Development of a chemiluminescent ELISA and a colloidal gold-based LFIA for TNT detection

To identify the explosive used in a terrorist attack, or to get an early sign of environmental pollution it is important to use simple and rapid assays able to detect analytes at low levels, possibly on-site. This is particular true for TNT (2,4,6-trinitrotoluene), one of the most employed explosives in the 20th century and at the same time, because its toxicity, a well known pollutant. In this work we describe the development of an indirect competitive ELISA with chemiluminescent detection (CL-ELISA) and of a lateral flow immunoassay (LFIA) based on colloidal gold nanoparticle labels. A commercially available monoclonal antibody was used and 13 specially synthesized conjugates were tested. We optimized the assay by determining the optimal concentration of monoclonal antibody and conjugates as well as the influence of various non specific parameters such as: the tolerance to organic solvents at different concentrations, the washing and competitive step time, and the cross-reactivity with related compounds. The CL-ELISA showed a good sensitivity (LOD and IC50 values in the ng mL-1 range) and a good reproducibility (CV value about 7%). It has been applied to real samples of various materials involved in a controlled explosion of an “Improvised Explosive Device”. Three extraction procedures have been tested on these samples, all employing methanol as the solvent. The Lateral Flow Immuno Assay (LFIA), developed by using the same immunoreagents, reached a Detection Limit of 1 µg mL-1 when tested on the same samples analysed by CL-ELISA.