Immunological Study of Responses to a Meningococcal Antigen Using a Multiplex Antibody Binding Assay

Patel, Hema (2014). Immunological Study of Responses to a Meningococcal Antigen Using a Multiplex Antibody Binding Assay. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f038

Abstract

Neisseria meningitidis is an important human pathogen and a major cause of septicaemia and meningitis worldwide. The immunodominant antigen of N. meningitidis is the highly variable OMP, PorA and detection of serum anti-PorA antibodies (IgG) is classically performed through use of the enzyme-linked immunosorbent assay (ELISA). Whilst sensitive and reliable, these methods are labour and reagent intensive particularly for detection of multiple anti-PorA antibodies within a single serum sample.

This thesis describes the development and evaluation of an assay for simultaneous detection of antibodies directed to eight serosubtypes of PorA. A panel of purified meningococcal proteins were developed including seven serosubtypes of PorA along with a variable regions 1 and 2 deleted mutant; three serotypes of PorB and five variants of FetA. PorA proteins were conjugated to fluorescent microsphere sets and assessed using serum from a range of sources including preclinical and clinical trials and carriage and seroepidemiology studies.

No evidence of microsphere interference was observed between monoplex and multiplex assays over a range of dilutions. The multiplex assay was specific; sensitive, with low limits of detection (<176.88 pg/ml); and reproducible for the measurement of serotype specific anti-PorA antibodies, with low intra- and inter-assay variability.

Increases in serum IgG against specific PorA serosubtypes were detected using the assay in serum from a range of sources. However with the complexity of adult serum, it is difficult to distinguish between pre-existing and induced responses and may result in what appear to be non-specific responses. Lack of a suitable human standard has hindered the quantification of antibodies and an arbitrary concentration was assigned for each PorA serosubtype. However, advantages of small sera volumes, high throughput of sera, simplicity of the assay, and the ability to extend the assay, make the PorA multiplex assay a viable alternative to the standard ELISA.

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