荧光免疫层析法汗液CRP快速检测试纸条的研发及不同保存条件对汗液CRP的影响

顾芳艳; 胡琛光; 李锦; 彭薇; 檀旭东; 桑培培; 周红辉; 田亚平; 张蔓丽

doi:10.12435/j.issn.2095-5227.2023.094

荧光免疫层析法汗液CRP快速检测试纸条的研发及不同保存条件对汗液CRP的影响

Development of a fluorescent immunochromatographic test strip for rapid detection of CRP in sweat and effect of different storage conditions on sweat CRP

摘要

摘要:
背景研究发现人体汗液C反应蛋白(C-reactive protein，CRP)与炎症有一定关联，但国内外未见关于汗液CRP快速检测和保存条件的研究。
目的利用荧光免疫层析技术研发一种快速半定量检测汗液CRP含量的试纸条并探讨人体汗液不同保存条件下CRP的变化。
方法利用双抗体夹心法研发汗液CRP试纸；于2021年5月24日7：00 - 16：00收集54例青年男性野外徒步50 km状态下的汗液，利用试纸条检测汗液CRP浓度并确定正常参考范围。取其中10例CRP相对高值汗液样本，设置不同保存温度和时间分别检测汗液CRP浓度变化。
结果 CRP抗原标准品和T/C值的标准曲线方程为Y=(7.5362 + 0.0105)/1 + (X/2186.5735)^-0.7549-0.0105，R²值为0.9999；空白限(LoB)为1.94 ng/mL；检出限(LoD)为5.94 ng/mL；用相对偏差的绝对值验证准确度，结果均＜10%；该测定结果与血清淀粉样蛋白A和降钙素原的交叉反应率分别为0和0.01%；重复性和实验室内精密度均＜10%；目标青年男性人群汗液CRP正常参考范围为≤25 ng/mL；不同温度(4℃、25℃、37℃)条件下汗液保存1 h，CRP检测结果差异无统计学意义；保存3 h后，4℃和25℃条件下CRP浓度显著下降，37℃条件下浓度变化不显著。
结论荧光免疫层析法快速半定量检测人汗液CRP的试纸条具有较好的准确度、特异性、重复性、实验室内精密度，应扩大样本量进一步研究，未来或可用于人汗液CRP含量快速检测。

Abstract:
Background Previous studies have found a certain association between C-reactive protein (CRP) in human sweat and inflammation, but there is no research on rapid detection and storage conditions for CRP in sweat both domestically and internationally.
Objective To develop a rapid semi-quantitative test strip for detecting CRP content in sweat using fluorescence immunochromatography technology and explore the changes in CRP in human sweat under different storage conditions.
Methods The sweat CRP test paper was developed by double antibody sandwich method. On May 24, 2021, from 07:00 to 16:00, sweat samples from 54 young men who were hiking for 50 kilometers in the field were collected. The sweat CRP concentration was measured using test strips and the normal reference range was determined. Taking 10 sweat samples with relatively high CRP values, and different storage temperatures and time were set to detect changes in sweat CRP concentration.
Results The standard curve equation for CRP antigen standard and T/C value was Y=(7.5362 + 0.0105)/1 + (X/2186.5735)^-0.7549 -0.0105, and the R² value was 0.9999. The blank limit (LoB) was 1.94ng/mL, and the detection limit (LoD) was 5.94ng/mL. The absolute value of relative deviation was used to verify the accuracy, and the results were all less than 10%. The cross-reactivity of the results with serum amyloid protein A (SAA) and procalcitonin (PCT) was 0.00% and 0.01%, respectively, and the repeatability and precision in the laboratory were ＜10%. The normal reference range of sweat CRP of target population was ≤ 25 ng/mL. When sweat was stored at different temperatures (4℃, 25℃, 37℃) for 1 hour, there was no significant difference in CRP test. After 3 hours, the concentration of CRP decreased significantly at 4℃ and 25℃, while at 37℃, the concentration change was not significant.
Conclusion The fluorescent immunochromatography test strip for rapid semi quantitative detection of human sweat CRP has good accuracy, specificity, repeatability, and laboratory precision, which could be used for rapid detection of human sweat CRP content in the future with further study and lager sample size.

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