title: Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).
abstract: Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.
authors.affiliation: Novartis Vaccines Institute for Global Health, Via Fiorentina 1, 53100, Siena, Italy.
authors.forename: Omar
authors.initials: O
authors.lastname: Rossi
authors.forename: Luana
authors.initials: L
authors.lastname: Maggiore
authors.forename: Francesca
authors.initials: F
authors.lastname: Necchi
authors.forename: Oliver
authors.initials: O
authors.lastname: Koeberling
authors.forename: Calman A
authors.initials: CA
authors.lastname: MacLennan
authors.forename: Allan
authors.initials: A
authors.lastname: Saul
authors.forename: Christiane
authors.initials: C
authors.lastname: Gerke
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D000942:Antigens, Bacterial / Q000032:analysis*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D003116:Color; D003124:Colorimetry / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005838:Genotype; D010641:Phenotype; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000032:analysis*
chemical_list: D000596:Amino Acids; D000942:Antigens, Bacterial; D012710:Serum Albumin, Bovine