A sensitive bioluminescent immunoassay for dinitrophenol and trinitrotoluene

Abstract

A bioluminescent immunoassay for measuring dinitrophenol and trinitrotoluene (TNT) has been developed. The DNP and TNT were covalently linked to firefly luciferase, resulting in a conjugate containing 1 mol of DNP or trinitrophenyl (TNP) per mole of luciferase. The conjugate retained 90% of its original catalytic activity. When the conjugate was incubated with immobillzed anti-TNT or anti-DNP and varying concentrations of free TNT or DNP-leucine, the amount of conjugate bound was inversely proportional to the concentration of the free compound. Using this procedure it is possible to detect 2.5 pmol of DNP-leucine and 1.0 pmol of TNT. If the TNP or DNP is linked to glucose-6-phosphate dehydrogenase instead of luciferase, much lower quantities of antigen can be detected. This is due to the fact that this enzyme has a large turnover number so that amplification is possible. The NADH produced is measured using immobilized bacterial NADH:FMN oxidoreductase and luciferase. With this procedure, 10 amol (10 −17 mol) of antigen can be measured. These procedures should be suitable for measuring any antigen.