Immunoassay fluorescent signal

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)...

None of the involved species are fluorescent. Therefore, for fluorescence signaling of citrate recognition, carboxyfluorescein is first added to the medium because binding to the receptor in the absence of citrate is possible and causes deprotonation of carboxyfluorescein, which results in high fluorescence. Citrate is then added, and because it has a better affinity for the receptor than carboxyfluorescein, it replaces the latter, which emits less fluorescence in the bulk solvent as a result of protonation. Note that this molecular sensor operates in a similar fashion to antibody-based biosensors in immunoassays. It was succes-fully tested on a variety of soft drinks. 

An immunosensor based on a competitive fluorescence energy-transfer immunoassay was reported by Anderson 105) for the measurement of phenytoin. Texas red-labeled antibody was incubated with a phenytoin derivative. On displacement of the derivative by the antigen, the change in the fluorescence signal was recorded. Detection limits approached 5 /iM with response times ranging from 5 to 30 min. 

An NIR biosensor coupled with an NIR fluorescent sandwich immunoassay has been developed. 109 The capture antibody was immobilized on the distal end of an optical fiber sensor. The probe was incubated in the corresponding antigen with consecutive incubation in an NIR-labeled sandwich antibody. The resulting NIR-labeled antibody sandwich was excited with the NIR beam of a laser diode, and a fluorescent signal that was directly proportional to the bound antigen was emitted. The sensitivity of the technique increased with increasing amounts of immobilized receptor. There are several factors involved in the preparation of the sandwich type biosensor. A schematic preparation of the sandwich optical fiber is shown in Figure 7.14. 

Fluoroimmunoassays comprise a subclass of extrinsic labehng methods where various selective antigen (Ag)- antibody (Ab) immunoassay fluorescent labeling schemes yield a emission signal. One common scheme involves an enzyme-linked immunosorbent assay (ELISA) depicted in Figure 11.2 where the free Ab is tagged with a fluorophore. Numerous analytes can be detected via these types of selective lock-and-key methods. ... 

Figure 6. (a) Fluorescence signal measured from three consecutive bead patterns after performing an immunoassay in stop-flow mode using a sample volume of 1,300 nL at different m-Ag antigen concentrations, (b) Integrated fluorescence obtained from fluorescent intensity profiles like shown in Fig. 6a for three different volumes of t-Ag solution exposing the bead patterns [17]. 

Instead of the optical labels, enzymes are also used as a labeling material [7]. ELISA is one of the most popular techniques in the conventional immunoassay and suitable in the microchip format. By using a fluorogenic reaction, the fluorescent signal can be taken at the downstream microchannel without interruption by microbeads. In the case of chromogenic reaction, a TLM is useful [8]. 

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