Antibodies detector

Fig. 5. (a) Antibody 2 is added to test solution where some or all of becomes complexed with target, T. (b) A fiber-optic probe containing covalently attached target is insetted into the solution. Unbound binds to probe, (c) The probe is inserted into solution containing en2yme-modified detector antibody which binds to probe if any is attached, (d) The probe is inserted into a solution producing a fluorescent compound, which is then... 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

Fig. 31 (A) Principle of a sandwich immunoassay using FDA particulate labels. The analyte is first immobilized by the capture antibody preadsorbed on the solid phase (a) and then exposed to antibody-coated microparticle labels (b). Every microparticle contains 108 FDA molecules. High signal amplification is achieved after solubilisation, release, and conversion of the precursor FDA into fluorescein molecules by the addition of DMSO and NaOH (c). (B) Calibration curves of IgG-FDA microcrystal labels with increasing surface coverage of detector antibody (a-d) compared with direct FITC-labeled detector antibody (e). The fluorescence signals increase with increasing IgG concentration. FDA microcrystals with a high IgG surface coverage (c,d) perform better than those with lower surface coverage (a,b). (Reprinted with permission from [189]. Copyright 2002 American Chemical Society)...

Figure 3.3.2 Format of sandwich ELISA for antigens (high molecular weight compounds). This format needs a capture antibody (on the microtitre plate surface) and a detector antibody, to which an enzyme (e.g. HRP) can be conjugated. Other possibilities would be that the detector antibody is labeled with a fluorophore or is biotinylated. The latter would use streptavidin-HRP in the detection step...

Assuring access to assay development toote Production/sourcing of capture and detector antibodies... 

In sandwich LBAs, both polyclonal antibodies (PAbs) and monoclonal antibodies (MAbs) are used as capture detector antibody pairs in various combinations. As MAbs recognize only a single epitope, they provide higher specificity than PAbs and are consequently more expensive to produce. Conversely, immunization of a single goat or sheep may provide a supply of PAbs sufficient for the execution of multiple studies over several years. PAbs can also provide higher sensitivity due to the presence of multiple antibodies binding to different epitope on the analyte molecule. 

A prototype method can be obtained from a pharmaceutical company s QC or discovery group, an academic institution, or some other source, although often the scientist may need to design and develop such a method. Checkerboard experiments [8,10] define the optimal concentration of coating material, detector antibody concentration, and approximate range of the calibration curve. A calibration curve generated in assay buffer serves as a reference to assess the suitability of blank matrix for spiking calibrators and QC samples. 

Perform a bridging assay in which the same antibody is used as both capture and detector antibody. If the assay signal in a predose sample is increased above the capture or detector antibody blank signal, interference should be suspected an increase in signal indicates likely presence of heterophilic antibodies cross-linking... 

Standard curve range components (coating plates and detector antibody) from <More than six concentrations> pg/mL in protein/... 

Incubation with FITC labeled detector antibody... 

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