Driskell Jeremy D, Kwarta Karen M, Lipert Robert J, Vorwald Ann, Neill John D, Ridpath Julia F, Porter Marc D
Iowa State University, Institute for Combinatorial Discovery, Department of Chemistry, The Ames Laboratory-USDOE, Ames, IA 50011-3020, United States.
J Virol Methods. 2006 Dec;138(1-2):160-9. doi: 10.1016/j.jviromet.2006.08.011. Epub 2006 Oct 10.
Immunosorbent assays are commonly employed as diagnostic tests in human healthcare, veterinary medicine and bioterrorism prevention. These assays, however, often require long incubation times, limiting sample throughput. As an approach to overcome this weakness, this paper examines the use of rotating capture substrates to increase the flux of antigen to the surface, thereby reducing the incubation time. To assess the capability of this approach, porcine parvovirus (PPV) was selectively extracted from solution by systematically varying the rotation rate of a gold substrate modified with a layer of anti-PPV monoclonal antibodies. The captured PPV were then directly imaged and quantified by atomic force microscopy. The benefits of substrate rotation are demonstrated by comparing an assay performed under stagnant conditions to one carried out with substrate rotation at 800 rpm, both for 10 min incubations at 25 degrees C. The use of rotation lowered the limit of detection to 3.4x10(4)TCID50/mL (approximately 80 fM) from 3.2x10(5)TCID50/mL (approximately 800 fM) under stagnant conditions. Results are also presented that show this strategy can be used: (1) to determine antigen concentrations without standards and (2) to establish the numerical relationship between quantal concentration units (e.g., 50% tissue culture infective dose (TCID50)) and quantitative concentration units (e.g., viruses/mL) The potential to broadly apply this technique to heterogeneous immunoassays is also briefly discussed.
免疫吸附测定法在人类医疗保健、兽医学和生物恐怖主义预防中通常用作诊断测试。然而，这些测定法通常需要较长的孵育时间，限制了样品通量。作为克服这一弱点的一种方法，本文研究了使用旋转捕获底物来增加抗原向表面的通量，从而缩短孵育时间。为了评估这种方法的能力，通过系统地改变用抗猪细小病毒（PPV）单克隆抗体层修饰的金底物的旋转速率，从溶液中选择性提取PPV。然后通过原子力显微镜对捕获的PPV进行直接成像和定量。通过比较在静态条件下进行的测定与在800 rpm的底物旋转条件下进行的测定（两者均在25℃下孵育10分钟），证明了底物旋转的益处。旋转的使用将检测限从静态条件下的3.2×10⁵TCID50/mL（约800 fM）降低到3.4×10⁴TCID50/mL（约80 fM）。还给出了结果，表明该策略可用于：（1）无需标准品即可确定抗原浓度，以及（2）建立定量浓度单位（例如，50%组织培养感染剂量（TCID50））与定量浓度单位（例如，病毒/mL）之间的数值关系。还简要讨论了将该技术广泛应用于异质免疫测定的潜力。
