Analytes in samples will compete with analytes immobilised on the membrane to bind to the detector antibody.
The capture line is normally analytes conjugated to a carrier protein immobilised on the membrane. And the detector reagent is typically colloidal gold labelled antibodies against the analyte. For smaller analyte (hapten), especially drugs of abuse and steroid-based ovulation prediction the competitive format assay is mainly used (Roda, Portanti, Mirasoli, Guardigli, & Pasini, 2003 Qian, & Bau, 2004). But in these applications, the method is based on the concepts of the double-antigen sandwich assay as its targets are mostly large molecule substances such as proteins. This assay depends on the transport of a reactant to its binding partner immobilised on the surfaces of the membrane (Birnbaum, Uden, Magusson, & Nilsson, 1992 Millipore, 1999 Paek, Lee, Cho, & Kim, 2000). The immunochromatographic strip assay has been a popular rapid diagnostic tool for detecting hormones (HCG), viruses (HIV, hepatitis B and C), bacteria, parasite antigens and β-agonist (Verheijen, Stouten, Cazemier, & Haasnoot, 1998 Zhang et al., 2006). There is still a need to develop a novel, rapid, simple assay for screening 19-NT on a large scale in endemic areas. However, these tests are all tedious, time-consuming and require a laboratory equipped with proper instruments and trained personnel (Horton, Swinburne, & Sullivan, 1991 Cooper, Currie, & Elliott, 2001 Yu, Ho, Leung, & Wan, 2005). In recent years, LC/MS/MS has been successfully applied to the analysis of ASs in various biological samples including urine from cows and horses, and bovine hair and kidney fat and used as confirmatory methods because of the high specificity of the information from the molecular structure of the analyte (Rapp & Meyer, 1989 Gaillard, Vayssette, & Pépin, 2000 Hewitt, Kearney, Currie, Young, & Kennedy, 2002 van Poucke & Peteghem, 2002). Until recently, the standard technique for steroid analysis has been gas chromatography mass spectrometry (GC/MS), but it requires the derivatisation of the ASs using silylation, acylation, or oxime/silylation reactions, according to the individual characteristics of the ASs (Bergemann & van Meyer, 1996 Casademont, Perez, & Garcia, 1996 Kuuranne et al., 2003), which has been the obstacle in the availability of this method (Stockl, de Sagher, Thienpont, Debruyckere, & Peteghem, 1994). So detecting the residue of 19-NT in urine is more reliable (Mathurin, Herrou, Bourgogne, Pascaud, & De Ceaurriz, 2001). 19-NT and its two major metabolites, 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) can be detected in urine, but 19-NA may be endogenous and produced naturally by animals. And it has been banned in food-producing livestock as a growth promoter in China since 2002 (Zhou, 2003). 17β-19-Nortestosterone (19-NT) is an exogenous anabolic steroid (AS) and its illegal use as a growth promoter for fattening livestock has been widely reported throughout the world.
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