开发一种用于检测小分子氨基脲的超灵敏夹心免疫分析法。

Development of an ultrasensitive sandwich immunoassay for detecting small molecule semicarbazide.

机构信息

Shenzhen Academy of Metrology and Quality Inspection, Shenzhen, PR China.

Department of Anesthesiology, Shenzhen University General Hospital, Shenzhen University, Shenzhen, PR China.

出版信息

Food Chem. 2023 Dec 15;429:136835. doi: 10.1016/j.foodchem.2023.136835. Epub 2023 Jul 13.

DOI:10.1016/j.foodchem.2023.136835

PMID:37463535

Abstract

Ultrasensitive sandwich immunoassays for detecting the small molecule semicarbazide (SEM) were developed based on derivatization. Several SEM derivatizing agents were synthesized by linking o-nitrobenzaldehyde (NBA) and biotin with dihydroxyalkanes (different lengths), which were then used to evaluate the distance effect of two epitopes. Sandwich ELISA for SEM derivatives was developed using an anti-SEM-NBA antibody and horseradish peroxidase-labeled avidin or anti-biotin antibody as a secondary conjugate. The advantageous distances of the two epitopes under the double-antibody sandwich and antibody-avidin sandwich modes were ≥12 and ≥13 Å, respectively. Under the distances, the sensitivities of the sandwich ELISA were no lower than those of competitive ELISA. The obtained optimal EC values were 11.2 pg/mL (double-antibody sandwich with the epitope distance ≥16 Å) and 7.3 pg/mL (antibody-avidin sandwich with the epitope distance ≥17 Å). Compared with competitive ELISA, the developed method achieved a 30-fold improvement in sensitivity, with simpler aquatic product pretreatment.

摘要

基于衍生化反应，开发了用于检测小分子氨基脲（SEM）的超灵敏夹心免疫分析方法。通过将邻硝基苯甲醛（NBA）和生物素与二羟链烷（不同长度）连接，合成了几种 SEM 衍生化试剂，然后用它们来评估两个表位的距离效应。使用抗 SEM-NBA 抗体和辣根过氧化物酶标记的亲和素或抗生物素抗体作为二级缀合物，开发了用于 SEM 衍生物的夹心 ELISA。在双抗体夹心和抗体亲和素夹心模式下，两个表位的有利距离分别为≥12 和≥13 Å。在这些距离下，夹心 ELISA 的灵敏度不低于竞争 ELISA。获得的最佳 EC 值分别为 11.2 pg/mL（双抗体夹心，表位距离≥16 Å）和 7.3 pg/mL（抗体亲和素夹心，表位距离≥17 Å）。与竞争 ELISA 相比，所开发的方法在简化水产品预处理的情况下，灵敏度提高了 30 倍。