title: Development of a large scale human complement source for use in bacterial immunoassays.
abstract: The serum bactericidal assay is the correlate of protection for meningococcal disease but the use and comparison of functional immunological assays for the assessment of meningococcal vaccines is complicated by the sourcing of human complement. This is due to high levels of immunity in the population acquired through natural meningococcal carriage and means that many individuals must be screened to find donors with suitably low bactericidal titres against the target strain. The use of different donors for each meningococcal strain means that comparisons of assay responses between strains and between laboratories is difficult. We have developed a method for IgG-depletion of 300 ml batches of pooled human lepirudin-derived plasma using Protein G sepharose affinity chromatography that retains complement activity. However, IgG-depletion also removed C1q. This was also eluted from the affinity matrix, concentrated and added to the complement source. The final complement source retained mean alternative pathway activity of 96.8% and total haemolytic activity of 84.2% in four batches. Complement components C3, C5, properdin and factor H were retained following the process and the IgG-depleted complement was shown to be suitable for use in antibody-mediated complement deposition and serum bactericidal activity assays against serogroup B meningococci. The generation of large IgG-depleted batches of pooled human plasma allows for the comparison of immunological responses to diverse meningococcal strain panels in large clinical trials.
authors.affiliation: Health Protection Agency, Porton Down, Salisbury, UK.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Brookes
authors.forename: Eeva
authors.initials: E
authors.lastname: Kuisma
authors.forename: Frances
authors.initials: F
authors.lastname: Alexander
authors.forename: Lauren
authors.initials: L
authors.lastname: Allen
authors.forename: Thomas
authors.initials: T
authors.lastname: Tipton
authors.forename: Sanjay
authors.initials: S
authors.lastname: Ram
authors.forename: Andrew
authors.initials: A
authors.lastname: Gorringe
authors.forename: Stephen
authors.initials: S
authors.lastname: Taylor
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000302:isolation & purification; D002846:Chromatography, Affinity; D015922:Complement C1q / Q000032:analysis; D003176:Complement C3 / Q000032:analysis; D003182:Complement C5 / Q000032:analysis; D017242:Complement Factor H / Q000032:analysis; D003170:Complement Pathway, Alternative; D003165:Complement System Proteins / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D006461:Hemolysis; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D009345:Neisseria meningitidis / Q000276:immunology*; D015203:Reproducibility of Results; D058248:Serum Bactericidal Antibody Assay / Q000379:methods*
chemical_list: D000907:Antibodies, Bacterial; C493495:CFH protein, human; D003176:Complement C3; D003182:Complement C5; D007074:Immunoglobulin G; D015922:Complement C1q; D017242:Complement Factor H; D003165:Complement System Proteins