Fluorescent probes antibody labeling with

FIAs can be based on steady-state intensity measurements without probe amplification, owing to the sensitivity of detection that is possible with fluorescence instrumentation, which exceeds that of spectrophotometers by two or three orders of magnitude. A sensitive fluorometer has been described for an estradiol assay(36) in which the limit of estradiol detection is 3 x KT11 M. Estradiol antibody labeled with rhodamine B is reacted with estradiol samples. Unreacted labeled antibody is removed with Sepharose-estradiol-casein beads, and the remaining fluorescence is directly proportional to the analyte concentration. The detection limit of rhodamine B on the same fluorometer is 5 x 1(T12 M. This instrument uses a 0.75 mW green helium-neon (HeNe) laser to irradiate the sample from above, at the air-liquid interface, to increase the light path and to decrease surface reflections. The sample compartment has a top-mounted photon trap, and a mirror mounted on the side of the sample compartment opposite the PMT to enhance detection. 

These assays resemble a hybrid of an immunohistochemistry assay and ELISA. Whole cells are fixed, for example with 3.7% formaldehyde, to MTPs permeabilized by repetitive washing with 0.1% Triton X-100, blocked with a protein solution, probed with primary antibodies (phospho-spe-cific, and non-phospho-specific), washed, and subsequently the secondary antibodies labeled with infrared fluorescent tags are added. After washing, these assays are read in a reader (such as the Odyssey or Aerius) designed for high sensitivity detection of two colors. The two colors are useful because one color can be used to accommodate a stain assigned as a total protein or cell number control or as an antibody to total protein, which allows for normalization. These assays may only require a single antibody versus the dual antibody sandwich required for ELISA. 

The fluorogenic molecules used to covalently label the antigens or the antibodies to be used in a fluorescence immunoassay are called fluorescent probes or labels. Fluorescent probes are small molecules whose fluorescent properties are altered subsequent to interactions with proteins or other macromolecules. 

There are a number of different probes available, including detection probes, which are labelled with a fluorescence marker or, for example, digoxigenin for a further linkage with an antibody-enzyme complex and then a later colorimetric reaction with a chromogenic substrate (such as nitro blue tetrazolium for alkaline phophatase) (Helentjaris McCreery, 1996 Kempf et al., 2000), Capture probes are used to bind the target sequence (RNA or DNA) to a plate or another surface. In most cases the probes are labelled with biotin to react with avidin, which is coated on a plate (Riley, Marshall, Coleman, 1986). 

Fluorescent labels, by contrast, can provide tremendous sensitivity due to their property of discrete emission of light upon excitation. Proteins, nucleic acids, and other molecules can be labeled with fluorescent probes to provide highly receptive reagents for numerous in vitro assay procedures. For instance, fluorescently tagged antibodies can be used to probe cells and tissues for the presence of particular antigens, and then detected through the use of fluorescence microscopy techniques. Since each probe has its own fluorescence emission character, more... 

Antibody molecules can be labeled with any one of more than a dozen different fluorescent probes currently available from commercial sources. Each probe option has its own characteristic spectral signals of excitation (or absorption) and emission (or fluorescence). Many derivatives of these fluorescent probes possess reactive functionalities convenient for covalently linking to antibodies and other molecules. Each of the main fluorophore families contains at least a few different choices in coupling chemistry to direct the modification reaction to selected functional groups on the molecule to be labeled. These choices include amine-reactive, sulfhydryl-reactive, and carbonyl-reactive. Examples of some of the more popular varieties of fluorescent probes can be found in Chapter 9. 

In addition to the wide range of commercial probes, many other fluorescent molecules have been synthesized and described in the literature. Only a handful, however, are generally used to label antibody molecules. Perhaps the most common fluorescent tags with application to immunoglobulin assays are reflected in the main derivatives produced by the prominent antibody manufacturing companies. These include derivatives of cyanine dyes, fluorescein, rhod-amine, Texas red, aminomethylcoumarin (AMCA), and phycoerythrin. Figure 20.16 shows the reaction of fluorescein isothiocyanate (FITC), one of the most common fluorescent probes, with an antibody molecule. 

There are literally hundreds of markers that are currently available for the mouse and human than can be used to characterize lymphoid and myeloid cells and subsets in primary and secondary lymphoid organs. Many of the markers expressed in mammals are highly conserved across species and have been designated as genetic clusters of differentiation (CD). CDs can be identified with fluorescently labeled monoclonal antibodies. As presented previously, when combined with other fluorescent probes, important information on intracellular biochemistry and cell function can be obtained. Many of the biochemical markers used by immunotoxicologists are common to other... 

It was shown by Creech and Jones (1) in 1940 that proteins, including antibodies, could be labeled with a fluorescent dye (phenylisocyanate) without biological or immunological effects to the intended target. In theory, fluorescent reporters (tracers, probes, antibodies, stains, and so on) can be used to detect or measure any cell constituent, provided that the tag reacts specifically and stoichiometrically with the cellular constituent in question (2). Today, the repertoire of fluorescent probes is expanding almost daily see Chapter 14). One area that has benefited from the ever-increasing number of fluorescent probes is flow cytometry. 

Fig. 28. Synthesis of labeled DNA probes. A Labeled DNA can be generated using different enzymes (Klenow fragment of DNA polymerase or a terminal transferase) to incorporate labeled nucleotides into specific DNA sequences. Probes can be labeled using radioactive nucleotides or nucleotides labeled with an immunogenic molecule such as biotin. B The labeled probe is then hybridized to the target nucleic acid, which is either bound to a membrane or in a tissue section or cell. An antibody is then used to detect the non-radioactively-labeled probe. C The antibody may be conjugated to a fluorescent or chemiluminescent dye, or an enzyme that produces a color reaction. The target nucleic acid is thus visualized.

Further application involved collected of fluorescence from dansyl-labeled bovine serum albumin via TIRF optics 150), TIRF-immunoassay for specific dye-labeled antibodies binding from the solution to an antigen-coated surface 151), and a viro-meter — a optical sensor for viruses treated with a fluorescent probe bound to the virus nucleic acid 152,153). 

The IFA is an immunoassay in which the antibody or antigen are labeled with a fluorescent probe. These can be direct or indirect (8, 41, 57). The IFA technique is usually used to locate cellular constituents. The commonly used fluorescent probes are rhodamine B isothiocyanate and fluorescein isothiocyanate. The sensitivity of the assay ranges in the ng/ml range. 

The high sample demands and low-throughput of LC-MS methods have led to the creation of a capillary electrophoresis (CE) platform for ABPP [48]. Proteomes are labeled with a fluorescent probe, digested with trypsin, and enriched with antifluorophore antibody resins. Use of CE coupled with laser-induced fluorescence (LIF) detection to analyze the enriched peptides resulted in far superior resolution to ID SDS-PAGE, particularly for enzymes that share similar molecular masses. Sensitivity limits of 0.05-0.1 pmol/mg proteome, negligible sample requirements (—0.01—0.1 pg proteome), and the ability to perform rapid CE runs in parallel with 96-channel instruments, make CE-based ABPP a potentially powerful technique. One drawback is that the identities of the probe-labeled proteins are not immediately apparent, and correlated LC-MS experiments must be performed to assign protein identities to the peaks on the CE readout. 

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