Synthesis of a Fluorescent Analog of Polychlorinated Biphenyls for Use in a Continuous Flow Immunosensor Assay

Abstract

A synthetic scheme has been developed for the preparation of a dye-labeled analog of polychlorinated biphenyls. The reaction of 2,3,5-trichlorophenol with 3-bromopropylamine hydrobromide under basic conditions was used to introduce a free primary amine group into the parent compound by formation of a stable ether linkage. Reaction of this amine with the succinimidyl ester of a sulfoindocyanine dye resulted in amide bond formation to produce a fluorescently-labeled product. The dye conjugate was used to charge a column containing immobilized antibodies against polychlorinated biphenyls. Upon application of samples containing various concentrations of polychlorinated biphenyls, the fluorescent analog was displaced from the column in amounts proportional to the concentration of analyte. Concentrations of polychlorinated biphenyl as low as 1 ppm were measurable using this system.

Immunochemical determination of xenobiotics with endocrine disrupting effects

“…In these assays the sample is incorporated in a carrier stream which enters in a reactor chamber where the immunological reaction takes place. Some FIIAs have been developed for the analysis of several pollutants such as pesticides [41,44,45,46] and other industrial pollutants such as PCBs [47], thereby demonstrating that FIIAs can be more precise and rapid than an ELISA offering automation with low cost.…”

Effect of lipid composition on phospholipase A2‐catalyzed membrane leakage in immobilized liposomes: Sensitization for polychlorinated biphenyls detection with antibody affinity column tandem with fluorescent liposome column

“…The detection limit was 100 times lower than that reported by Charles et al [3] using a conjugate of tetrachlorophenol and fluorescent dye, Cy5.29 for competitive binding. In the method of Charles et al, one fluorescent signal was caused by one unbound hapten, but in this method, numbers of fluorescent dyes can be released by enzymatic dye leakage.…”

“…Thus, many immunoassays have been developed as alternative methods to determine such small compounds. Continuous-flow fluorescent immunoassays have been developed for rapid detection of small molecules, e.g., 2,4-dinitrophenol [2] and PCBs [3] in which the detection limits were about 26 ng/mL and 1 mg/mL, respectively. The enzyme-linked immunosorbent assay offered rapid and sensitive characterizations for PCB contamination, and achieved detec-tion level of 5 mg PCB /mL from soil sample or 1 ng/mL from liquid sample [4,5].…”

Ligand Displacement Fluorescence Immunoassay for Gentamicin and Human IgG#

“…By combining LD-FIA and a continuous flow system, a number of rapid assays have been developed for detecting explosives and environmental pollutants (Kusterbeck et al 1990;Ligler et al 1993;Charles et al 1995;Narang et al 1997aNarang et al , 1997bRabbany et al 1998Rabbany et al , 2000. The activity of the labeled antibodies could then be measured spectrophotometrically for enzyme labels or in a fluorometer for fluorescent labels.…”

“…More recently the technique from ligand displacement fluorescence immunoassay, (LD-FIA) was further developed by Ligler et al using flow systems into sensitive, continuous and real time analytical methods (Kusterbeck et al 1990;Ligler et al 1993;Charles et al 1995;Narang et al 1997aNarang et al , 1997bRabbany et al 1998Rabbany et al , 2000. Several solid-phases have been used in the development of flow LD-FIA such as Sepharose 4B (Kusterbeck et al 1990;Ligler et al 1993); Emphaze beads, a synthetic polymeric beads containing amine-reactive azalactone groups (Charles et al 1995); microcapillaries (Narang et al 1997a(Narang et al , 1997b; porous membranes (Rabbany et al 1998(Rabbany et al , 2000.…”