Immunodetection Approaches and High-Performance Immunoaffinity Chromatography for an Analogue of Bovine Growth Hormone Releasing Factor at Trace Levels
✍ Scribed by Richard A. Strong; Byung-Yun Cho; Daniel H. Fisher; John Nappier; Ira S. Krull
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 1017 KB
- Volume
- 10
- Category
- Article
- ISSN
- 0269-3879
No coin nor oath required. For personal study only.
✦ Synopsis
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 phosphatase (ALP) was incubated with the bCHRF analogue to preform a complex between the antigen (Ag) and the antibody-enzyme (Ab-En) conjugate. The complex was then injected onto a cartridge containing m 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' femtomole, 30 p.p.b.). This approach was compared against a method in which the antigedAbEn complex was iNeckd onto an immobilized Ab affinity cartridge to form an antibody-antigen conjugate sandwich and subsequent stopflow incubation with substrate.