Gel-based immunoassay for non-instrumental detection of pyrene in water samples

A new qualitative immunologically based tube test for non-instrumental detection of pyrene (PYR) in water samples was developed. The method combines the pre-concentration of analyte by immunoextraction and its detection by immunoassay using Sepharose 4B-immobilized IgG-fraction of a polyclonal anti-PYR antiserum (immunoaffinity gel) and 1-pyrenebutyric acid-horseradish peroxidase conjugate (PYR-BA-HRP). The immunoaffinity gel was placed in a standard 1-ml SPE column through which a 10-ml aliquot of water sample spiked with 10% acetonitrile was passed. Following, free antibody binding sites were detected by application of PYR-BA-HRP. Four minutes after addition of the chromogenic substrate the results were visually evaluated by occurring or stayed away blue colour development for negative and positive samples, respectively. Total time for assay was about 15 min for six samples. Under optimized conditions a cut-off level for pyrene of 0.04 ng ml^-1 was found. At this defined concentration, a set of spiked samples (n = 175) was analyzed and very low rates of false negatives (1.2%) and false positives (4.6%) determined which fulfils the requirement set by Commission Decision 2002/657/EC for a screening method. No interference by other PAH compounds like naphthalene, fluoranthene, phenanthrene, anthracene, and benzo[a]pyrene at a concentration of 20 ng ml^-1, i.e., 500-fold excess compared to the defined cut-off level was observed. Different water types like surface water, tap water, bottled water, and melted snow were analyzed for PYR contamination by the proposed method and results confirmed by HPLC-FLD.