RESUMO
Determination of protein concentration in vaccines containing aluminum salt adjuvant typically necessitates desorption of the protein prior to analysis. Here we describe a method based on the intrinsic fluorescence of tyrosine and tryptophan that requires no desorption of proteins. Adjuvanted formulations of three model Bordetella pertussis antigens were excited at 280â¯nm and their emission spectra collected from 290 to 400â¯nm. Emission spectra of protein antigens in the presence of aluminum salt adjuvants were able to be detected, the effects of adjuvants on the spectra were analyzed, and linear regressions were calculated. The fluorescence method proved to be very sensitive with a limit of quantification between 0.4 and 4.4⯵g/mL and limit of linearity between 100 and 200⯵g/mL, across the formulations tested. The fluorescence method was found to be influenced by adjuvant presence, type of adjuvant, adjuvant concentration, buffer and pH conditions. The method also demonstrated ability to monitor the percent adsorption of antigens to the adjuvants. Furthermore, intrinsic fluorescence showed good correlation with micro-Kjeldahl elemental assay in quantifying protein concentration. Being a non-invasive, quick and sensitive method, intrinsic fluorescence has the potential to be utilized as a high throughput tool for vaccine development and conceivably implemented in-line, using in-line fluorimeters, to monitor antigen concentration during formulation processing.
Assuntos
Adjuvantes Imunológicos/química , Hidróxido de Alumínio/química , Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Bordetella pertussis/química , Medições Luminescentes/métodos , Adesinas Bacterianas/análise , Adesinas Bacterianas/química , Proteínas da Membrana Bacteriana Externa/análise , Proteínas da Membrana Bacteriana Externa/química , Fímbrias Bacterianas/química , Fluorescência , Humanos , Triptofano/química , Tirosina/química , Vacinas/imunologia , Fatores de Virulência de Bordetella/análise , Fatores de Virulência de Bordetella/químicaRESUMO
degP-deficient strains of Escherichia coli grown in M-9 medium supplemented with ZnCl2 expressed the recombinant S1 subunit of pertussis toxin (rS1) in a form electrophoretically identical to the authentic S1 subunit. Subcellular fractionation showed that the full-length form of rS1 was membrane associated, while proteolytic fragments of rS1 were present in the periplasm. rS1 was extracted from outer membrane preparations with 8 M urea and purified by gel filtration chromatography. Purified rS1 ADP-ribosylated transducin at a similar molar efficiency relative to authentic pertussis toxin and, when associated with the native B oligomer of pertussis toxin, elicited Chinese hamster ovary cell clustering.