Engström Henrik A, Andersson Per Ola, Gregorius Klaus, Ohlson Sten
School of Pure and Applied Natural Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden.
J Immunol Methods. 2008 Apr 20;333(1-2):107-14. doi: 10.1016/j.jim.2008.01.021. Epub 2008 Feb 20.
In this report we have evaluated the potential of using fluorescence/Förster resonance energy transfer (FRET) in a competitive immunosensor for continuous monitoring of the carbohydrate hapten maltose. The cyanine dyes Cy5 and Cy5.5 were used as a donor-acceptor pair by conjugation to maltose-labeled bovine serum albumin (BSA) and the monoclonal antibody IgG 39.5, giving Cy5-BSA-maltotriitol (3.1/1/18) and Cy5.5-mAb39.5 (2.2/1), respectively. This antibody with weak affinity towards maltose showed full reversibility to both the free maltose and the maltose-labeled conjugate. It allowed us to measure continuously the maltose content by monitoring the FRET signal change over time due to displacement of Cy5-BSA-maltotriitol from Cy5.5-mAb39.5 inside a semipermeable capsule. A near 22% total increase was seen in the fluorescence intensity ratio I(670)/I(700) in the presence of maltose, with a calculated EC(50)=1.87+/-0.13 mM (R(2)=0.9984) from the sigmoidal dose-response curve at 25 degrees C. Specificity of the immunosensor was shown with the structural analog to maltose, cellobiose, and it generated no detectable response. A minor drift in the sensor baseline was seen with 0.4% per 24 h, which was in the same magnitude as the signal-to-noise ratio, during the 4 weeks of measurements. The immunosensor was applied to crude samples of oat drinks for direct quantification of the maltose content. Overall, this work demonstrates the potential to use an immunosensor based on weakly binding antibodies and FRET technology for remote and non-invasive carbohydrate monitoring.
在本报告中，我们评估了在竞争性免疫传感器中使用荧光/福斯特共振能量转移（FRET）对碳水化合物半抗原麦芽糖进行连续监测的潜力。花青染料Cy5和Cy5.5通过与麦芽糖标记的牛血清白蛋白（BSA）和单克隆抗体IgG 39.5偶联，分别用作供体-受体对，得到Cy5-BSA-麦芽三糖醇（3.1/1/18）和Cy5.5-mAb39.5（2.2/1）。这种对麦芽糖亲和力较弱的抗体对游离麦芽糖和麦芽糖标记的缀合物均表现出完全可逆性。通过监测由于半透性胶囊内Cy5-BSA-麦芽三糖醇从Cy5.5-mAb39.5上的置换导致的FRET信号随时间的变化，我们能够连续测量麦芽糖含量。在存在麦芽糖的情况下，荧光强度比I(670)/I(700)总体增加了近22%，在25℃下从S形剂量-反应曲线计算得到的EC(50)=1.87±0.13 mM（R(2)=0.9984）。免疫传感器对麦芽糖的结构类似物纤维二糖表现出特异性，未产生可检测到的响应。在4周的测量过程中，传感器基线出现了每24小时0.4%的轻微漂移，这与信噪比处于相同量级。该免疫传感器应用于燕麦饮料的粗样品，用于直接定量麦芽糖含量。总体而言，这项工作证明了使用基于弱结合抗体和FRET技术的免疫传感器进行远程和非侵入性碳水化合物监测的潜力。
