血红蛋白驼源纳米抗体的筛选与表征

Screening and characterization of camelid-derived nanobodies against hemoglobin

摘要:

血红蛋白是红细胞内的主要蛋白质，负责在血液中运输氧气。从贫血的诊断到血液疾病的筛查，血红蛋白浓度的测定在医疗诊断和健康管理中发挥着不可替代的作用。基于抗原-抗体相互作用的免疫学检测方法具有高灵敏度和准确性。为了开发具有高特异性和高亲和力的抗体，提高血红蛋白检测的准确性，本研究将人血红蛋白免疫双峰驼并构建噬菌体展示纳米抗体文库，应用固相筛选法进行抗体筛选，并探明筛选得到的纳米抗体与血红蛋白的结合活性。首先，将人血红蛋白免疫双峰驼，并构建了库容量为2.85×10⁸菌落形成单位(colony forming unit,CFU)的纳米抗体文库。其次，经过3轮“吸附-洗脱-富集”实验淘选，得到10条血红蛋白特异性纳米抗体序列，并对其进行真核表达。最后，使用酶联免疫吸附实验和生物膜干涉技术表征纳米抗体的理化稳定性、结合活性和特异性。结果表明，纳米抗体在20–40℃温度范围内具有较好的稳定性；当pH值为7.0时，纳米抗体结合活性最高；纳米抗体可耐受10%的甲醇溶液。其中，纳米抗体VHH-12与血红蛋白结合活性最高，其半数最大效应浓度值(half maximal effective concentration,EC₅₀)为10.63 nmol/L，平衡解离常数(equilibrium dissociation constant,K_D)为2.94×10^–7 mol/L。VHH-12与卵清蛋白、牛血清蛋白等8种蛋白质均无交叉反应性，与猪、羊、兔、牛血红蛋白表现出一定的交叉反应性。本研究成功淘选出1条血红蛋白特异性高亲和力纳米抗体，有望应用于疾病诊断和健康监测。

Abstract:

Hemoglobin, the principal protein in red blood cells, is crucial for oxygen transport in the bloodstream. The quantification of hemoglobin concentration is indispensable in medical diagnostics and health management, which encompass the diagnosis of anemia and the screening of various blood disorders. Immunological methods, based on antigen-antibody interactions, are distinguished by their high sensitivity and accuracy. Consequently, it is necessary to develop hemoglobin-specific antibodies characterized by high specificity and affinity to enhance detection accuracy. In this study, we immunized a Bactrian camel (Camelus bactrianus) with human hemoglobin and subsequently constructed a nanobody library. Utilizing a solid-phase screening method, we selected nanobodies and evaluated the binding activity of the screened nanobodies to hemoglobin. Initially, human hemoglobin was used to immunize a Bactrian camel. Following four immunization sessions, blood was withdrawn from the jugular vein, and a nanobody library with a capacity of 2.85×10⁸ colony forming units (CFU) was generated. Subsequently, ten hemoglobin-specific nanobody sequences were identified through three rounds of adsorption-elution-enrichment assays, and these nanobodies were subjected to eukaryotic expression. Finally, enzyme-linked immunosorbent assay and biolayer interferometry were employed to evaluate the stability, binding activity, and specificity of these nanobodies. The results demonstrated that the nanobodies maintained robust binding activity within the temperature range of 20–40 ℃ and exhibited the highest binding activity at pH 7.0. Furthermore, the nanobodies were capable of tolerating a 10% methanol solution. Notably, among the nanobodies tested, VHH-12 displayed the highest binding activity to hemoglobin, with a half maximal effective concentration (EC₅₀) of 10.63 nmol/L and a equilibrium dissociation constant (K_D) of 2.94×10^–7 mol/L. VHH-12 exhibited no cross-reactivity with a panel of eight proteins, such as ovalbumin and bovine serum albumin, while demonstrating partial cross-reactivity with hemoglobin derived from porcine, goat, rabbit, and bovine sources. In this study, a hemoglobin-specific high-affinity nanobody was successfully isolated, demonstrating potential applications in disease diagnosis and health monitoring.