Research Article: A novel flow cytometry-based assay for the quantification of antibody-dependent pneumococcal agglutination

Date Published: March 13, 2017

Publisher: Public Library of Science

Author(s): Marrit N. Habets, Saskia van Selm, Christa E. van der Gaast—de Jongh, Dimitri A. Diavatopoulos, Marien I. de Jonge, Paulo Lee Ho.


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.

Partial Text

Nasopharyngeal colonization with the respiratory pathogen Streptococcus pneumoniae is a prerequisite for the development of pneumococcal disease. Following dissemination of bacteria to the ear, lung, bloodstream or brain, otitis media, pneumonia, sepsis or meningitis may develop, respectively. Several mucosal defense mechanisms, such as antibody-mediated opsonization and opsonophagocytosis by phagocytes, have been suggested to be important in the reduction or complete prevention of colonization [1,2]. Recently, Roche et al. (2015) showed that the presence of agglutinating antibodies on the mucosal surface plays an important role in the prevention of pneumococcal colonization in a mouse model of colonization and transmission [3]. The agglutinating properties of antibodies raised against novel vaccine candidates might therefore be predictive for efficacy, and would be an important parameter to include in clinical trials.

Antibody-mediated agglutination of pneumococci has recently been shown to be an important factor in the prevention of pneumococcal colonization [3]. While bacterial agglutination may be analyzed using methods such as light microscopy, they are generally not very suitable for quantification and/or high-throughput analysis. Thus, we present a novel flow cytometry-based assay to quantify the pneumococcal agglutinating capacity of serum. This assay can be easily adapted to incorporate other strains and/or other bacterial species and allows screening of serum samples in a high-throughput manner. Finally, we show that both anti-capsule and anti-protein antibodies are able to induce agglutination.




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