title: Flow cytometry: an alternative method for direct quantification of antigens adsorbed to aluminum hydroxide adjuvant.
abstract: Flow cytometry (FC) has been widely used in biological research; however, its use for vaccine characterization has been very limited. Here we describe the development of an FC method for the direct quantification of two Neisseria meningitidis vaccine antigens, in mono- and multivalent formulations, while still adsorbed on aluminum hydroxide (AH) suspension. The antibody-based method is specific and sensitive. Because FC allows microscopic particle examination, the entire aluminum suspension carrying adsorbed antigen(s) can be analyzed directly. In addition to determining antigen concentration and identity, the assay is able to determine the distribution of the antigens on AH. High correlation coefficients (r(2)) were routinely achieved for a broad range of antigen doses from 0 to 150 μg/dose. Traditional assays for quantitative and qualitative antigen characterization on AH particles involve either complete aluminum dissolution or antigen desorption from the adjuvant. Because our direct method uses the whole AH suspension, the cumbersome steps used by traditional methods are not required. Those steps are often inefficient in desorbing the antigens and in some cases can lead to protein denaturation. We believe that this novel FC-based assay could circumvent some of the complex and tedious antigen-adjuvant desorption methods.
authors.affiliation: Novartis Vaccines and Diagnostics, Cambridge, MA 02139, USA. mildred.ugozzoli@novartis.com
authors.forename: Mildred
authors.initials: M
authors.lastname: Ugozzoli
authors.forename: Donatello
authors.initials: D
authors.lastname: Laera
authors.forename: Sandra
authors.initials: S
authors.lastname: Nuti
authors.forename: David A G
authors.initials: DA
authors.lastname: Skibinski
authors.forename: Simone
authors.initials: S
authors.lastname: Bufali
authors.forename: Chiara
authors.initials: C
authors.lastname: Sammicheli
authors.forename: Simona
authors.initials: S
authors.lastname: Tavarini
authors.forename: Manmohan
authors.initials: M
authors.lastname: Singh
authors.forename: Derek T
authors.initials: DT
authors.lastname: O'Hagan
mesh_terms: D000276:Adjuvants, Immunologic / Q000737:chemistry*; D000327:Adsorption; D000536:Aluminum Hydroxide / Q000737:chemistry*; D000956:Antigens, Viral / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D015151:Immunoblotting; D008589:Meningococcal Infections / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D022401:Meningococcal Vaccines / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D009345:Neisseria meningitidis / Q000276:immunology / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; D000956:Antigens, Viral; D022401:Meningococcal Vaccines; D000536:Aluminum Hydroxide