RESUMEN
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.
Asunto(s)
Ácido 2,4-Diclorofenoxiacético/análogos & derivados , Ácido 2,4-Diclorofenoxiacético/análisis , Anticuerpos/química , Haptenos/química , Herbicidas/análisis , Inmunoensayo/métodos , Residuos de Plaguicidas/análisis , Proteínas/química , Ácido 2,4-Diclorofenoxiacético/inmunología , Animales , Especificidad de Anticuerpos , Unión Competitiva , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Herbicidas/inmunología , Indicadores y Reactivos , Conejos , Albúmina Sérica Bovina/química , Espectrometría de Fluorescencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The present study describes a lateral-flow-based dipstick immunoassay format using a novel hapten-protein-gold conjugate for the rapid screening of atrazine in water samples. The immunoassay is based on the competitive inhibition, in which a newly developed hapten-protein-gold conjugate competes with the free antigen present in the sample, for the limited antibody binding sites available at test zone on dipstick membrane, housed in a plastic cartridge. The tracer used as the detection reagent was prepared by first conjugating hapten (a derivative of atrazine) molecules to a carrier protein (bovine serum albumin) via its surface lysine residues and then linking colloidal gold nanoparticles to the hapten-protein conjugate via cysteine residues of the carrier protein. The developed conjugate showed a high level of stability as it did not show any significant loss of activity even after 8 weeks of storage at ambient conditions. The color developed due to conjugate, based on competitive inhibition approach, is correlated with the concentration of atrazine sample. The sensitivity of the developed dipstick was enhanced by gold nanoparticles, as an amplification tag, presenting detection limit of atrazine in standard water samples down to 1.0 ppb level. The kit could serve as a rapid screening methodology for visual screening of atrazine contamination of water samples within 5 min of analysis time, and, when coupled with a portable colorimeter, as an inexpensive semi-quantitative assay. The method reported can be useful for screening a large number of pesticides samples in a very short time in the field.