Analytica chimica acta · 2025
A facile and sensitive protein immune polyacrylamide gel electrophoresis with online fluorescent imaging.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Zou Rui, Yu Zixian, Gao Jing, Zhang Qiang, Cao Yiren, Lin Xiaoyi, Peng Yibing, Liu Weiwen, Wu Jianguang, Cao Chengxi
Аннотация
BACKGROUND: Polyacrylamide gel electrophoresis (PAGE) has been widely used for separation and detection of protein since 1950s. However, its inability to facilely and specifically detect target protein in complex sample remains a significant limitation. Traditional approaches for specific protein detection, such as western blotting (WB) or immunofixation electrophoresis (IFE), are often hampered by complex procedures, time-consuming processing, and semi-quantitative assay. A simple, rapid, and quantitative method for specific protein detection and analysis remains an urgent need in both research and clinical applications.
RESULTS: Herein, we have developed a novel method of immune PAGE with online fluorescence imaging (PAGE-FI) without pre-purification of target protein for selective target antigen detection by using anti-human epidermal growth factor receptor 2 (anti-HER2 mAb) and anti-human IgG antibody labeled with fluorescein isothiocyanate (anti-human Ab-FITC) as the model antigen and antibody, respectively. In the method, anti-HER2 antigen and anti-human Ab-FITC were mixed together and incubated for more than 1 h, then the mixture sample were separated and imaged by using an independently-invented PAGE-FI system, and the real-time monitoring could be carried out on the separation of anti-human Ab-FITC and immune complexes of anti-HER2∼anti-human Ab-FITC. The method demonstrates excellent analytical performance with good selectivity, wide linear range (10-10000 ng), high sensitivity (LOD = 5 ng), good stability (RSD <5.21 %) and reliable recovery (95-105 %). Moreover, the method utilizes cost-effective anti-human Ab-FITC and completes the whole process within 1.5 h. Finally, the developed method was briefly used for the analyses of myeloma subtype ntibodies in complex serum samples.
SIGNIFICANCE: This work illuminated a highly promising immune PAGE for the detection of target analyte with high selectivity, simplicity, sensitivity, stability, rapidity and low cost but no treatment of pre-purification of the target, indicating great potential for diverse applications of clinical diagnosis, antibody screening, and investigation on affinity interaction of protein-protein and protein-nucleic acid etc.
RESULTS: Herein, we have developed a novel method of immune PAGE with online fluorescence imaging (PAGE-FI) without pre-purification of target protein for selective target antigen detection by using anti-human epidermal growth factor receptor 2 (anti-HER2 mAb) and anti-human IgG antibody labeled with fluorescein isothiocyanate (anti-human Ab-FITC) as the model antigen and antibody, respectively. In the method, anti-HER2 antigen and anti-human Ab-FITC were mixed together and incubated for more than 1 h, then the mixture sample were separated and imaged by using an independently-invented PAGE-FI system, and the real-time monitoring could be carried out on the separation of anti-human Ab-FITC and immune complexes of anti-HER2∼anti-human Ab-FITC. The method demonstrates excellent analytical performance with good selectivity, wide linear range (10-10000 ng), high sensitivity (LOD = 5 ng), good stability (RSD <5.21 %) and reliable recovery (95-105 %). Moreover, the method utilizes cost-effective anti-human Ab-FITC and completes the whole process within 1.5 h. Finally, the developed method was briefly used for the analyses of myeloma subtype ntibodies in complex serum samples.
SIGNIFICANCE: This work illuminated a highly promising immune PAGE for the detection of target analyte with high selectivity, simplicity, sensitivity, stability, rapidity and low cost but no treatment of pre-purification of the target, indicating great potential for diverse applications of clinical diagnosis, antibody screening, and investigation on affinity interaction of protein-protein and protein-nucleic acid etc.