Casarin Elisabetta, Lucchese Laura, Grazioli Santina, Facchin Sonia, Realdon Nicola, Brocchi Emiliana, Morpurgo Margherita, Nardelli Stefano
University of Padova, Department of Pharmaceutical and Pharmacological Sciences Padova, Italy.
Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), Legnaro (PD), Italy.
PLoS One. 2016 Jan 13;11(1):e0145912. doi: 10.1371/journal.pone.0145912. eCollection 2016.
Diagnostic tests for veterinary surveillance programs should be efficient, easy to use and, possibly, economical. In this context, classic Enzyme linked ImmunoSorbent Assay (ELISA) remains the most common analytical platform employed for serological analyses. The analysis of pooled samples instead of individual ones is a common procedure that permits to certify, with one single test, entire herds as "disease-free". However, diagnostic tests for pooled samples need to be particularly sensitive, especially when the levels of disease markers are low, as in the case of anti-BoHV1 antibodies in milk as markers of Infectious Bovine Rhinotracheitis (IBR) disease. The avidin-nucleic-acid-nanoassembly (ANANAS) is a novel kind of signal amplification platform for immunodiagnostics based on colloidal poly-avidin nanoparticles that, using model analytes, was shown to strongly increase ELISA test performance as compared to monomeric avidin. Here, for the first time, we applied the ANANAS reagent integration in a real diagnostic context. The monoclonal 1G10 anti-bovine IgG1 antibody was biotinylated and integrated with the ANANAS reagents for indirect IBR diagnosis from pooled milk mimicking tank samples from herds with IBR prevalence between 1 to 8%. The sensitivity and specificity of the ANANAS integrated method was compared to that of a classic test based on the same 1G10 antibody directly linked to horseradish peroxidase, and a commercial IDEXX kit recently introduced in the market. ANANAS integration increased by 5-fold the sensitivity of the 1G10 mAb-based conventional ELISA without loosing specificity. When compared to the commercial kit, the 1G10-ANANAS integrated method was capable to detect the presence of anti-BHV1 antibodies from bulk milk of gE antibody positive animals with 2-fold higher sensitivity and similar specificity. The results demonstrate the potentials of this new amplification technology, which permits improving current classic ELISA sensitivity limits without the need for new hardware investments.
用于兽医监测项目的诊断测试应具备高效、易用且可能经济的特点。在这种情况下，经典的酶联免疫吸附测定（ELISA）仍然是血清学分析中最常用的分析平台。分析混合样本而非单个样本是一种常见的程序，通过一次测试就能证明整个畜群“无病”。然而，针对混合样本的诊断测试需要特别灵敏，尤其是当疾病标志物水平较低时，如牛奶中的抗牛疱疹病毒1型（BoHV1）抗体作为牛传染性鼻气管炎（IBR）疾病的标志物时。抗生物素蛋白 - 核酸 - 纳米组装体（ANANAS）是一种基于胶体多抗生物素蛋白纳米颗粒的新型免疫诊断信号放大平台，使用模型分析物时，与单体抗生物素蛋白相比，它能显著提高ELISA测试性能。在此，我们首次将ANANAS试剂整合应用于实际诊断环境中。将单克隆1G10抗牛IgG1抗体进行生物素化，并与ANANAS试剂整合，用于从模拟IBR患病率在1%至8%的畜群储奶罐样本的混合牛奶中间接诊断IBR。将ANANAS整合方法的灵敏度和特异性与基于直接连接辣根过氧化物酶的相同1G10抗体的经典测试以及市场上最近推出的商业IDEXX试剂盒进行了比较。ANANAS整合使基于1G10单克隆抗体的传统ELISA的灵敏度提高了5倍，且不失特异性。与商业试剂盒相比，1G10 - ANANAS整合方法能够以高2倍的灵敏度和相似的特异性检测gE抗体阳性动物的散装牛奶中抗BHV1抗体的存在。结果证明了这种新的放大技术的潜力，它无需新的硬件投资就能提高当前经典ELISA的灵敏度极限。
