SI-traceable calibration-free analysis for the active concentration of G2-EPSPS protein using surface plasmon...

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 100μLmin^-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 × 10⁶M^-1s^-1, 5.79 ± 0.06 ×10^-3s^-1, and 2.65 ± 0.06 × 10^-9M, 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 1.5-8nM. The appropriate concentration range of G2-EPSPS in the proposed method was determined to be 1.5-8nM. 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.