Characterization of hapten-protein conjugates: antibody generation and immunoassay development for chlorophenoxyacetic acid pesticides.
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.
Boro
Robin C
RC
Institute of Microbial Technology (CSIR), Sector 39-A, Chandigarh 160 036, India.
Singh
K Vikas
KV
Suri
C Raman
CR
D015084:2,4-Dichlorophenoxyacetic Acid / Q000031:analogs & derivatives* / Q000032:analysis* / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000918:Antibody Specificity; D001667:Binding, Competitive; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D006241:Haptens / Q000737:chemistry*; D006540:Herbicides / Q000032:analysis* / Q000276:immunology; D007118:Immunoassay / Q000379:methods*; D007202:Indicators and Reagents; D010573:Pesticide Residues / Q000032:analysis*; D011506:Proteins / Q000737:chemistry*; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000906:Antibodies; D006241:Haptens; D006540:Herbicides; D007202:Indicators and Reagents; D010573:Pesticide Residues; D011506:Proteins; D015084:2,4-Dichlorophenoxyacetic Acid; D012710:Serum Albumin, Bovine