Preparation of antibodies and development of enzyme-linked immunosorbent assay for nonylphenol

Abstract

Polyclonal rabbit antibodies against nonylphenol (NP), the main product of nonionic surfactants destruction, were obtained and applied for immunoenzyme assay (ELISA). Two approaches to immunogen synthesis were compared. The first was direct coupling of a mixture of NP isomers to the carrier protein by Mannich reaction. The second was formation of amide bonds between 7-(p-hydroxyphenyl)heptanoic acid (HHA), a linear carboxylated analog of NP, and the carrier protein. Anti-HHA antibodies showed a low affinity to technical NP, whereas with Mannich synthesis it was possible to generate antibodies specific to branched NP molecules. An indirect competitive ELISA was developed based on the anti-NP antibodies. The detection limit of the analysis is 10 ng/mL, with a total duration of around 3 h. The developed ELISA can be applied for group-specific determination of nonionic surfactants and their toxic metabolites. The possibility of NP detection in environmental water matrices using the proposed ELISA without loss of sensitivity is explored.

Keywords:

• Immunoassay
• ELISA
• Nonylphenol
• Nonionic surfactants
• Endocrine disruptors

Acknowledgements

The present research was supported by the INCO-Copernicus Foundation (grant # ICA2-CT-2001-10007), INTAS Foundation (grant # 00-00870) and Russian Government Program ‘Investigations and Elaborations in the Priority Fields of Science and Technology’ (contract # 43.073.1.1.2505).