Sapan C V, Lundblad R L, Price N C
NABI, 5800 Park of Commerce Boulevarde, NW, Boca Raton, FL 33487, USA.
Biotechnol Appl Biochem. 1999 Apr;29(2):99-108.
There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.
在过去20年中，用于测定蛋白质浓度的比色测定技术的数量有所增加。随着新技术的出现，这导致了灵敏度和准确性的明显提高。本综述考虑了这些进展，重点是此类技术在生物制药分析中的潜在用途。所综述的技术包括考马斯亮蓝G-250染料结合法（Bradford法）、Lowry法、二辛可宁酸法和双缩脲法。结果表明，每种测定方法在灵敏度、操作简便性、文献认可度、准确性和重现性/变异系数/实验室间差异方面都有优缺点。本文还对使用几种测定方法分析相同样本群体的情况进行了比较。有人提出，在使用显色蛋白质测定法来表征生物制药时，最关键的问题是选择用于校准测定的标准品；标准品必须能够代表样品，这一点至关重要。如果从蛋白质组成的角度无法使标准品与样品匹配，那么最好使用对蛋白质组成不敏感的测定方法，如微量凯氏定氮法、定量氨基酸分析或双缩脲法。在复杂混合物中，专注于蛋白质测定的通用方法可能不合适，而使用与特定感兴趣蛋白质相关的特定方法（使用特定测定法或基于抗体的方法）可能更具信息价值。关键在于，无论采用何种方法作为给定蛋白质的“金标准”，都需要常规使用该方法进行校准。
