Battaglia Federica, Bonelli Francesca, Sgorbini Micaela, Intorre Luigi, Minunni Maria, Scarano Simona, Meucci Valentina
Department of Chemistry "Ugo Schiff", University of Florence, 50019 Sesto Fiorentino, FI, Italy.
Department of Veterinary Science, University of Pisa, 56122 Via Livornese, PI, Italy.
Anal Methods. 2022 Dec 22;15(1):27-35. doi: 10.1039/d2ay01175a.
In this study, a new sandwich-type immunoenzymatic assay, based on a molecularly imprinted polymer (MIP) as an artificial antibody (pseudo-ELISA), was developed for the determination of procalcitonin (PCT) in veterinary species. The quantification of PCT in human medicine represents the state of the art for the diagnosis of sepsis; instead the clinical studies on the relevance of PCT as a sepsis predictor in veterinary patients are few, likely due to the total absence of validated assays. MIPs have been widely used as antibody mimics for important applications, and MIP-based sandwich assays have emerged as promising analytical tools for the detection of disease biomarkers. Herein, a polynorepinephrine (PNE)-based imprinted film was directly synthesized on the well surface of a 96-well plate. Subsequently, based on a commercial ELISA kit, the PCT quantification was accomplished a colorimetric sandwich assay by replacing the capture antibody of the kit with the PNE-based MIP. This method was performed to detect canine and equine PCT in buffer and in plasma samples. Under optimal conditions, the results obtained in plasma samples showed a limit of detection (LOD) of 5.87 ng mL and a reproducibility (CV) of 10.0% for canine samples, while a LOD = 4.46 ng mL and CV = 7.61% were obtained for equine samples.
在本研究中，开发了一种基于分子印迹聚合物（MIP）作为人工抗体的新型夹心式免疫酶测定法（伪酶联免疫吸附测定），用于测定兽医学中的降钙素原（PCT）。在人类医学中，PCT的定量是脓毒症诊断的最新技术；相反，关于PCT作为兽医患者脓毒症预测指标相关性的临床研究很少，这可能是由于完全缺乏经过验证的检测方法。分子印迹聚合物已被广泛用作重要应用中的抗体模拟物，基于分子印迹聚合物的夹心测定法已成为检测疾病生物标志物的有前途的分析工具。在此，基于聚去甲肾上腺素（PNE）的印迹膜直接在96孔板的孔表面合成。随后，基于商业酶联免疫吸附测定试剂盒，通过用基于PNE的分子印迹聚合物替代试剂盒的捕获抗体，以比色夹心测定法完成了PCT定量。该方法用于检测缓冲液和血浆样品中的犬和马PCT。在最佳条件下，血浆样品的检测结果显示，犬类样品的检测限（LOD）为5.87 ng/mL，重现性（CV）为10.0%，而马类样品的检测限为4.46 ng/mL，CV为7.61%。
