Tools for site-specific antibody immobilization for immunoassays

抗体，最常见的是免疫球蛋白Gs(IGG)，被广泛用于研究和诊断分析 以其广泛的靶点、高度的特异性和被证明的有效性。在许多这样的“免疫分析”中(例如 酶标法、生物传感器等)，抗体必须固定在表面。敏感度、稳定性和 在这种情况下使用的抗体的寿命高度依赖于这些分子的取向。 研究表明，正确的方向可以导致敏感度提高数十倍甚至1000倍。2-5 使用特定部位固定化技术的巨大好处，它们很少在商业上采用 免疫分析。这在很大程度上是由于与网站相关的复杂性、缺点和经济障碍- 1最近，我们开发了一种简单、快速和有效的方法来定位- 特异地共价固定在表面的天然免疫球蛋白。我们的方法依赖于使用低分子 重量，具有紫外线活性的非天然氨基酸的蛋白A和G的单价亚结构域， 苯甲酰苯丙氨酸(BPA)，在Fc结合域中。暴露在无毒的长波紫外线下 在光照下，BPA被激活，并在抗体结合域和抗体之间形成共价键。 重组产生的光反应性抗体结合域(PAbBds)已与 表达蛋白连接(EPL)，允许将几乎任何所需的化学处理引入到 蛋白纯化过程中pAbBDS的C-末端。这实现了pAbBD的特定部位附着 广泛的表面上。总体而言，pAbBDS用于定点固定抗体的用途是 成本效益高、易于扩展、适应高通量工艺，并可利用蛋白质生产 商业上可行的技术。该提案的主要目标是直接获取数据 展示了我们的技术在免疫分析应用中的价值。我们相信，这将开启新的 免疫分析行业的合作机会和商业伙伴关系。具体目标是 建议是：目标1：优化定点固定抗体的条件 微孔板；目的2：量化位点特异性免疫球蛋白固定化对免疫分析灵敏度的影响 检测和动态范围。

Antibodies, most commonly Immunoglobulin Gs (IgGs), are widely used in research and diagnostic assays due to their wide array of targets, high specificity and proven efficacy. In many of these “immunoassays” (e.g. ELISA's, biosensors, etc.), antibodies must be immobilized onto surfaces. The sensitivity, stability and longevity of antibodies used in this context are highly dependent on orienation of these molecules.1 Numerous work have shown that correct orienation can result in tens to even a 1000-fold increase in sensitivity.2-5 Despite the enormous benefit of using site-specific immobilization techniques, they are rarely adopted in commercial immunoassays. This is largely due to the complexity, shortcomings and economic hurdles associated with site- specific immobilization techniques.1 Recently, we developed a simple, rapid, and efficient approach to site- specifically and covalently immobilize native IgG on surfaces. Our approach relies on the use of low-molecular weight, monovalent subdomains of Protein A and G that possess a UV-active non-natural amino acid, benzoylphenyalanine (BPA), within the Fc-binding domains. Upon exposure to innocuous long wavelength UV light, the BPA is activated and forms a covalent link between the antibody-binding domain and the antibody. The recombinant production of photoreactive antibody-binding domains (pAbBDs) has been combined with expressed protein ligation (EPL) to allow for the introduction of nearly any desirable chemical handle onto the C-terminus of the pAbBDs during protein purification. This enables the site-specific attachment of the pAbBDs to a wide range of surfaces. Overall, the use of pAbBDs for the site-specific immobilization of antibodies is cost-effective, easily scalable, amenable to high-throughput processes, and utilizes protein production techniques that are commercially viable. The primary goal of this proposal is to acquire data that directly demonstrates the value of our technology in immunoassay applications. We believe that this will open up new opportunities for collaboration and commercial partnerships in the immunoassay industry. The specific aims for the proposal are: Aim 1: Optimize the conditions for the site-specific immobilization of antibodies in a microplate; Aim 2: Quantify the effect of site-specific IgG immobilization on immunoassay sensitivity, limit of detection, and dynamic range.