Antibodies labelled

EIA was originally developed as a histological technique to localize specific ceUular sites using the specificity of an immunological reaction (23). The resulting fluorescent antibodies can be detected in animal tissues at levels as low as 1 /tg/mL of body fluid. Eluorophore-labeled antibodies have also been used widely for flow cytometry appHcations using fluorescein antibodies to cell surface markers to detect and quantify specific cells (24). 

Radioisotopes have become very important ia the practice of modem medicine, for both diagnosis and treatment. Some diagnoses are done by injecting a radionucHde ia a biochemical form such that it goes to a particular organ, and the measured radiation then allows the functional level of that organ to be determined. A common treatment is to expose a portion of the body, for example a tumor, to radiation from a radioisotope with the source either internal or external to the body. Another usage iavolves radioactively labeled antibodies (see Immunoassay). 

Keep, P. A, Searle, F., Begent, R. H. J., Barrat, G. M., Boden, J., Bagshawe, K. D., and Ryman, B. E. (1983). Clearance of injected radioactively labelled antibodies to tumor products by liposome-bound second antibodies, Oncodev. Biol. Med., 4, 273-280. 

The use of labelled antibodies suggests their presence in the amygdala, septum, hypothalamus and cerebellum. However, little is known about these receptors, mainly because of the shortage, until recently, of selective ligands, their low density and the limited distribution of their mRNA in the brain. 

CD36 or GP IV is a marker that is expressed on both resting and activated platelets. Labeled antibodies can be used that exhibit increased binding to activated platelets. 

Fig. 21. The N2S4 ligand diaminotetrathiol N,N,N, N -tetrakis-(2-mercaptoethyl)ethylene-diamme used to label antibodies...

The commercially available dicyanomethylene squaraine dye Seta-670-mono-NHS showed extremely low blinking effects and good photostability when used in single-molecule studies of multiple-fluorophore labeled antibodies [113]. Seta-670-mono-NHS and Seta-635-NH-mono-NHS were covalently labeled to antibodies and used in a surface-enhanced immunoassay [114]. From the fluorescence intensity and lifetime changes determined for a surface that had been coated with silver nanoparticles, both labeled compounds exhibited a 15- to 20-fold... 

Fig. 13.3. Fluorescence on the surface of H. contortus intestinal cells following incubation of transverse sections of the worm with fluorescein-labelled antibody probes.

Analyte is bound, allowing some labeled antibodies to link to the surface... 

Taylor CR, Burns J. The demonstration of plasma cells and other immunoglobulin containing cells in formalin-fixed, paraffin-embedded tissues using peroxidase labelled antibody. J. Clin. Pathol. 1974 27 14-20. 

The tuneable nature of the evanescent field penetration depth is critical to the effective operation of this sensor as it facilitates surface-specific excitation of fluorescence. This means that only those fluorophores attached to the surface via the antibody-antigen-labelled antibody recognition event... 

Figure 20. Image of fluorescence recorded from Cy5-labelled antibodies attached to (A) plain glass surface (B) surface containing metallic nanoislands.

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

Figure 7.21 Dendrimers that are fluorescently labeled as well as biotinylated create enhanced detection reagents for use in (strept)avidin-biotin-based assays. Large complexes containing multiple fluorescent dendrimers can bind to antigens and form a highly sensitive detection system that exceeds the detection capability of fluorescently labeled antibodies.

Figure 9.51 Time-resolved FRET assay systems involve energy transfer between the lanthanide chelate and an organic dye that are brought together as two labeled molecules bind to an analyte. In this illustration, an antibody labeled with a lanthanide chelate is used along with a Cy5-labeled antibody to detect a protein target in solution. Excitation of the lanthanide label results in energy transfer and excitation of the cyanine dye only if they are held within close enough proximity to allow efficient FRET to occur. Under these conditions, excitation of the lanthanide chelate results in cyanine dye emission, which will not occur if the labeled antibodies have not bound to a target.

Organic fluorescent dyes with the appropriate spectral properties also can be paired with lanthanide chelates in FRET systems. For instance, many rhodamine dyes and the cyanine dye Cy5 have ideal excitation wavelengths for receiving energy from a nearby europium chelate. The LeadSeeker assay system from GE Healthcare incorporates various Cy5-labeled antibodies for developing specific analyte assays. In addition, if using a terbium chelate as the donor, then a Cy3 fluorescent dye can be used in assays as the acceptor. 

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