Institute of Agricultural Engineering, ARO, the Volcani Center, Bet Dagan 50250, Israel.
Biomolecules. 2019 Aug 14;9(8):372. doi: 10.3390/biom9080372.
Bovine mastitis (BM) is a frequent disease in the dairy industry that causes staggering economical losses due to decreased milk production and increased health care costs. Traditionally, BM detection depends on the efficacy and reliability of analytical techniques that measure somatic cell counts (SCC), detect pathogens, and reveal inflammatory status. Herein, we demonstrate the detection of bovine haptoglobin, a well-documented acute phase protein for evaluating BM clinical status, by utilizing hemoglobin-binding capacity within luminol chemiluminescence (CL) system. The resulting haptoglobin-hemoglobin complex reduces the CL signal proportionally to inherent haptoglobin concentrations. Different sizes of cross-linked gold nanoparticles (GNPs) were examined for enhanced CL (eCL) signal amplification, presenting over 30-fold emitted radiation enhancement for optimized size within real milk samples with respect to nanoparticle-free assay. The eCL values were proportionally related to nanoparticle size and content, influenced by SCC and pathogen type (e.g., and coagulase-negative staphylococci). The optimized bioassay showed a broad linear response (1 pg mL-10 µg mL) and minute detection limit of 0.19 pg mL, while presenting quantitative performance in agreement with commercial ELISA kit. Finally, the resulting optimized eCL concept offers an efficient label-free detection of haptoglobin biomarker, offering means to diagnose the severity of the associated diseases.
奶牛乳腺炎（BM）是奶牛养殖业中一种常见疾病，由于产奶量下降和医疗保健成本增加，给该行业造成了巨大的经济损失。传统上，BM 的检测依赖于测量体细胞计数（SCC）、检测病原体和揭示炎症状态的分析技术的功效和可靠性。在这里，我们展示了利用化学发光（CL）系统中的血红蛋白结合能力来检测牛触珠蛋白的方法，触珠蛋白是一种经过充分验证的评估 BM 临床状况的急性期蛋白。所得的触珠蛋白-血红蛋白复合物会使 CL 信号按固有触珠蛋白浓度成比例降低。我们研究了不同大小的交联金纳米颗粒（GNPs），以增强 CL（eCL）信号放大，在真实牛奶样本中，对于无纳米颗粒的测定，优化尺寸的 GNPs 呈现出超过 30 倍的发射辐射增强。eCL 值与纳米颗粒的尺寸和含量成正比，受 SCC 和病原体类型（例如 和凝固酶阴性葡萄球菌）的影响。优化后的生物测定法表现出宽的线性响应（1 pg mL-10 µg mL）和检测限低至 0.19 pg mL，同时与商业 ELISA 试剂盒的定量性能一致。最后，所得的优化 eCL 概念提供了一种高效的无标记触珠蛋白生物标志物检测方法，为诊断相关疾病的严重程度提供了手段。
