[A protein-specific quantitative detection method based on polyacrylamide gel electrophoresis and online fluorescence imaging].

Specific protein detection plays a crucial role in biological analysis and clinical diagnostics， serving as an essential tool for disease diagnosis， therapeutic monitoring， and biological research. However， conventional methods such as immunofixation electrophoresis （IFE） and western blotting （WB） suffer from complex workflows， time-consuming operations， and limited quantification capabilities owing to intricate staining and de-staining procedures. In addition， these traditional immunological detection methods require extensive manual handling and specialized expertise， while low levels of automation restrict their applicability to high-throughput or large-scale analysis scenarios. Moreover， the multistep nature of these methods increases the risk of experimental errors and compromises quantification accuracy.Herein， we present a quantitative protein immune polyacrylamide gel electrophoresis （PAGE） detection method that combines immune-recognition principles with online fluorescence imaging technology， thereby offering a rapid and specific approach for quantifying target proteins. The developed method exploits the specificity of fluorescently labeled antibodies and the separation capability of PAGE， with formaldehyde crosslinking used to stabilize antigen-antibody complexes under the denaturing conditions of electrophoresis， thereby ensuring reliable quantification. The entire experimental workflow can be completed within 1.5 h and consists of three main steps. Firstly， the target protein is incubated with fluorescently labeled antibodies at room temperature for 0.5 h to form immune complexes， after which they are crosslinked using formaldehyde. The cross-linked samples are then loaded onto polyacrylamide gels and separated under optimized electrophoresis conditions （120 V， 15 min； 150 V， 15 min； 200 V， 15 min）. Electrophoretic separation is finally monitored in real-time using an online fluorescence imaging system， which enables direct visualization of protein migration and eliminates the need for post-separation processing. Three distinct bands are observed on the precast gel following immune PAGE separation： the immune complexes at the uppermost position， the free fluorescent antibodies in the middle， and other proteins at the bottom. ImageJ software is used to analyze the electrophoresis pattern， and quantification is achieved based on the linear relationship between the fluorescence intensity of the free antibody and the mass concentration of the target protein. We systematically validated the performance of the method using human transferrin （TRF） as the model antigen protein and fluorescein-isothiocyanate-labeled （FITC-labeled） anti-TRF IgG antibody （anti-TRF IgG-FITC） as the detection probe， which involved analyzing three key aspects： the necessity of the formaldehyde-crosslinking step for maintaining immune complex stability， antibody recognition specificity in complex samples， and the linear correlation between the fluorescence signal and the mass concentration of the target protein. The method exhibited excellent analytical performance， with a linear range that extended between 5.0 and 200.0 mg/L and a correlation coefficient （