Jangulova Assem N, Taranova Nadezhda A, Dzantiev Boris B, Akanova Zhannara Zh, Bulashev Aitbay K
Department of Veterinary Medicine, Faculty of Veterinary and Livestock Technology, S. Seifullin Kazakh Agrotechnical Research University, 62 Zhenis Avenue, Astana 010011, Kazakhstan.
Immunobiochemistry Laboratory, A.N. Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, 33, Leninsky Avenue, Moscow 119071, Russia.
Vet World. 2024 Nov;17(11):2527-2536. doi: 10.14202/vetworld.2024.2527-2536. Epub 2024 Nov 13.
In animal husbandry, antibiotics are frequently used as growth promoters, as well as for illness prevention and treatment. They are considered important toxic and allergenic contaminants of food and a serious risk factor for the spread of antibiotic resistance. National and international regulatory authorities have established limits on the permissible residue of antibiotics in food. Immunochromatographic test strips are the most efficient tools for the simple and rapid control of antibiotics for food safety. In these tests, each antibody specific to a particular antibiotic is labeled with a marker, which complicates the manufacturing technology and increases the cost of the test. This study aimed to develop a multiplex immunochromatographic assay (ICA) to determine streptomycin (STR), tetracycline (TC), and chloramphenicol (CAP) residues in milk by indirect labeling of antibiotic-specific antibodies.
Test strips were composed using 15 μm pore size CNPC nitrocellulose membranes, GFB-R4 separation, and AP045 adsorption pads. The applied reactants include TC and STR conjugates with bovine serum albumin, and CAP-soybean trypsin inhibitor conjugate; anti-TC, anti-STR, and anti-CAP mouse monoclonal antibodies; goat anti-mouse immunoglobulin G (GAMI) conjugated with gold nanoparticles (GNPs) and staphylococcal protein A. Milk samples were collected from cows and goats that had not been injected with any antibiotics. STR and TC/CAP at concentrations of 0.27-600 ng/mL and 0.04-30 ng/mL were added to skim milk, respectively. Milk samples were tested by ICA and calibration curves were constructed to determine the sensitivity of the assay for each antibiotic used.
A multiplex ICA of three antibiotic residues in milk was achieved through labeling of immune complexes using a single reagent, GNPs-GAMI. The visual limits of detection (LOD) were 600 ng/mL, 10 ng/mL, and 30 ng/mL for STR, TC, and CAP in cow and goat milk, respectively. Instrumental LODs gave higher sensitivity when analyzed goat milk to STR, TC, and CAP (1.2, 0.05, and 1.3 ng/mL) than cows' milk (7.27, 0.96, and 2.07 ng/mL, respectively).
The developed approach for manufacturing multiplex ICA tests for the detection of antibiotic residues in milk does not involve labeling specific antibodies and is implemented using only GNP conjugates with anti-species antibodies.
在畜牧业中，抗生素常被用作生长促进剂，以及用于疾病预防和治疗。它们被视为食品中重要的有毒和致敏污染物，以及抗生素耐药性传播的严重风险因素。国家和国际监管机构已对食品中抗生素的允许残留量设定了限制。免疫层析试纸条是用于食品安全中抗生素简单快速检测的最有效工具。在这些检测中，每种针对特定抗生素的抗体都用一种标记物进行标记，这使制造技术变得复杂并增加了检测成本。本研究旨在通过间接标记抗生素特异性抗体开发一种多重免疫层析分析方法（ICA），以测定牛奶中的链霉素（STR）、四环素（TC）和氯霉素（CAP）残留量。
试纸条使用孔径为15μm的CNPC硝酸纤维素膜、GFB - R4分离膜和AP045吸附垫制成。所应用的反应物包括与牛血清白蛋白偶联的TC和STR，以及CAP - 大豆胰蛋白酶抑制剂偶联物；抗TC、抗STR和抗CAP小鼠单克隆抗体；与金纳米颗粒（GNP）偶联的山羊抗小鼠免疫球蛋白G（GAMI）和葡萄球菌蛋白A。从未注射任何抗生素的奶牛和山羊采集牛奶样本。分别向脱脂牛奶中添加浓度为0.27 - 600 ng/mL的STR和0.04 - 30 ng/mL的TC/CAP。通过ICA对牛奶样本进行检测，并构建校准曲线以确定所使用的每种抗生素检测方法的灵敏度。
通过使用单一试剂GNP - GAMI标记免疫复合物，实现了牛奶中三种抗生素残留的多重ICA检测。牛和山羊奶中STR、TC和CAP的目视检测限（LOD）分别为600 ng/mL、10 ng/mL和30 ng/mL。对山羊奶中STR、TC和CAP进行仪器分析时，仪器检测限（分别为1.2、0.05和1.3 ng/mL）比牛奶（分别为7.27、0.96和2.07 ng/mL）具有更高的灵敏度。
所开发的用于制造检测牛奶中抗生素残留的多重ICA检测方法不涉及标记特异性抗体，仅使用与抗物种抗体偶联的GNP来实现。
