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

Abstract

To identify the explosive used in a terrorist attack, or to obtain 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 particularly true for TNT (2,4,6-trinitrotoluene), one of the most employed explosives in the 20th century and at the same time, because of 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 and the influence of various non-specific factors such as: tolerance to organic solvents at different concentrations, the washing and competitive step time, and the cross-reactivity with related compounds. The sensitivity and reproducibility of the CL-ELISA were good (LOD and IC50 values in the ng mL−1 range, and 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 were tested on these samples, all employing methanol as the solvent. The lateral flow immunoassay (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.

LFIA of TNT standard at different concentrations: 1) No TNT; 2) 100 g mL−1 TNT; 3) 1 g mL−1 TNT;4) 0.1 g mL−1 TNT.

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Acknowledgments

This work was supported by grants from the Ministry of University and University of Bologna (PRIN 2006033429: “New Analytical Tools for Security and Criminal Investigations: Trace Detection and Identification of Explosives and Related Compounds”) and “Fundamental Oriented Research” and Grant 08-03-90301 of the Russian Foundation for Basic Research “New strategy for robust and sensitive chemiluminescent immuno-bio-technology for detection of chlorinated herbicides in environment”.

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Correspondence to S. Girotti.

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Girotti, S., Eremin, S., Montoya, A. et al. Development of a chemiluminescent ELISA and a colloidal gold-based LFIA for TNT detection. Anal Bioanal Chem 396, 687–695 (2010). https://doi.org/10.1007/s00216-009-3264-0

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Keywords

  • 2,4,6-Trinitrotoluene (TNT)
  • Chemiluminescence
  • ELISA
  • LFIA
  • Immunoassay