Carter Robert M, Blake Robert C, Nguyen Trong D, Bostanian Levon A
College of Pharmacy, Division of Basic Pharmaceutical Sciences, Xavier University of Louisiana, 1 Drexel Drive, New Orleans, LA 70125, USA.
Biosens Bioelectron. 2003 Jan;18(1):69-72. doi: 10.1016/s0956-5663(02)00107-0.
A fluorescent biosensor assay has been developed for near real-time detection of 2,4-dinitrophenol (DNP). The assay was based on fluorescent detection principles that allow for the analysis of antibody/antigen interactions in solution using the KinExA immunoassay instrument. Our KinExA consisted of a capillary flow observation cell containing a microporous screen that maintains a compact capture antigen-coated bead bed. The bead bed was comprised of polymethylmethacrylate (PMMA) beads coated with dinitrophenol-human serum albumin (DNP-HSA) conjugate. Phosphate buffered saline (PBS) solutions, containing various concentrations of free DNP, were incubated for 30 min with mouse anti-DNP monoclonal antibody to equilibrium. Solutions containing the DNP-monoclonal antibody complex and possible excess free antibodies were then passed over DNP-HSA labeled beads. The free monoclonal anti-DNP antibody, if available, was then bound to the DNP-HSA fixed on the beads. The system was then flushed with excess PBS to remove unbound reactants in the bead bed. The beads were then subjected to brief contact with PBS solutions containing goat anti-mouse fluorescein isothiocyanate (FITC)-labeled secondary antibody, once again, followed by a short PBS flush. The fluorescence was recorded during the addition of the FITC labeled secondary antibody to the bead bed through the final PBS flushing with the KinExA. The amount of DNP detected could then be determined from the fluorescent slopes that were generated or by the remaining fluorescence that was retained on the beads after final PBS flushing of the system. This assay has been able to detect a minimum of 5 ng/ml of DNP in solution and can be adapted for other analytes of interest simply by changing the capture antigen and antibody pairs.
已开发出一种荧光生物传感器检测方法，用于近实时检测2,4-二硝基苯酚（DNP）。该检测方法基于荧光检测原理，可使用KinExA免疫分析仪器分析溶液中的抗体/抗原相互作用。我们的KinExA由一个毛细管流动观察池组成，该池包含一个微孔筛，用于维持紧密的捕获抗原包被珠床。珠床由涂有二硝基苯酚-人血清白蛋白（DNP-HSA）偶联物的聚甲基丙烯酸甲酯（PMMA）珠组成。将含有不同浓度游离DNP的磷酸盐缓冲盐水（PBS）溶液与小鼠抗DNP单克隆抗体孵育30分钟至平衡。然后将含有DNP-单克隆抗体复合物和可能过量的游离抗体的溶液通过DNP-HSA标记的珠子。如果有游离的单克隆抗DNP抗体，它会与固定在珠子上的DNP-HSA结合。然后用过量的PBS冲洗系统，以去除珠床中未结合的反应物。然后使珠子与含有山羊抗小鼠异硫氰酸荧光素（FITC）标记的二抗的PBS溶液短暂接触，再次冲洗后，再用PBS短暂冲洗。在通过KinExA向珠床中加入FITC标记的二抗直至最终用PBS冲洗的过程中记录荧光。然后可以根据产生的荧光斜率或系统最终用PBS冲洗后珠子上保留的剩余荧光来确定检测到的DNP量。该检测方法能够检测溶液中低至5 ng/ml的DNP，并且只需改变捕获抗原和抗体对，就可适用于其他感兴趣的分析物。
