A facile and sensitive protein immune polyacrylamide gel electrophoresis with online fluorescent imaging.

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.