Characterization of Hapten–Protein Conjugates: Antibody Generation and Immunoassay Development for Pesticides Monitoring

Abstract

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 IC50 values equal to 12 and 70 ng mL−1 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−1, respectively. The developed immunoassay format could be used as convenient quantitative tools for sensitive and specific screening of pesticides in samples.

Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries

“…An important step in anti-hapten antibody development is verification of the success of conjugation and the conjugation degree of the hapten to carrier proteins. The use of mass spectrometer MALDI-TOF (Matrix-assisted laser desorption/ionization, time-of-flight) has been described previously [ 26 ] as being able to meet these needs, and a similar approach was used here. The MALDI-TOF results revealed that the MPA-AP and MPA-HSA had conjugation degrees of ~5 and ~29 MPA/protein, respectively.…”

Chemical Modification of Aptamers for Increased Binding Affinity in Diagnostic Applications: Current Status and Future Prospects

“…Indeed, the most prominent method for generating antibodies relies on the immunization of animals with an immunogenic analyte. Small analytes are typically non-immunogenic due to their low molecular weight, thus, rendering the generation of suitable antibodies difficult, and while methods have been developed to remediate this drawback, these procedures are costly and time consuming [ 13 , 14 ]. Aptamers, on the other hand, do not suffer from this limitation and can be generated against a wide range of analytes, such as ions [ 15 ], small molecules [ 16 ], proteins [ 17 ], viruses [ 18 ], bacteria [ 19 ] and even whole cells [ 20 ].…”

In Vivo Production of RNA Aptamers and Nanoparticles: Problems and Prospects

“…The substance is introduced to the animals to generate an immune response—immunization. However, not all compounds are immunogenic, requiring high-cost and laborious methods to obtain suitable antibodies [ 30 , 31 , 32 ]. In contrast, aptamers can practically be developed for all sorts of chemicals, ranging from single ions to microorganisms [ 6 , 7 , 33 , 34 ].…”