ABSTRACT
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.
Subject(s)
2,4-Dichlorophenoxyacetic Acid/analogs & derivatives , 2,4-Dichlorophenoxyacetic Acid/analysis , Antibodies/chemistry , Haptens/chemistry , Herbicides/analysis , Immunoassay/methods , Pesticide Residues/analysis , Proteins/chemistry , 2,4-Dichlorophenoxyacetic Acid/immunology , Animals , Antibody Specificity , Binding, Competitive , Cross Reactions , Electrophoresis, Polyacrylamide Gel , Herbicides/immunology , Indicators and Reagents , Rabbits , Serum Albumin, Bovine/chemistry , Spectrometry, Fluorescence , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
A novel immunoassay format employing direct coating of small molecular hapten on microtiter plates is reported for the detection of atrazine and 2,4-dichlorophenoxyacetic (2,4-D). In this assay, the polystyrene surface of microtiter plates was first treated with an acid to generate -NO2 groups on the surface. Acid treated plates were further treated with 3-aminoprpyltriethoxysilane (APTES) to functionalize the plate surface with amino groups for covalent linkage to small molecular hapten with carboxyl groups. The modified plates showed significantly high antibody binding in comparison to plates coated with hapten-carrier protein conjugates and presented excellent stability as a function of the buffer pH and reaction time. The developed assay employing direct hapten coated plates and using affinity purified atrazine and 2,4-D antibodies demonstrated very high sensitivity, IC50 values for atrazine and 2,4-D equal to 0.8 ng mL(-1) and 7 ng mL(-1), respectively. The assay could detect atrazine and 2,4-D levels in standard water samples even at a very low concentration upto 0.02 and 0.7 ng mL(-1) respectively in the optimum working range between 0.01 and 1000 ng mL(-1) with good signal reproducibility (p values: 0.091 and 0.224 for atrazine and 2,4-D, respectively). The developed immunoassay format could be used as convenient quantitative tool for the sensitive screening of pesticides in samples.
