Tools for site-specific antibody immobilization for immunoassays

抗体，最常见的是免疫球蛋白G（IgG），广泛用于研究和诊断测定，这是由于 其广泛的靶点、高度特异性和经证实的功效。在许多这些“免疫测定”（例如， ELISA、生物传感器等），抗体必须固定在表面上。灵敏度、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，正确的方向可以使灵敏度提高数十倍甚至1000倍。2 -5 尽管使用特定地点固定技术具有巨大好处，但它们很少在商业中采用 免疫测定。这在很大程度上是由于复杂性，缺点和经济障碍与网站- 特异性固定化技术。1最近，我们开发了一种简单、快速和有效的方法， 特异性和共价结合表面上的天然IgG。我们的方法依赖于使用低分子 重量，蛋白A和G的单价亚结构域，其具有UV活性非天然氨基酸， 苯甲酰苯丙氨酸（BPA），在Fc结合结构域内。暴露在无害的长波长紫外线下 当BPA被激活并在抗体结合结构域和抗体之间形成共价连接时， 光反应性抗体结合结构域（pAbBD）的重组生产已经与 表达蛋白连接（EPL），以允许将几乎任何期望的化学手柄引入到细胞上。 在蛋白质纯化过程中pAbBD的C-末端。这使得pAbBD的位点特异性连接成为可能 到各种各样的表面。总之，使用pAbBD用于抗体的位点特异性固定是有利的。 具有成本效益，易于扩展，适用于高通量过程，并利用蛋白质生产 商业上可行的技术。该提案的主要目标是获取数据， 证明了我们的技术在免疫分析应用中的价值。我们相信，这将开辟新的 在免疫分析行业的合作和商业伙伴关系的机会。具体目标为 目的1：优化抗体定点固定的条件， 微孔板;目的2：定量位点特异性IgG固定对免疫测定灵敏度、 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.