title: A novel flow cytometry-based assay for the quantification of antibody-dependent pneumococcal agglutination.
abstract: The respiratory pathogen Streptococcus pneumoniae is a major cause of diseases such as otitis media, pneumonia, sepsis and meningitis. The first step towards infection is colonization of the nasopharynx. Recently, it was shown that agglutinating antibodies play an important role in the prevention of mucosal colonization with S. pneumoniae. Here, we present a novel method to quantify antibody-dependent pneumococcal agglutination in a high-throughput manner using flow cytometry. We found that the concentration of agglutinating antibodies against pneumococcal capsule are directly correlated with changes in the size and complexity of bacterial aggregates, as measured by flow cytometry and confirmed by light microscopy. Using the increase in size, we determined the agglutination index. The cutoff value was set by measuring a series of non-agglutinating antibodies. With this method, we show that not only anti-polysaccharide capsule antibodies are able to induce agglutination but that also anti-PspA protein antibodies have agglutinating capabilities. In conclusion, we have described and validated a novel method to quantify pneumococcal agglutination, which can be used to screen sera from murine or human vaccination studies, in a high-throughput manner.
authors.affiliation: Laboratory of Pediatric Infectious Diseases, Department of Pediatrics, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.
authors.forename: Marrit N
authors.initials: MN
authors.lastname: Habets
authors.affiliation: Laboratory of Pediatric Infectious Diseases, Department of Pediatrics, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.
authors.forename: Saskia
authors.identifier: http://orcid.org/0000-0002-9121-1043
authors.initials: S
authors.lastname: van Selm
authors.affiliation: Laboratory of Pediatric Infectious Diseases, Department of Pediatrics, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.
authors.forename: Christa E
authors.initials: CE
authors.lastname: van der Gaast-de Jongh
authors.affiliation: Laboratory of Pediatric Infectious Diseases, Department of Pediatrics, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.
authors.forename: Dimitri A
authors.initials: DA
authors.lastname: Diavatopoulos
authors.affiliation: Laboratory of Pediatric Infectious Diseases, Department of Pediatrics, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.
authors.forename: Marien I
authors.initials: MI
authors.lastname: de Jonge
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006360:Heat-Shock Proteins / Q000276:immunology; D051379:Mice; D013296:Streptococcus pneumoniae / Q000276:immunology / Q000502:physiology*
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; C069816:phage shock protein, Bacteria