Development of Microplate Immunoenzyme Determination of Nonylphenol with Magnetic Sample Concentration

IF 1 4区生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Applied Biochemistry and Microbiology Pub Date : 2024-05-16 DOI:10.1134/S0003683824603536

A. N. Berlina, L. V. Barshevskaya, K. V. Serebrennikova, N. S. Komova, A. V. Zherdev, B. B. Dzantiev

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Abstract

Nonylphenol is an aromatic organic compound that has an estrogen-like effect and has a negative effect on the human endocrine system. A method has been developed for the competitive determination of nonylphenol using magnetic particles, rabbit antiserum, nonylphenol conjugate with soybean trypsin inhibitor (STI) and biotin. The principle of the analysis is the formation of immune complexes on the surface of magnetite particles due to covalent immobilization of protein G through the oriented immobilization of polyclonal antibodies from rabbit serum during a competitive reaction between the free analyte (nonylphenol) and the bound one (as part of the nonylphenol-STI-biotin conjugate) for the binding sites of specific antibodies. The detection of formed immune complexes is proposed to be carried out using a streptavidin-polyperoxidase conjugate, which makes it possible to achieve a nine-fold gain in the level of the analytical signal. The developed ELISA using magnetite particles allows us to achieve a detection limit of nonylphenol at the level of 3.8 ng/mL, which is 14.5 times lower in comparison with the classic competitive ELISA (55 ng/mL). Based on the results of the experimental work, the optimized volume of the test sample was 500 μL, which makes it possible to concentrate low-contaminated samples by 17 times.

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利用磁性样品浓度开发壬基酚微孔板免疫酶测定法

壬基酚是一种芳香族有机化合物，具有类似雌激素的作用，对人体内分泌系统有负面影响。本研究采用磁性颗粒、兔抗血清、壬基酚与大豆胰蛋白酶抑制剂（STI）共轭物和生物素，开发了一种竞争性测定壬基酚的方法。分析原理是在游离分析物（壬基酚）和结合分析物（作为壬基酚-STI-生物素共轭物的一部分）对特异性抗体结合位点的竞争反应中，通过兔血清多克隆抗体的定向固定，在磁铁矿颗粒表面通过共价固定蛋白 G 形成免疫复合物。建议使用链霉亲和素-聚过氧化物酶共轭物来检测形成的免疫复合物，这样可以使分析信号水平提高九倍。使用磁铁矿颗粒开发的酶联免疫吸附法可使壬基酚的检测限达到 3.8 纳克/毫升，与传统的竞争性酶联免疫吸附法（55 纳克/毫升）相比降低了 14.5 倍。根据实验结果，测试样品的最佳体积为 500 μL，这使得低污染样品的浓缩倍数提高了 17 倍。

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来源期刊

Applied Biochemistry and Microbiology 生物-生物工程与应用微生物

CiteScore

1.70

自引率

12.50%

发文量

审稿时长

6-12 weeks

期刊介绍： Applied Biochemistry and Microbiology is an international peer reviewed journal that publishes original articles on biochemistry and microbiology that have or may have practical applications. The studies include: enzymes and mechanisms of enzymatic reactions, biosynthesis of low and high molecular physiologically active compounds; the studies of their structure and properties; biogenesis and pathways of their regulation; metabolism of producers of biologically active compounds, biocatalysis in organic synthesis, applied genetics of microorganisms, applied enzymology; protein and metabolic engineering, biochemical bases of phytoimmunity, applied aspects of biochemical and immunochemical analysis; biodegradation of xenobiotics; biosensors; biomedical research (without clinical studies). Along with experimental works, the journal publishes descriptions of novel research techniques and reviews on selected topics.