PDF(Pubmed)
Abstract：
Diversity in specificity of polyclonal antibody (pAb) responses is extensively investigated in vaccine efficacy or immunological evaluations, but the heterogeneity in antibody avidity is rarely probed as convenient tools are lacking. Here we have developed a polyclonal antibodies avidity resolution tool (PAART) for use with label-free techniques, such as surface plasmon resonance and biolayer interferometry, that can monitor pAb-antigen interactions in real time to measure dissociation rate constant (kd ) for defining avidity. PAART utilizes a sum of exponentials model to fit the dissociation time-courses of pAb-antigens interactions and resolve multiple kd contributing to the overall dissociation. Each kd value of pAb dissociation resolved by PAART corresponds to a group of antibodies with similar avidity. PAART is designed to identify the minimum number of exponentials required to explain the dissociation course and guards against overfitting of data by parsimony selection of best model using Akaike information criterion. Validation of PAART was performed using binary mixtures of monoclonal antibodies of same specificity but differing in kd of the interaction with their epitope. We applied PAART to examine the heterogeneity in avidities of pAb from malaria and typhoid vaccinees, and individuals living with HIV-1 that naturally control the viral load. In many cases, two to three kd were dissected indicating the heterogeneity of pAb avidities. We showcase examples of affinity maturation of vaccine induced pAb responses at component level and enhanced resolution of heterogeneity in avidity when antigen-binding fragments (Fab) are used instead of polyclonal IgG antibodies. The utility of PAART can be manifold in examining circulating pAb characteristics and could inform vaccine strategies aimed to guide the host humoral immune response.
摘要：
多克隆抗体（pAb）反应特异性的多样性在疫苗效力或免疫学评估中得到了广泛的研究。但是由于缺乏方便的工具，很少研究抗体亲和力的异质性。在这里，我们已经开发了多克隆抗体亲和力分辨率工具（PAART）用于无标记技术，如表面等离子体共振和生物层干涉，可以实时监测pAb-抗原相互作用以测量解离速率常数(kd)来定义亲合力。PAART利用指数模型的总和来拟合pAb-抗原相互作用的解离时间进程，并解析有助于整体解离的多个kd。通过PAART解析的pAb解离的每个kd值对应于具有相似亲合力的一组抗体。PAART旨在确定解释分离过程所需的最小指数数，并通过使用Akaike信息准则对最佳模型进行简约选择来防止数据过度拟合。使用具有相同特异性但与它们的表位相互作用的kd不同的单克隆抗体的二元混合物进行PAART的验证。我们应用PAART来检查来自疟疾和伤寒疫苗的pAb亲和力的异质性，以及自然控制病毒载量的HIV-1感染者。在许多情况下,解剖两到三kd，表明pAb亲和力的异质性。当使用抗原结合片段（Fab）代替多克隆IgG抗体时，我们展示了在组分水平上疫苗诱导的pAb应答的亲和力成熟以及亲和力异质性的增强分辨率的例子。PAART的效用在检查循环pAb特性方面可以是多方面的，并且可以为旨在指导宿主体液免疫应答的疫苗策略提供信息。
