Fluorescent-labelled hapten

In the competitive format for the determination of haptens, sample haptens, and fluorescent-labeled haptens compete for the binding sites at the immobilized antibodies (in limited amount) (Scheme lA). Fluorescence can be measured either at the solid phase or in solution. 

Visualizing more than one epitope on one section can be accomplished by different fluorescence labeling or different sizes of colloidal gold coupled to primary or secondary antibodies. Primary antibodies from different species and adequate secondary antibodies labeled differently can be used. In case of primary antibodies from the same species, the hapten technique can be applied. A hapten is a small molecule that can be bound to antibodies dinitrophenol and arsinilate are typically used as haptens. Again, adequate secondary antibodies labeled differently can be used (14,17,32). A collection of protocols for multiple immu-nolabeling has been described by Beesley (37). 

After mixing, add 5 of GU-labeled hapten (about 0.125 absorbance units at 245 nm), and monitor the increase in fluorescence over 2-3 min. 

The rapid expansion of lectin-based applications for the detection and quantification of glycoconjugates has been led by the development of commercially available, purified and chemically derivatized lectins, and in some cases, anti-lectin antibodies. Over 50 purified plant lectins are sold commercially by a number of producers and vendors, with this number growing annually. Equally important is the ease by which investigators can obtain lectins labeled with various fluorescent dyes, haptenic moieties, biotin, and radioactive atoms, as well as conjugated to enzymes and solid-phase supports. These derivatized lectins are useful for either direct or indirect detection and quantification techniques, or for the physical separation of particulate-bound or soluble glycoconjugates. Table 4 lists many of the commercially available lectin reagents and sources. 

An example for a homogeneous assay system is the polarization fluoroimmunoassay (PFIA). PFIA measures the increased polarization of fluorescence when a fluorophore-labeled hapten (tracer) is bound by a specific Ab, and the decreased signal when free analyte competes... 

Several modes of detecting a single base extension have been investigated, including measuring the incorporation of fluorescent, haptenated, or radioactive ddNTPs [6, 7] or gel electrophoresis based-detection of fluorescent primers extended by non-fluorescent nucleotides [8]. Recently, the Applied Biosystems SNapshot lYimer Extension Kit was introduced. In this assay, a primer is extended by one or more fluorescent- labelled dideoxynucleotides with subsequent detection in a fluorescent-based DNA sequencer. Several primers can be analysed within one lane of the DNA sequencer. 

Among homogeneous fluoroimmunoassays based on conventional fluorimetry, substrate-labeled fluoro-immunoassay is another alternative for both hapten and protein determination using a fluorescent label. As in other homogeneous competitive immunoassays, the sensitivity is limited by the serum background fluorescence and the limited amount of tracer that can be used. 

The substrate is cleaved by the enzyme-labeled hapten in the immunocomplex at the surface. The fluorescence can be measured in solution or at the surface. For the determination of high molecular mass antigens a two-site sandwich assay can be applied. A variation represents the double antibody assay, whereby the second antibody, which is directed against the hapten-specific antibody, is enzyme-labeled. Reagent-excess based assays are mainly used for the determination of antibodies in a noncompetitive format. In Scheme 6, a procedure for an indirect ELISA for the determination of antibodies is described. 

Laser-induced fluorescence polarization (LIFP) has been used as a detection technique in immunocapillary electrophoresis analysis of haptens. Polarization of fluorescence depends on the molecular size of the molecule. Small molecules exhibit low fluorescence polarization. LIFP was used to distinguish the peak of the fluorescent hapten-Ab complex from the peak of the excess of free fluorescent hapten in competitive assays [124]. Besides, the dependence of LIFP on molecular size has made it possible to perform quantitation without separating free from bound labeled hapten [125]. An attempt to use LIFP to quantitate staphylococcal enterotoxin A (28 kDa) did not show an increase of polarization upon formation of the Ag-Ab complex due to the high molecular weight of the Ag [126]. Formation of the complex could be identified by measuring the LIFP peak while varying the Ag/Ab ratio. 

Another approach to this persistent problem relies on haptenylation of primary antibodies. Hapten (e.g., biotin, digoxigenin or any fluorophore) can be covalently bound to the antibody via A-hydroxysuccinimide esters (NHS-ES) (see Sect. 2.1), or conjugated employing monovalent IgG Fc-specific Fab fragments (see Sect. 2.2). Haptenylated primary antibodies can be subsequently visualized with the use of secondary antibodies recognizing the corresponding hapten (Fig. 8.5). Fluorophore-labeled primary antibodies can be directly visualized in a fluorescent microscope. 

Probes can be differently labeled with hapten labels, for example carboxyfluorescein (6-FAM), digoxigenin (DIG) and biotin can be bound to LNA oligos. The choice of probe label depends on experimental design and the techniques available in the laboratory. The hapten label provides a template for crucial signal amplification since the FITC label on the oligo itself is not sufficient to allow detection in standard epifluorescence. In this study, the fluorescence signal was obtained with the TSA-FITC substrate, which allowed detection of miR-21 and miR-205. 

Self (S4) first proposed the concept of noncompetitive assay for haptens utilizing an adequate combination of an a-type and a jS-type anti-idiotype antibody, in which he used the term, selective antibody for the a-type antibodies. Then, Barnard and Cohen (Bl) applied this assay principle for the determination of serum E2, naming the assay system an idiometric assay. Figure 12A illustrates the assay procedure of the idiometric assay of E2. The target hapten is captured by excess anti-E2 antibody immobilized on microtiter strips by incubation at room temperature for 1 h (step i). After washing the strips, the /3-type anti-idiotype antibody was added in order to saturate (or block) the unoccupied paratope of the anti-E2 antibody (incubation, room temperature for 30 min) (step ii). The a-type anti-idiotype antibody, which has been labeled with a europium chelate (H4), was then added to the plate and incubated at room temperature for a further 2 h (step iii). Finally, fluorescence intensity due to bound europium was measured with a time-resolved fluorometer. Because of large steric hindrance around the bound jS-type antibody (MW 150,000), the labeled a-type antibody would. 

PCR-ELISA [37]) were described. The immobilized hapten-labeled amplification product was subsequently detected by an antibody-enzyme conjugate similar to conventional ELISA. By choice of a chemiluminescence- or fluorescence-inducing substrate, the sensitivity of the IPCR essay is further enhanced [37]. A comparison of different detection methods is given schematically in Fig. 6 typical results are compared in Fig. 5. 

Fluorescence may by induced using labeled mono- or polyclonal antibodies raised against the compound of interest. This technique has been used successfully to detect the presence of okadaic acid in cultures of Prorocentrum lima, and, further, to estimate quantities of the compound in individual cells.110 Immunofluoresence in combination with thin layer chromatography and ELISA techniques have also been used to detect multiple haptens in mycotoxin families.111112... 

The utihzation of fluorescence dyes for analytical measurements enhances the sensitivity for the detection of the molecules of interest. First, Cronick and Little made use of evanescent wave excitation for a fluorescence immunoassay, in 1975. By using totally internally reflected light, they excited the fluorescence of a fluorescein-labeled antibody which has become bound to a hapten-protein conjugate adsorbed on a quartz-plate in an antibody solution [41]. Contrary to the label-free high-refractive-index sensors where the mass of the molecule of interest is... 

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