Beijing University of Chemical Technology, No. 15 North Third Ring Road, Chaoyang District, Beijing, 100029, China.
National Institute of Metrology, No. 18 North Third Ring Road, Chaoyang District, Beijing, 100029, China.
Anal Bioanal Chem. 2020 May;412(12):2777-2784. doi: 10.1007/s00216-020-02504-z. Epub 2020 Feb 20.
The activity of proteins rather than the concentration of proteins in biopharmaceutical and in vitro diagnostics are often the primary focus. Nonetheless, development of a calibration-free concentration analysis (CFCA) approach that accurately quantifies the concentration of proteins based on molecular interactions with specific monoclonal antibodies and without the requirement of external calibrators would be beneficial to diagnostics. Generally, only analytes that interact with the antibody (Ab) are quantified by CFCA. Moreover, protein concentrations measured by CFCA usually vary when different Abs are used, and are lower than those obtained by amino acid analysis because any non-native state population of the target protein is not captured by the Ab. To achieve comparable results between CFCA and traditional amino acid analysis (AAA), an Ab that recognizes the target protein irrespective of its conformation should be used. In this report, three different monoclonal antibodies were used to quantify purified human myoglobin in solution by CFCA. The concentrations obtain by the Abs (i.e., 2.985, 2.912, 3.032 mg mL) were comparable with that obtained by AAA. Moreover, isotope dilution mass spectrometry (IDMS) gave a human myoglobin concentration of 2.851 mg mL, which is also in agreement with the results from CFCA. The performance of CFCA was evaluated by measuring various parameters, including within-day and between-day precision. The results demonstrated that the active concentration measured by CFCA is comparable with that of IDMS when the appropriate Ab is used. Recommended procedures for performing the new CFCA approach are provided. This study shows that CFCA represents a primary method for accurate protein concentration determination, which should aid the development of certified reference materials. Graphical abstract.
蛋白质的活性而非浓度通常是生物制药和体外诊断的主要关注点。然而，开发一种无校准浓度分析（CFCA）方法，该方法基于与特定单克隆抗体的分子相互作用，而无需外部校准品，即可准确定量蛋白质浓度，这将对诊断学有益。通常，只有与抗体（Ab）相互作用的分析物才会被 CFCA 定量。此外，由于 CFCA 测量的蛋白质浓度不受任何非天然状态的靶蛋白的影响，因此使用不同的 Ab 时，CFCA 测量的蛋白质浓度通常会有所不同，并且低于通过氨基酸分析获得的浓度。为了在 CFCA 和传统的氨基酸分析（AAA）之间实现可比的结果，应该使用一种能够识别目标蛋白无论其构象如何的 Ab。在本报告中，使用三种不同的单克隆抗体通过 CFCA 定量溶液中纯化的人肌红蛋白。Ab 获得的浓度（即 2.985、2.912、3.032 mg mL）与 AAA 获得的浓度相当。此外，同位素稀释质谱法（IDMS）给出的人肌红蛋白浓度为 2.851 mg mL，这也与 CFCA 的结果一致。通过测量各种参数，包括日内和日间精密度，对 CFCA 的性能进行了评估。结果表明，当使用适当的 Ab 时，CFCA 测量的活性浓度与 IDMS 的结果相当。提供了执行新的 CFCA 方法的建议程序。本研究表明，CFCA 代表了一种准确测定蛋白质浓度的主要方法，这将有助于有证参考物质的开发。
