Boro Robin C, Singh K Vikas, Suri C Raman
Institute of Microbial Technology (CSIR), Sector 39-A, Chandigarh 160 036, India.
J AOAC Int. 2009 Nov-Dec;92(6):1773-9.
The generation of specific and sensitive antibodies against small molecules is greatly dependent upon the characteristics of the hapten-protein conjugates. In this 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 for two different chlorophenoxyacetic acid pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenoxybutyric acid (2,4-DB), coupled to carrier protein. Highly sensitive anti-2,4-D and anti-2,4-DB antibodies were obtained using these well-characterized hapten-protein conjugates. The generated antibodies were used in an immunoassay format demonstrating inhibitory concentration (IC50) values equal to 30 and 7 ng/mL for 2,4-D and 2,4-DB, respectively. Linearity was observed in the concentration range between 0.1-500 nglmL with LODs around 4 and 3 ng/mL for 2,4-D and 2,4-DB, respectively, in standard water samples. The proposed method was successfully applied for the determination of the extent of hapten-protein conjugation to produce specific antibodies for immunoassay development against pesticides.
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-蛋白质缀合物的特性。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-蛋白质缀合物。该方法基于半抗原-蛋白质缀合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两种不同的氯苯氧基乙酸类农药（2,4-二氯苯氧基乙酸（2,4-D）和2,4-二氯苯氧基丁酸（2,4-DB））与载体蛋白偶联后的半抗原-蛋白质缀合密度。使用这些表征良好的半抗原-蛋白质缀合物获得了高灵敏度的抗2,4-D和抗2,4-DB抗体。所产生的抗体用于免疫分析形式，显示2,4-D和2,4-DB的抑制浓度（IC50）值分别等于30和7 ng/mL。在标准水样中，2,4-D和2,4-DB在0.1 - 500 ng/mL的浓度范围内观察到线性，检测限分别约为4和3 ng/mL。所提出的方法成功应用于测定半抗原-蛋白质缀合程度，以产生用于针对农药免疫分析开发的特异性抗体。
