Preparation and utilization of an immunoadsorption column for aflatoxin B1 composed of nanobodies and pre-crosslinked agarose microspheres

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B Pub Date : 2025-05-18 DOI:10.1016/j.jchromb.2025.124656

Xiao Ding , Shuo Wang , Wei Wang , Jingjing Sun , Chengcheng Jiang , Jianhua Hao

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引用次数: 0

Abstract

Recent studies on immunoaffinity chromatography (IAC) columns for aflatoxin B₁ (AFB₁) have not provided a comprehensive preparation procedure or explored their capacity, reusability, and stability. In this study, we outline a preparation process for an AFB₁ immune affinity column filler (AFB₁-IAC) for extracting AFB₁ from food sources like rice, peanuts, and soybeans. Using commercially available IAC is often limited due to high costs, low capacity, slow flow rates, and poor reusability. To overcome these issues, we developed succinic anhydride (SA)-modified agarose pre-crosslinked microspheres (SA-A) activated with 1,1′‑carbonyldiimidazole (CDI). The activated microspheres were coupled with the anti-AFB₁ nanobody Nb02, creating an immunoaffinity packing called CDI-SA-A-Nb02 (D1-IAC). After verification, the results indicated that the column capacity of D1-IAC was about 4.67 times that of several commercial IAC brands. Additionally, the maximum flow rate at a pressure of 0.04 MPa was 25 % greater than specific commercial IAC, and the recovery rate remained above 80 % after being reused five times. This method is considered effective for extracting and detecting AFB₁ in food products. The preparation process for D1-IAC shows great consistency, and the column's performance is better than that of available immunoaffinity columns. This study explores using an IAC to extract and purify AFB₁, which is then quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

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纳米体与预交联琼脂糖微球组成黄曲霉毒素B1免疫吸附柱的制备及应用

最近对黄曲霉毒素B1 （AFB1）免疫亲和层析（IAC）色谱柱的研究尚未提供全面的制备工艺或探索其容量、可重复使用性和稳定性。在这项研究中，我们概述了一种AFB1免疫亲和柱填料（AFB1- iac）的制备工艺，用于从大米、花生和大豆等食物来源中提取AFB1。由于成本高、容量低、流量慢和可重用性差，使用商业上可用的IAC通常受到限制。为了克服这些问题，我们开发了丁二酸酐（SA）修饰琼脂糖预交联微球（SA- a），并以1,1′-羰基二咪唑（CDI）活化。将活化的微球与抗afb1纳米体Nb02偶联，形成称为CDI-SA-A-Nb02 （D1-IAC）的免疫亲和包装。经验证，D1-IAC的柱容量约为几种商业IAC品牌的4.67倍。0.04 MPa压力下的最大流量比特定的商用IAC高25%，重复使用5次后回收率仍保持在80%以上。该方法对食品中AFB1的提取和检测是有效的。D1-IAC的制备工艺具有较好的一致性，色谱柱的性能优于现有的免疫亲和柱。本研究探索了利用IAC提取和纯化AFB1，然后使用高效液相色谱-串联质谱（HPLC-MS/MS）进行定量。

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来源期刊

Journal of Chromatography B 医学-分析化学

CiteScore

5.60

自引率

3.30%

发文量

306

审稿时长

44 days

期刊介绍： The Journal of Chromatography B publishes papers on developments in separation science relevant to biology and biomedical research including both fundamental advances and applications. Analytical techniques which may be considered include the various facets of chromatography, electrophoresis and related methods, affinity and immunoaffinity-based methodologies, hyphenated and other multi-dimensional techniques, and microanalytical approaches. The journal also considers articles reporting developments in sample preparation, detection techniques including mass spectrometry, and data handling and analysis. Developments related to preparative separations for the isolation and purification of components of biological systems may be published, including chromatographic and electrophoretic methods, affinity separations, field flow fractionation and other preparative approaches. Applications to the analysis of biological systems and samples will be considered when the analytical science contains a significant element of novelty, e.g. a new approach to the separation of a compound, novel combination of analytical techniques, or significantly improved analytical performance.