Active proteins play important roles in the function regulation of human bodies and attract much interest for use in pharmaceuticals and clinical diagnostics. However, the lack of primary methods to analyze active proteins means there is currently no metrology standard for active protein measurement. In recent years, calibration-free concentration analysis (CFCA), which is based on surface plasmon resonance (SPR) technology, has been proposed to determine the active concentration of proteins that have specific binding activity with a binding partner without any higher order standards. The CFCA experiment observes the changes of binding rates at totally different two flow rates and uses the known diffusion coefficient of an analyte to calculate the active concentration of proteins, theoretically required, the binding process have to be under diffusion-limited conditions. Measuring the active concentration of G2-EPSPS protein by CFCA was proposed in this study. This method involves optimization of the regeneration buffer and preparation of chip surfaces for appropriate reaction conditions by immobilizing ligands (G2-EPSPS antibodies) on sensor chips (CM5) via amine coupling. The active concentration of G2-EPSPS was then determined by injection of G2-EPSPS protein samples and running buffer over immobilized and reference chip surfaces at two different flow rates (5 and 100 μL min⁻¹). The active concentration of G2-EPSPS was obtained after analyzing these sensorgrams with the 1:1 model. Using the determined active concentration of G2-EPSPS, the association, dissociation, and equilibrium constants of G2-EPSPS and its antibody were determined to be 2.18 ± 0.03 × 10⁶ M⁻¹ s⁻¹, 5.79 ± 0.06 ×10⁻³ s⁻¹, and 2.65 ± 0.06 × 10⁻⁹ M, respectively. The performance of the proposed method was evaluated. The within-day precisions were from 3.26% to 4.59%, and the between-day precision was 8.36%. The recovery rate of the method was from 97.46% to 104.34% in the concentration range of 1.5–8 nM. The appropriate concentration range of G2-EPSPS in the proposed method was determined to be 1.5–8 nM. The active G2-EPSPS protein concentration determined by our method was only 17.82% of that obtained by isotope dilution mass spectrometry, showing the active protein was only a small part of the total G2-EPSPS protein. The measurement principle of the proposed method can be clearly described by equations and the measurement result can be expressed in SI units. Therefore, the proposed method shows promise to become a primary method for active protein concentration measurement, which can benefit the development of certified reference materials for active proteins.

活性蛋白在人体功能调节中起着重要作用，并在药物和临床诊断中引起了极大的兴趣。但是，缺乏分析活性蛋白的主要方法，意味着目前尚无用于测量活性蛋白的计量标准。近年来，已经提出了基于表面等离振子共振（SPR）技术的无标定浓度分析（CFCA），用于确定与结合配偶体具有特定结合活性的蛋白质的有效浓度，而无需任何更高级别的标准。CFCA实验观察两种完全不同流速下的结合速率变化，并使用分析物的已知扩散系数来计算理论上所需的蛋白质活性浓度，结合过程必须在扩散受限的条件下进行。这项研究提出了通过CFCA测定G2-EPSPS蛋白的活性浓度的方法。该方法包括通过胺偶联将配体（G2-EPSPS抗体）固定在传感器芯片（CM5）上，优化再生缓冲液并为适当的反应条件准备芯片表面。然后通过以两种不同的流速（分别为5和100μLmin）注入G2-EPSPS蛋白样品并在固定和参考芯片表面上运行缓冲液，测定G2-EPSPS的活性浓度。该方法包括通过胺偶联将配体（G2-EPSPS抗体）固定在传感器芯片（CM5）上，优化再生缓冲液并为适当的反应条件准备芯片表面。然后通过以两种不同的流速（分别为5和100μLmin）注入G2-EPSPS蛋白样品并在固定和参考芯片表面上运行缓冲液，测定G2-EPSPS的活性浓度。该方法包括通过胺偶联将配体（G2-EPSPS抗体）固定在传感器芯片（CM5）上，优化再生缓冲液并为适当的反应条件准备芯片表面。然后通过以两种不同的流速（分别为5和100μLmin）注入G2-EPSPS蛋白样品并在固定和参考芯片表面上运行缓冲液，测定G2-EPSPS的活性浓度。^-1）。用1：1模型分析这些传感图后，获得了G2-EPSPS的活性浓度。使用G2-EPSPS，该结合，解离，和G2-EPSPS及其抗体的平衡常数的确定的活性浓度被确定为2.18±0.03×10 ⁶ 中号^-1 小号^-1，5.79±0.06×10 ^-3 小号^-1和2.65±0.06×10 ^-9 M分别。评价了所提出方法的性能。日内精确度为3.26％至4.59％，日间精确度为8.36％。在1.5–8 nM的浓度范围内，该方法的回收率为97.46％至104.34％。在所提出的方法中，G2-EPSPS的适当浓度范围确定为1.5–8 nM。通过我们的方法测定的活性G2-EPSPS蛋白浓度仅为同位素稀释质谱法获得的浓度的17.82％，表明活性蛋白仅占总G2-EPSPS蛋白的一小部分。所提出的方法的测量原理可以通过方程式清楚地描述，并且测量结果可以用SI单位表示。因此，所提出的方法有望成为活性蛋白质浓度测量的主要方法，