High throughput measurement of antibody and complement mediated immunity to meningococcal disease

Brookes, Charlotte (2012). High throughput measurement of antibody and complement mediated immunity to meningococcal disease. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000d58a


The accepted correlate of protection for meningococcal disease is serum bactericidal activity. The importance of bactericidal activity was established by Goldschneider et al. (1969) who demonstrated an inverse relationship between disease incidence and bactericidal titres of ≥1:4. The importance of antibody and complement mediated immune mechanisms is also emphasised by the increased susceptibility of complement deficient individuals to meningococcal disease.

However, the absence of bactericidal activity does not necessarily indicate susceptibility and protection has been demonstrated in the absence of bactericidal activity. This has indicated the importance of other protection mechanisms such as opsonophagocytosis.

In this study a high throughput antibody-mediated complement deposition assay has been developed to measure the deposition of both C3b/iC3b and C5b-9 onto Neisseria meningitidis. Antibody mediated C5b-9 deposition measured in this assay has been shown to correlate highly with bactericidal activity for several strains and C3b/iC3b deposition has been shown to correlate with opsonophagocytosis. This assay has also been shown to be reproducible. Measurement of antibody mediated C5b-9 and C3b/iC3b deposition will not be a replacement for opsonic killing assays or the accepted correlate SBA, but could be used as a tool to evaluate large panels of sera against multiple strains due to the very low serum volumes required.

A respiratory burst assay has also been developed to measure both the uptake of fluorescently-stained bacteria and the induction of a respiratory burst response. This assay was optimised with respect to the reagents used to measure the respiratory burst response, bacterial stain and the assay buffers.

Complement is an important reagent used in immunoassays to evaluate antibody mediated immunity to N. meningitidis. Due to the high levels of nasopharyngeal carriage in adults it is difficult to obtain a complement source without cross- reactive bactericidal activity, often resulting in the requirement for a source from a different individual for each strain. An IgG-depleted complement source has been developed for use in immunoassays. The method developed has been shown to be reproducible and effective at removing the IgG whilst retaining functional complement and will be a valuable tool for assessing immunity to N. meningitidis.

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