Single-Cell Analysis of B Cell/Antibody Cross-Reactivity Using a Novel Multicolor FluoroSpot Assay

• Published in: The Journal of immunology (1950) Vol. 195; no. 7; pp. 3490 - 3496
• Main Authors: Hadjilaou, Alexandros, Green, Angela M, Coloma, Josefina, Harris, Eva
• Format: Journal Article
• Language: English
• Published: United States 01.10.2015
• Subjects: Aedes; Animals; Antibodies, Monoclonal - immunology; Antibodies, Viral - immunology; Antigens, Viral - analysis; Antigens, Viral - immunology; B-Lymphocytes - immunology; Cells, Cultured; Cross Reactions - immunology; Dengue - immunology; Dengue - virology; Dengue Vaccines - immunology; Dengue virus; Dengue Virus - classification; Dengue Virus - genetics; Dengue Virus - immunology; Enzyme-Linked Immunospot Assay - methods; Humans; Single-Cell Analysis; Vaccination; Viral Envelope Proteins - immunology
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.1500918

Dengue is a major public health problem globally. It is caused by four antigenically distinct serotypes of dengue virus (DENV1-4), and although serotype-specific and strongly neutralizing cross-reactive immune responses against the four DENV serotypes are thought to be protective, subneutralizing Abs can contribute to increased disease severity upon secondary infection with a different DENV serotype. Understanding the breadth of the immune response in natural DENV infections and in vaccinees is crucial for determining the correlates of protection or disease severity. Transformation of B cell populations to generate mAbs and ELISPOT assays have been used to determine B cell and Ab specificity to DENV; however, both methods have technical limitations. We therefore modified the conventional ELISPOT to develop a Quad-Color FluoroSpot to provide a means of examining B cell/Ab serotype specificity and cross-reactivity on a single-cell basis. Abs secreted by B cells are captured by an Fc-specific Ab on a filter plate. Subsequently, standardized concentrations of all four DENV serotypes are added to allow equal stoichiometry for Ag binding. After washing, the spots, representing individual B cells, are visualized using four fluorescently labeled DENV serotype-specific detection mAbs. This method can be used to better understand the breadth and magnitude of B cell responses following primary and secondary DENV infection or vaccination and their role as immune correlates of protection from subsequent DENV infections. Furthermore, the Quad-Color FluoroSpot assay can be applied to other diseases caused by multiple pathogen serotypes in which determining the serotype or subtype-specific B cell response is important.