Departamento de Bioquímica y Biología Molecular, Edificio "Severo Ochoa" Planta Baja, Campus de Rabanales, Universidad de Córdoba, Córdoba, Spain.
Campus de Excelencia Internacional CeiA3, Córdoba, Spain.
Methods Mol Biol. 2021;2344:151-161. doi: 10.1007/978-1-0716-1562-1_11.
Infectious diseases represent a major cause of morbidity and mortality worldwide. Early detection of infections is capital for managing life-threatening cases. So far, traditional diagnostic methods such as microbiological cultures are slow and, sometimes, inaccurate. In the molecular era, high-throughput techniques are essential for providing tools that are able to diagnose in a fast and reliable way, as well as they can be used for monitoring the humoral response of groups of people in a program of epidemiological surveillance when an outbreak occurs, or when a vaccine is being evaluated. Antigen-based protein microarrays are an ideal means for these purposes, as they can carry up to thousands of protein antigens from pathogenic sources and be probed with sera from different human groups (acute or chronic infected people, convalescent, controls). For the diagnosis of bacterial infections, the best antigens are in principle the surface proteins, as they have the highest chances to raise an effective immune response. Here we describe a general protocol for fabricating a glass slide-based protein microarray using recombinant bacterial surface antigens, according to our own expertise in the study of pneumococcal disease. The probing with human sera aims to evaluate differences between diseased and healthy people, in order to discover discriminating antigens that can be used, after appropriate validation, in further easy-to-use formats such as immunostrips.
传染病是全球发病率和死亡率的主要原因。早期发现感染对于治疗危及生命的病例至关重要。到目前为止，传统的诊断方法（如微生物培养）速度较慢，有时也不够准确。在分子时代，高通量技术对于提供能够快速可靠地诊断的工具至关重要，并且当发生疫情或评估疫苗时，它们也可以用于监测人群的体液反应。基于抗原的蛋白质微阵列是实现这些目标的理想手段，因为它们可以携带来自病原体的多达数千种蛋白质抗原，并可以用来自不同人群（急性或慢性感染者、恢复期、对照者）的血清进行探测。对于细菌感染的诊断，原则上最好的抗原是表面蛋白，因为它们最有可能引起有效的免疫反应。在这里，我们根据我们在肺炎球菌病研究方面的专业知识，描述了一种使用重组细菌表面抗原制作玻片基蛋白质微阵列的一般方案。用人类血清探测的目的是评估患病者和健康者之间的差异，以便发现有区别的抗原，这些抗原在经过适当验证后，可以进一步用于更易于使用的格式，如免疫条。
