Comparison of Colorimetric Assays with Quantitative Amino Acid Analysis for Protein Quantification of Generalized Modules for Membrane Antigens (GMMA)

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• 作者：Omar Rossi (1)
  Luana Maggiore (1)
  Francesca Necchi (1)
  Oliver Koeberling (1)
  Calman A. MacLennan (1)
  Allan Saul (1)
  Christiane Gerke (1)
  
  1. Novartis Vaccines Institute for Global Health ; Via Fiorentina 1 ; 53100 ; Siena ; Italy
  
• 关键词：Protein quantification ; Generalized Modules for Membrane Antigens (GMMA) ; Outer Membrane Vesicles (OMV) ; Quantitative amino acid analysis ; Colorimetric protein assay ; Lowry assay ; Bradford assay ; Non ; Interfering assay
• 刊名：Molecular Biotechnology
• 出版年：2015
• 出版时间：January 2015
• 年：2015
• 卷：57
• 期：1
• 页码：84-93
• 全文大小：1,078 KB
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• 刊物主题：Biotechnology; Biochemistry, general; Cell Biology; Protein Science; Biological Techniques; Human Genetics;
• 出版者：Springer US
• ISSN：1559-0305

文摘

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.