利用功能化金纳米颗粒偶联抗体进行布鲁氏菌检测的快速直接鉴别凝集试验

IF 4.1 Q2 MATERIALS SCIENCE, MULTIDISCIPLINARY
R. Hans, P. Yadav, M. Zaman, Rajaram Poolla, D. Thavaselvam
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引用次数: 0

摘要

布鲁氏菌病是世界上传播最广和最严重的人畜共患疾病,影响牲畜、森林野生动物、海洋生物和人类。它是通过属于布鲁氏菌属的阿尔法变形菌获得的,被归类为潜在的生物威胁剂。在这项研究中,我们开发了一种快速和直接的基于差异全细胞(WC)凝集的现场检测方法。采用亲和层析技术纯化重组外膜(rommp28)蛋白衍生的布鲁氏菌特异性小鼠IgG多克隆抗体(pAbs),并与功能化金纳米颗粒(AuNPs)偶联进行快速凝集。32 kDa蛋白的阳性免疫印迹分析显示rommp28 - pab具有特异性免疫反应性。对于AuNPs的合成,优化了传统的“Turkevich法”,在金前驱体浓度< 1 mM时,可获得50纳米大小的颗粒。采用紫外可见分光光度法、傅里叶变换红外光谱法(FT-IR)、拉曼光谱法、x射线衍射法(XRD)、扫描电镜法(SEM)、透射电镜法(TEM)、动态光散射法(DLS)、ζ电位法(ζ, ZP)和荧光光谱法对其理化性质进行了分析。然后用N-(3-二甲氨基丙基)-N′-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)功能化这些AuNPs,制备改性的羧化AuNPs。为了与布鲁氏菌rom28 IgG pab生物偶联,制备了抗体偶联的功能化AuNP结构体,并使用具有强N-H变形的FT-IR分析对其进行了表征。随后,将这些生物偶联的AuNPs用于直接差分玻片凝集试验,检测限为104 CFU mL−1。与标准双抗体夹心ELISA (S-ELISA)相比,该方法的灵敏度较高,检测限为103 CFU mL - 1,检测范围为102-108 CFU mL - 1。基于与一系列密切相关的细菌物种的特异性评估,未观察到种内交叉反应。综上所述,利用生物偶联功能化AuNPs获得的凝集试验检测布鲁氏菌的灵敏度和特异性均提高了≥98%。因此,在疾病早期诊断的情况下,它具有简单、可靠、成本低、反应时间短的优点,可作为直接WC检测细菌的替代快速方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A rapid direct-differential agglutination assay for Brucella detection using antibodies conjugated with functionalized gold nanoparticles
Brucellosis is the most widespread and serious zoonotic disease worldwide which affects livestock, sylvatic wildlife, marine dwellers, and humans. It is acquired through Alphaproteobacteria which belong to the genus Brucella and is categorized as a potential bio-threat agent. In this study, we developed a rapid and direct differential whole cell (WC) agglutination-based assay for its on-field detection. The recombinant outer membrane (rOmp28) protein-derived specific mice IgG polyclonal antibodies (pAbs) of Brucella were purified using affinity chromatography and conjugated with functionalized gold nanoparticles (AuNPs) for rapid agglutination. A positive blot of 32 kDa protein revealed specific immuno-reactivity of rOmp28-pAbs using immunoblot analysis. For the synthesis of AuNPs, the conventional “Turkevich method” was optimized at a concentration < 1 mM of gold precursor for obtaining 50-nm-sized particles. Also, their physico-chemical characteristics were analyzed using UV-visible spectrophotometry, Fourier transform infra-red spectroscopy (FT-IR), Raman spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential (ζ, ZP), and fluorescence spectroscopy. Furthermore, these AuNPs were functionalized with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to prepare modified carboxylated AuNPs. For bioconjugation with Brucella rOmp28 IgG pAbs, antibody-conjugated functionalized AuNP constructs were prepared and characterized using FT-IR analysis with strong N–H deformations. Subsequently, these bioconjugated AuNPs were used to develop a direct-differential slide agglutination assay with a detection limit of 104 CFU mL−1. The sensitivity of this assay was compared with standard double-antibody sandwich ELISA (S-ELISA) using rOmp28 IgG pAbs with an LOD of 103 CFU mL−1 and a detection range of 102–108 CFU mL−1. No intraspecies cross-reactivity was observed based on evaluation of its specificity with a battery of closely related bacterial species. In conclusion, the increased sensitivity and specificity of the developed agglutination assay obtained using bioconjugated functionalized AuNPs is ≥ 98% for the detection of Brucella. Therefore, it can be used as an alternate rapid method of direct WC detection of bacteria as it is simple, robust, and cost-effective, with minimal time of reaction in the case of early disease diagnosis.
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来源期刊
Frontiers in Nanotechnology
Frontiers in Nanotechnology Engineering-Electrical and Electronic Engineering
CiteScore
7.10
自引率
0.00%
发文量
96
审稿时长
13 weeks
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