Determining factor of enzyme conjugates, bridge heterology and analytical variables of immunogens in prednisolone ELISA

IF 5.4 Q1 CHEMISTRY, ANALYTICAL

Sensing and Bio-Sensing Research Pub Date : 2024-11-02 DOI:10.1016/j.sbsr.2024.100700

Dinesh Kumar , Harinder Singh Oberoi , Subash Chandra Sonkar , Bidhan Chandra Koner , T.G. Shrivastav

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Abstract

In this study, adipic acid dihydrazide (ADH), carbohydrazide (CH), ethylenediamine (EDA), and urea (U) were used as spacer molecules, covalently linking prednisolone (PSL) to carrier proteins for immunogen preparation, and PSL to enzymes for enzyme conjugate preparation using N-hydroxysuccinimide (NHS)-mediated carbodiimide reactions. The resulting immunogens were used to generate antiserum in New Zealand white rabbits. Antibodies produced against immunogens with various spacers were tested for immunoreactivity with enzyme conjugates, both with and without spacers, in a total of twenty different combinations. All combinations demonstrated binding and were subsequently evaluated through displacement studies. Sensitivity and specificity tests revealed that the combination of PSL-21-HS-U-BSA-antibody with PSL-21-HS-HRP enzyme conjugate exhibited the best sensitivity (0.032 ng/mL) and limited cross-reactivity with other steroids. This combination was further examined for analytical parameters, showing recovery rates of 93.87–101.19 % for PSL from spiked human serum samples, with intra- and inter-assay CVs of <8.88 %. The serum PSL values obtained by this method showed strong correlation with a commercially available ELISA kit (r² = 0.97, n = 78).

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泼尼松龙酶联免疫吸附试验中酶共轭物、桥异性和免疫原分析变量的决定因素

本研究使用己二酸二酰肼（ADH）、碳酰肼（CH）、乙二胺（EDA）和尿素（U）作为间隔分子，将泼尼松龙（PSL）与载体蛋白共价连接以制备免疫原，并使用 N-羟基琥珀酰亚胺（NHS）介导的碳化二亚胺反应将 PSL 与酶共价连接以制备酶结合物。得到的免疫原用于在新西兰白兔身上产生抗血清。针对带有各种间隔物的免疫原产生的抗体与酶结合物进行了免疫反应性测试，包括带间隔物和不带间隔物的酶结合物，共有二十种不同的组合。所有组合都显示出了结合性，随后通过置换研究对其进行了评估。灵敏度和特异性测试表明，PSL-21-HS-U-BSA-抗体与 PSL-21-HS-HRP 酶结合物的组合灵敏度最高（0.032 ng/mL），与其他类固醇的交叉反应有限。对这一组合的分析参数进行了进一步检测，结果显示，从添加的人体血清样本中提取 PSL 的回收率为 93.87-101.19%，测定内和测定间的 CV 为 8.88%。该方法获得的血清 PSL 值与市售的酶联免疫吸附试剂盒显示出很强的相关性（r2 = 0.97，n = 78）。

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来源期刊

Sensing and Bio-Sensing Research Engineering-Electrical and Electronic Engineering

CiteScore

10.70

自引率

3.80%

发文量

审稿时长

87 days

期刊介绍： Sensing and Bio-Sensing Research is an open access journal dedicated to the research, design, development, and application of bio-sensing and sensing technologies. The editors will accept research papers, reviews, field trials, and validation studies that are of significant relevance. These submissions should describe new concepts, enhance understanding of the field, or offer insights into the practical application, manufacturing, and commercialization of bio-sensing and sensing technologies. The journal covers a wide range of topics, including sensing principles and mechanisms, new materials development for transducers and recognition components, fabrication technology, and various types of sensors such as optical, electrochemical, mass-sensitive, gas, biosensors, and more. It also includes environmental, process control, and biomedical applications, signal processing, chemometrics, optoelectronic, mechanical, thermal, and magnetic sensors, as well as interface electronics. Additionally, it covers sensor systems and applications, µTAS (Micro Total Analysis Systems), development of solid-state devices for transducing physical signals, and analytical devices incorporating biological materials.