CONTINUOUS FLOW IMMUNOASSAY: USE OF A NOVEL TRIFUNCTIONAL CARRIER MOLECULE FOR THE SYNTHESIS OF FLUOROPHORE-LABELED ANTIGENS

Abstract

We developed a fluorescent immunosensor operating in continuous flow and capable of detecting low molecular weight antigens. The approach differs from previously described continuous flow assays by not requiring incubation steps or the introduction of reagents following the loading of the sample into the system. Detection of the antigen is rapid, occurring within three minutes in the system described. The assay is based on the binding of labeled antigen to an immobilized antibody, with subsequent displacement of the labeled antigen when antigen is present in the buffer flow. In order to increase the sensitivity of the assay, we developed a novel trifunctional carrier molecule for the fluorescent labeling of the antigen. The backbone of the carrier consists of the 21 amino acid residues of the insulin A-chain, which provides a single site (terminal amino group) for covalent coupling of the antigen, three carboxyl groups for the attachment of fluorophores, and four sulfhydryl groups for derivatization with hydrophilic residues to compensate for the hydrophobic effect of the fluorophores. In this study, the model antigen 2,4-dinitrophenol (DNP) was coupled to the terminal amino group, the sulfhydryl groups were oxidized to S-sulfonates, and the carboxyl groups were derivatized with fluorescein using carbohydrazide as spacer. The properties of the DNP-insulin A-chainfluorescein conjugate (DNP-Ins-Fl) were compared to those of a DNP derivative labeled with a single fluorescein residue via a small lysine spacer (DNP-Lys-Fl). At equimolar concentrations the DNP-Ins-Fl generated a 2.6-fold higher fluorescent signal than the DNP-Lys-Fl, and exhibited a three-fold lower nonspecific adsorption to immobilized nonimmune IgG. Due to these properties of DNP-Ins-Fl,as little as 50 pmol of DNP-lysine could be detected in the fluorescent continuous flow immunoassay.