Noncompetitive flow injection immunoassay for a hapten, .alpha.-(difluoromethyl)ornithine

Abstract

A noncompetitive flow injection immunoassay method has been developed to assay small haptens. In this assay the sample containing the hapten is incubated with excess enzyme-labeled monovalent antibody for a brief period. The excess antibody is then separated from the bound antibody by eluting through an antigen-immobilized immunoaffinity column. The enzyme label of the eluting antibody-hapten complex is fluorometrically detected. The applicability of the method is demonstrated by assaying alpha-(difluoromethyl)ornithine (DFMO), an anticancer drug in human plasma samples. The assay is sensitive enough to detect 200 amol of DFMO. Interferences from other similar endogenous amines have been eliminated by selective immunoaffinity purification of the antibodies.

Immunodetection Approaches and High-Performance Immunoaffinity Chromatography for an Analogue of Bovine Growth Hormone Releasing Factor at Trace Levels

“…Wilsons group (Gunaratna and Wilson, 1993) demonstrated a linear response region twicc the dynamic range as was found here and a 300-fold reduction in the limit of detection. This was not entirely unexpected since that study used Fab fragments labelled with horseradish peroxidase and avoided many of the disadvantages already discussed.…”

“…2, and was based upon previous reports (Gunaratna and Wilson, 1993;Freytag er al., 1984). In this scheme, Ag was incubated with an excess of Ab-En conjugate off-line.…”

“…The instrumental set-up is illustrated in Fig. 2 and is analogous to that described by Gunaratna and Wilson (1993 2, except that the immobilized Ag affinity column had been removed. Aliquots of the freshly purified Ab-En conjugate stock solution were serially diluted with BSA sample buffer.…”

Recent Progress in Noncompetitive Hapten Immunoassays: A Review

“…After which, target analyte is passed through the membrane, and the displacement of labeled antigen is monitored downstream. This method has been successfully applied to the detection of numerous hapten molecules and satisfactory sensitivity have been obtained: 10pM for thyroxine (Arefyev et al, 1990); 20 pM for -(difluoromethyl) ornithine (Gunaratna et al, 1993) and 40pM for digoxigenin (Lovgren et al, 1997). The most inviting advantage of this method is the ability to realize automatic detection.…”

Flow Injection-Immunometric Assay of 17α-Hydroxyprogesterone Using Fluorescein-Labeled Fab Fragment

“…The continuous flow analysis using immobilized antigen to remove unbound labeled antibody after the competitive reaction with antigen has also been developed, but a highly purified monospecific antibody such as monoclonal antibody is required to minimize the background level. 3,4 If we employ a reactor consisting of labeled antibody bound on immobilized antigen, the analyte bound with labeled antibody in an eluent from the reactor can be determined successively (Fig. 1).…”