Development of a Luminex based competitive immunoassay for 2,4,6-trinitrotoluene (TNT).
Previously, a displacement immunoassay for 2,4,6-trinitrotoluene (TNT) was demonstrated using the Luminex 100. The work presented utilized this same specialized flow cytometer to demonstrate a highly sensitive and rapid competitive immunoassay for TNT. This required a TNT analog to be attached to the microsphere surface. Various linkers were evaluated; bovine serum albumin provided over 3 times more binding sites in comparison to various shorter diamine linkers. For this assay TNB-coated microspheres were added to samples; then biotinylated anti-TNT antibody and the reporter molecule, Streptavidin-R-Phycoerythrin, were added. In the absence of TNT, a highly fluorescent complex was formed on the surface of the microsphere. The presence of TNT resulted in dose-dependent decreased fluorescence. Various anti-TNT antibodies were evaluated; Mab 30-1 gave the strongest response, yielding the lowest limit of detection (<1.0 ng/mL) and a dynamic range up to 1 microg/mL. Other factors such as reaction time, cross reactivity to other nitro-compounds, evaluation of acetone extracts of TNT contaminated soils, testing in environmental matrices such as fresh water and seawater were all completed. Finally, a multiplex assay for TNT and three protein toxins was successfully conducted using the competitive format.
Anderson
George P
GP
Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, Washington, DC, USA. george.anderson@nrl.navy.mil
Lamar
Jacqueline D
JD
Charles
Paul T
PT
D000911:Antibodies, Monoclonal / Q000737:chemistry; D004784:Environmental Monitoring / Q000379:methods; D005456:Fluorescent Dyes; D007118:Immunoassay / Q000379:methods*; D007834:Lasers; D007840:Latex; D008863:Microspheres; D014303:Trinitrotoluene / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D007840:Latex; D014303:Trinitrotoluene