Direct hapten coated immunoassay format for the detection of atrazine and 2,4-dichlorophenoxyacetic acid herbicides

• Published in: Analytica chimica acta Vol. 607; no. 1; pp. 92 - 99
• Main Authors: Kaur, Jasdeep, Boro, Robin C., Wangoo, Nishima, Singh, Kumar Rajesh, Suri, C. Raman
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 21.01.2008; Elsevier
• Subjects: 2,4-Dichlorophenoxyacetic acid; 2,4-Dichlorophenoxyacetic Acid - analysis; 2,4-Dichlorophenoxyacetic Acid - immunology; Analytical chemistry; Animals; Antibody Specificity; Atrazine; Atrazine - analysis; Atrazine - immunology; Carrier Proteins - immunology; Carrier Proteins - metabolism; Cattle; Chemistry; Enzyme-Linked Immunosorbent Assay - methods; Exact sciences and technology; Haptens - immunology; Herbicides - analysis; Herbicides - immunology; Immunoassay; Immunoglobulin G - immunology; Miscellaneous; Polystyrene surface; Rabbits; Silanization; Immunoassay; Silanization; Polystyrene surface; 2,4-Dichlorophenoxyacetic acid; Atrazine; Water; Chemical analysis; Stability; 2,4-D; Microtiter plate; Antibody; Pesticides; Coatings; Hapten; Herbicide; Protein; Immunological method; Sensitivity; Reproducibility; Nitrogen dioxide; Comparative study
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2007.11.017

A novel immunoassay format employing direct coating of small molecular hapten on microtiter plates is reported for the detection of atrazine and 2,4-dichlorophenoxyacetic (2,4-D). In this assay, the polystyrene surface of microtiter plates was first treated with an acid to generate –NO 2 groups on the surface. Acid treated plates were further treated with 3-aminoprpyltriethoxysilane (APTES) to functionalize the plate surface with amino groups for covalent linkage to small molecular hapten with carboxyl groups. The modified plates showed significantly high antibody binding in comparison to plates coated with hapten–carrier protein conjugates and presented excellent stability as a function of the buffer pH and reaction time. The developed assay employing direct hapten coated plates and using affinity purified atrazine and 2,4-D antibodies demonstrated very high sensitivity, IC 50 values for atrazine and 2,4-D equal to 0.8 ng mL −1 and 7 ng mL −1, respectively. The assay could detect atrazine and 2,4-D levels in standard water samples even at a very low concentration upto 0.02 and 0.7 ng mL −1 respectively in the optimum working range between 0.01 and 1000 ng mL −1 with good signal reproducibility ( p values: 0.091 and 0.224 for atrazine and 2,4-D, respectively). The developed immunoassay format could be used as convenient quantitative tool for the sensitive screening of pesticides in samples.