,与壳聚糖的氨基共价结合形成席夫碱,修饰于纸基微孔表面,随后包被抗体通过EDC/Sulfo-NHS化学交联方式固定于修饰后的纸基微孔表面,建立新型p-ELISA。以检测甲胎蛋白(Alpha-fetoprotein,AFP)为例,与商业化ELISA进行比较,考察新型p-ELISA的分析性能,确认方法的可行性。结果PDMS印章法制备纸基微孔板大小均一性好且无渗漏现象。基于化学交联法固定抗体的新型p-ELISA线性范围为0.08~20.00 ng/mL,最低检测限为0.035 ng/mL,测定血清中AFP的回收率为96.30%~104.00%,批内变异系数(coefficient of variation,CV)为7.66%~8.68%,批间CV为7.78%~10.49%。新型p-ELISA与商业化ELISA同时检测不同浓度AFP样本,检测结果差异无统计学意义(P>0.05)。结论成功建立了一种低成本、准确和高灵敏度的新型p-ELISA,可为临床疾病诊断提供简单、有效的检测。

Objective To establish and evaluate a new paper-based enzyme-linked immunosorbent assay(paper-based ELISA) with high sensitivity. Methods Polydimethylsiloxane(PDMS) seal method was used to prepare paper based microporous plates, and the surface structure of paper based microporous plates was characterized by scanning electron microscopy. Oxidized the hydroxyl group on the surface of the paper micropores to the aldehyde group with sodium periodate oxidant and linked it to amino group of the chitosan covalently to form a Schiff base. Then the antibody was fixed to the surface of the modified paper micropores through method EDC/Sulfo-NHS to establish a novel paper based P-ELISA. In this study, AFP was taken as an example to compare with commercial ELISA, and the analytical ability of the new paper-based ELISA was investigated to verify the feasibility of the method. Results The size uniformity of the paper-based microporous plates prepared by PDMS seal method was good and there was no leakage. The linear range of the novel paper-based ELISA method based on the chemical cross-linking for fixing antibody was 0.08 to 20.00 ng/mL, and the minimum detection limit was 0.035 ng/mL. The recoveries of alpha-fetoprotein in serum were 96.30% to 104.00%, the coefficient of variation in the experiment was 7.66% to 8.68%, and the coefficient of variation between experiments range from 7.78% to 10.49%. AFP samples were detected by the new P-ELISA method and the commercial ELISA method at the same time, and there was no significant difference in AFP detection results between the two methods(P>0.05). Conclusion In this study, we established a low-cost, accurate and highly sensitive paper-based ELISA assay successfully, which provides a simple and effective detection method for clinical disease diagnosis.