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 IC₅₀value) 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.

Keywords:

• Enzyme immunoassay
• atrazine
• polyclonal antibodies
• immunogen
• environmental samples
• correlation