Preparation of antibodies and development of an enzyme immunoassay for determination of atrazine in environmental samples

Abstract

An indirect competitive enzyme-linked immunosorbent assay (ELISA) has been developed and optimized for atrazine determination in soil at different depths (0-10, 10-20, and 20-30 cm) before and after 48 h of application, corn shoot and cow milk samples collected from Dina farm, Egypt. This assay was based on a specific polyclonal antibodies (PAb) raised by immunizing New Zealand rabbits with an immunogen prepared by coupling 3-{4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine-2-yl} thiopropanoic acid to bovine serum albumin (BSA) via N-hydroxysuccinimide (NHS) active ester method. The sensitivity (estimated as IC₅₀ value) was 17.5 μg mL⁻¹ with a detection limit of 0.1 ng mL⁻¹. The maximum atrazine concentration was found in soil especially in the deepest layer (325 and 890 μg kg⁻¹ before and after application, respectively). Atrazine concentration in corn shoot was 333.28, μg kg⁻¹ dry plant, while there was no detectable amount in milk. All samples screened by ELISA were validated by gas chromatography mass spectrometer procedure (GC/MS). Good correlation was achieved between the two methods (r = 0.997 for soil and 0.9814 for plant). This study demonstrates the utility and convenience of the simple, practical and cost-effective ELISA method in the laboratory for analysis of environmental samples. The method is ideal for the rapid screening of large numbers of samples in laboratories where access to GC/MS facilities, is limited or lacking.

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

“…In many previous studies, polyclonal antisera as such have been used for estimating the levels of different pesticides [16]. However, only few groups have reported the use of purified antibodies for pesticides detection assay [17]. The present study demonstrates the efficient purification of antibodies with high yield using a combination of protein A sepharose column followed by passing over carrier protein column which resulted in total recovery of 90 % having around 75–80 % anti-hapten antibodies.…”

Development of a Highly Sensitive and Specific Immunoassay for Determining Chrysoidine, A Banned Dye, in Soybean Milk Film

“…The chrysoidine derivative bearing a carboxylic acid group at the end of the spacer was activated by the active ester method and then covalently coupled with a carrier protein (BSA or OVA) [ 21 ]. The conjugates of hapten-BSA and hapten-OVA were used as immunogen and coating antigen, respectively.…”

Development of a Model Immunoassay Utilizing Monoclonal Antibodies of Different Specificities for Quantitative Determination of Dieldrin and Heptachlors in Their Mixtures

“…The current general analysis methods of dieldrin and heptachlors comprise gas chromatography–mass spectrometry (GC-MS), GC–electron capture detection (GC-ECD), and high-performance liquid chromatography with UV detection (HPLC-UV), which identify the target substances with high accuracy and sensitivity. − However, these instrumental analysis methods require expensive equipment for laboratory testing and highly skilled operators, and they are not suitable for high-throughput analysis in comprehensive monitoring studies. In comparison, immunoassays are relatively simple and sensitive methods that can be used in high-throughput analyses. − Immunoassay development requires the production of antibodies of desired specificity and their incorporation into an appropriate assay. Several studies have been published on the analysis of chlorinated cyclodiene pesticides by immunoassays, but the analytes probed in these works were mostly endosulfan and its metabolites.…”