Immunodetection approaches and high-performance immunoaffinity chromatography for an analogue of bovine growth hormone releasing factor at trace levels.

Abstract

An indirect detection method using high-performance immunoaffinity chromatography (HPIAC) was used to measure low levels of an analogue of bovine Growth Hormone Releasing Factor (bGHRF). An antibody (Ab) labelled with alkaline phosphate (ALP) was incubated with the bGHRF analogue to perform a complex between the antigen (Ag) and the antibody-enzyme (Ab-En) conjugate. The complex was then injected onto a cartridge containing an immobilized Ag affinity support. Species which were not recognized by the affinity cartridge, i.e. eluted, were then directly combined, via a connecting tee, with a buffer containing a substrate. Incubation proceeded on-line, inside a knitted reactor coil, under conditions of constant flow. The subsequent generation of a fluorescently active substrate product was detected by conventional means. The assay described has a linear response region from 1.0 to 25 ng of the bGHRF analogue and a limit of detection of 0.60 ng (1.7 x 10(2) femtomole, 30 p.p.b.). This approach was compared against a method in the antigen/Ab-En complex was injected onto a immobilized Ab affinity cartridge to form an antibody-antigen conjugate sandwich and subsequent stop-flow incubation with substrate.