Use of L-lysine fluorescence derivatives as tracers to enhance the performance of polarization fluoroimmunoassays. A study using two herbicides as model antigens.
Fluorescence polarization immunoassay (FPIA) is a convenient homogeneous assay, the use of which is restricted in environmental analysis by low sensitivity and matrix effects. We selected the herbicides 2,4D and 2,4,5T to synthesize new L-lysine-based fluorescent tracers using solid-phase chemistry. In addition, three different immunogens of 2,4,5T were prepared for immunization and antibody production. The new tracers and antibodies were adapted to FPIA. Tracers with the hapten attached to the alpha-aminogroup of L-lysine and fluorescein to the e-amino group exhibited at least a 5-fold increased sensitivity when compared to the previously reported ethylenediamine-based tracer (2,4D-EDA-F). The isomeric structure (hapten attached to the e-amino and fluorescein to the alpha-amino group) appeared 7.6 times less sensitive, and all other alternative structures exhibited even lower sensitivities. This observation was confirmed against the monoclonal anti-2,4D antibody E2/G2 and polyclonal anti-2,4,5T antibodies. The affinity constant of 2,4D-EDA-F with E2/G2 was 8.1 times higher when compared with the new tracer, suggesting the more specific nature of the L-lysine-based tracer, the use of which leads to a more sensitive assay. This type of tracer could improve performance and lower substantially the detection limits of FPIAs.
Hatzidakis
George I
GI
Laboratory of Toxicology, Medical School, University of Crete, Heraklion, Greece. ghatzi@imbb.forth.gr
Tsatsakis
Aristidis M
AM
Krambovitis
Elias K
EK
Spyros
Apostolos
A
Eremin
Sergei A
SA
D015085:2,4,5-Trichlorophenoxyacetic Acid / Q000032:analysis; D015084:2,4-Dichlorophenoxyacetic Acid / Q000032:analysis; D000818:Animals; D000941:Antigens / Q000032:analysis; D016231:Fluorescence Polarization Immunoassay / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006540:Herbicides / Q000032:analysis*; D007202:Indicators and Reagents; D008239:Lysine / Q000737:chemistry*; D011817:Rabbits; D012710:Serum Albumin, Bovine
D000941:Antigens; D005456:Fluorescent Dyes; D006540:Herbicides; D007202:Indicators and Reagents; D015084:2,4-Dichlorophenoxyacetic Acid; D012710:Serum Albumin, Bovine; D015085:2,4,5-Trichlorophenoxyacetic Acid; D008239:Lysine