Antibodies biotin analysis

Immunosensors have been developed commercially mostly for medical purposes but would appear to have considerable potential for food analysis. The Pharmacia company has developed an optical biosensor, which is a fully automated continuous-flow system which exploits the phenomenon of surface plasmon resonance (SPR) to detect and measure biomolecular interactions. The technique has been validated for determination of folic acid and biotin in fortified foods (Indyk, 2000 Bostrom and Lindeberg, 2000), and more recently for vitamin Bi2. This type of technique has great potential for application to a wide range of food additives but its advance will be linked to the availability of specific antibodies or other receptors for the various additives. It should be possible to analyse a whole range of additives by multi-channel continuous flow systems with further miniaturisation. 

Add 100-p.L aliquots of the antibody dilutions in duplicate to separate 0.9 mL aliquots of the HABA-avidin or streptavidin solution and stir at room temperature for 5-10 min. If the DOL of some of the samples is very high, a precipitate may form during the incubation that should be removed by centrifugation prior to spectrophotomet-ric analysis. The decrease in A500 for each sample should then be recorded. The average value from each pair of samples is used to determine the nmol of biotin in the various aliquots of antibody... 

The concept of linking PCR and ELISA for ultrasensitive protein analysis soon sparked the imagination of laboratory scientists, and they developed a method that separates the concept of antibody-DNA linkage from hard-to-obtain protein chimeras. An easy way to establish the desired coupling was accessible with the streptavidin/biotin binding system The tetravalent protein STV, from Streptomyces avidii [19], derived, for example, as a recombinant core protein with optimized properties [20], or as its egg-yolk... 

Fig. 4. Schematic diagram of the steps in the automated PCR/OLA procedure performed with a robotic workstation. The assay contains three steps (1) DNA target amplification (2) analysis of target nucleotide sequences with biotin (B)-labeled and digoxigenin (D)-labeled oligonucleotide probes and T4 DNA ligase (L) and (3) capture of the biotin-labeled probes on streptavidin (SA)-coated microtiter wells and analysis for covalently linked digoxigenin by using an ELISA procedure with alkaline phosphatase (AP)-conjugated antidigoxigenin (aD) antibodies and a substrate (S). Reprinted with the permission of Nickerson el al. (N2) and the Proc. Natl. Acad. Sci. (U.SA.).

The ABC detection system has been shown to be more sensitive than most other detection system (5, 6), primarily because of the large size of the preformed ABC complexes, which result in amplification of the signals. Alternative detection systems for immunohis-tochemical analysis include the peroxidase-antiperoxidase (PAP) (1) and the alkaline phosphatase-antialkaline phosphatase(APAAP) systems (7) (see Chapter 24). More recently, newer commercially available polymer-based detection systems have been developed that combine the secondary antibody and detection enzyme into a single reagent. Polymer-based detection systems overcome problems that may occur with biotin-link based systems, have excellent sensitivity, and can be applied to frozen section immunohistochemistry. Polymer-based detection systems are discussed in other chapters of the manual. (XREF). 

Armexin A1 emerged as a promising antigen for the radiolabeled antibody-based imaging and therapy of cancer [100]. In our laboratory, we use terminal perfusion protocols featuring active esters of biotin for the selective chemical labeling of accessible proteins in vascular structures. Biotinylated proteins are then purified from different organs (collected separately) and are submitted to a comparative proteomic analysis [106]. 

Thus, in this assay, rapid method has eliminated the two steps that are normally included in the conventional electrochemical genosensor assay the denaturation of the PCR amplicon and its hybridization. Here, we merely immobilized the biotin- and fluorescein-labeled PCR amplicon on a streptavidin-modified SPC, followed by incubation with HRP-conjugated anti-fluorescein antibody, and the direct detection of the amperometric signal [8]. However, there is a need to incorporate PCR and electrochemical analysis into a single device for this method to be fully usable for field applications [13]. 

Antibodies are immunoreagents. Considering the ligand-receptor basis for immunochemistry, avidin and biotin are often considered immunochemical reagents. The literature is replete with assays that employ immunological methods to increase sensitivity or specificity of nucleic acid analysis. 

---