Rajesh Kumar, Rana K Vikas, Suri C Raman
Department of Biotechnology, Shoolini Institute of Life Science, Solan, Himachal Pradesh India.
Bionanoscience. 2013 Jun;3(2):137-144. doi: 10.1007/s12668-013-0083-8.
The generation of specific and sensitive antibodies against small molecules is greatly dependent upon the characteristics of the hapten-protein conjugates. In the present study, we report a new fluorescence-based method for the characterization of hapten-protein conjugates. The method is based on an effect promoted by hapten-protein conjugation density upon the fluorescence intensity of the intrinsic tryptophan chromophore molecules of the protein. The proposed methodology is applied to quantify the hapten-protein conjugation density of two different class of pesticides (atrazine and 2,4-dichlorophenoxyacetic acid in this study) coupled to carrier protein. The study proved useful for monitoring the course of hapten-protein conjugation for the production of specific antibodies against small molecules. Well-characterized hapten-protein conjugates enabled obtaining highly sensitive anti-atrazine and anti-2,4-D antibodies with IC values equal to 12 and 70 ng mL for atrazine and 2,4-D respectively. These antibodies were used for developing a fluorescence-based immunoassays format demonstrating a detection limit of atrazine and 2,4-D in standard water samples 2 and 7 ng mL, respectively. The developed immunoassay format could be used as convenient quantitative tools for sensitive and specific screening of pesticides in samples.
针对小分子产生特异性和灵敏性抗体在很大程度上取决于半抗原 - 蛋白质偶联物的特性。在本研究中，我们报告了一种基于荧光的新方法来表征半抗原 - 蛋白质偶联物。该方法基于半抗原 - 蛋白质偶联密度对蛋白质中固有色氨酸发色团分子荧光强度的促进作用。所提出的方法用于量化与载体蛋白偶联的两类不同农药（本研究中的莠去津和2,4 - 二氯苯氧乙酸）的半抗原 - 蛋白质偶联密度。该研究被证明有助于监测针对小分子产生特异性抗体时半抗原 - 蛋白质偶联的过程。表征良好的半抗原 - 蛋白质偶联物能够分别获得对莠去津和2,4 - D的IC值等于12和70 ng/mL的高灵敏抗莠去津和抗2,4 - D抗体。这些抗体用于开发一种基于荧光的免疫分析形式，其在标准水样中对莠去津和2,4 - D的检测限分别为2和7 ng/mL。所开发的免疫分析形式可作为方便的定量工具，用于对样品中的农药进行灵敏且特异的筛选。
