title: Development of an opsonin inhibition assay for evaluation of complex polysaccharide protective epitopes.
abstract: The induction of opsonic antibodies directed against capsular polysaccharides (Ps) is an important mechanism by which immunization protects against the development of invasive pneumococcal (Pn) infection. In preparing Pn vaccines, it is necessary to compare different manufacturing lots of capsular Ps, or to compare oligosaccharides used for conjugate vaccines with native capsular Ps, in order to insure that important epitopes of the Ps are maintained. We have developed an opsonic-antibody inhibition assay (OIA) to compare the functional epitopes of different capsular Ps preparations in vitro. Components of the OIA are primary neutrophils, rabbit complement (C'), and type-specific antibody (Ab). After conditions for optimal opsonic killing were determined for each Pn serotype, anti-Pn Ab was pre-incubated with different dilutions of purified capsular Ps, then added to the OIA mix. Plotting the % bacteria killed versus Ps concentration (log transformed) yielded a linear curve that was used to quantify the concentration of capsular Ps which inhibited the bacteria killing by 50% (IC50). The IC50 was determined for 8 Pn Ps types. These ranged between 6 ng/ml for type 6B and 1268 ng/ml for type 23F. Importantly OIA curves were statistically identical for two different manufacturing lots of capsular Ps for the 8 Pn Ps types. We conclude that differences among capsular Ps used for Pn vaccines could be detected with an OIA assay and these differences may predict the ability of Ps preparations to induce functionally active antibody when formulated into vaccines.
authors.affiliation: Merck Research Labs, Merck Inc., WP26-253, West Point, PA 19486, USA. tessie_mcneely@merck.com
authors.forename: Tessie
authors.initials: T
authors.lastname: McNeely
authors.forename: Shengyuan
authors.initials: S
authors.lastname: Luo
authors.forename: Walt
authors.initials: W
authors.lastname: Manger
authors.forename: Wayne
authors.initials: W
authors.lastname: Herber
authors.forename: Tim
authors.initials: T
authors.lastname: Schofield
authors.forename: Charles
authors.initials: C
authors.lastname: Tan
authors.forename: Kathy
authors.initials: K
authors.lastname: Newman
authors.forename: Jerald
authors.initials: J
authors.lastname: Sadoff
authors.forename: John
authors.initials: J
authors.lastname: Donnelly
authors.forename: Alan
authors.initials: A
authors.lastname: Cross
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D000942:Antigens, Bacterial / Q000032:analysis / Q000276:immunology; D016667:Bacterial Capsules / Q000032:analysis / Q000276:immunology*; D015169:Colony Count, Microbial; D003165:Complement System Proteins; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D005260:Female; D006801:Humans; D007158:Immunologic Techniques; D020128:Inhibitory Concentration 50; D008253:Macaca mulatta; D008297:Male; D009504:Neutrophils / Q000276:immunology; D009895:Opsonin Proteins / Q000276:immunology*; D010587:Phagocytosis; D013296:Streptococcus pneumoniae / Q000276:immunology*
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D000939:Epitopes; D009895:Opsonin Proteins; D003165:Complement System Proteins