A continuous flow immunoassay for rapid and sensitive detection of small molecules

An immunosensor operating in continuous flow and capable of detecting low molecular weight antigens is described. The approach differs from previously described continuous flow assays by not requiring incubation steps or the introduction of additional reagents following the loading of the sample into the system. Detection of the antigen is rapid, occurring within 3 min 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. Signal detection occurs downstream of the antigen recognition event. In this study, the hapten 2,4-dinitrophenol (DNP) as DNP-lysine was used as model antigen. To generate a labeled antigen, DNP was coupled to the terminal amino group of insulin A chain (tetra S-sulfonate form) which provides two tyrosine residues for the introduction of an 125I-label (DNP-Ins-125I) or three carboxyl groups for the attachment of three fluorescein residues (DNP-Ins-Fl). The radiolabeled antigen was used to establish assay conditions. Subsequently, fluorescein was substituted for the radioisotope label in order to develop an assay independent of the restrictions associated with isotopes. Using this flow immunoassay, we were able to detect DNP-lysine down to a detection limit of 143 nM (29 pmol/200 microliters) using DNP-Ins-125I or DNP-Ins-Fl as labeled antigen. The density of immobilized antibody and the flow rate were identified to be critical parameters for the sensitivity of the assay.