Fluorescence-tagged antibodies

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... 

Because of their characteristic distributions, IF proteins are useful in the diagnosis and treatment of certain tumors. In a tumor, cells lose their normal appearance, and thus their origin cannot be identified by their morphology. However, tumor cells retain many of the differentiated properties of the cells from which they are derived, Including the expression of particular IF proteins. With the use of fluorescence-tagged antibodies specific for those IF proteins, diagnosticians can often determine whether a tumor originated in epithelial, mesenchymal, or neuronal tissue. 

Flow cytometry has been at the forefront of immunophenotyping (see Chapter 3.2). Immunophenotyping is the term used to describe the application of fluorescently tagged antibodies to discriminate and quantitate the various constituent cells of the immune system by flow cytometry. Immunophenotyping cells on the basis of cell surface markers can be thought of as the flrst generation application of flow cytometry in immunotoxicology. 

Cover slip the slides and treat thereafter as if they have been labeled with a fluorescently tagged antibody (see Section 3 7.1, step 5 and Notes 40 and 41). 

In addition to wavelength and time-resolved fluorescence techniques, polarization fluorescence can yield important information about an analyte." This is especially true when differentiating between chiral compounds. Combining, for example, a fluorescently tagged antibody immunoassay with polarization detection allows for very sensitive detection limits of chiral enantiomers. Laser-induced fluorescence polarization (LIFP) has been used to detect concentrations as low as 0.9 nM of an antibody-boimd cyclosporine A (an immunosuppressive drug) in human blood. A conventional single channel fluorescence detector can be easily modified to perform such measurements, simply by adding the appropriate polarization filters. 

Alternatively, fluorescently tagged antibodies can replace the first layers of B4F and streptavidin (Fig. 4c). See Note 4. 

Fig. 4 Functional LAPAP patterns, (a) Biotinylated peptides can be used. Since streptavidin has four binding sites, beyond steric effects, approximately three peptides will react with each molecule, (b) Primary and biotinylated secondary antibodies represent an alternative approach for full protein LAPAP patterns. Adapted from [11]. (c) Fluorescently tagged antibodies can replace B4F and be combined with primary antibodies to bind full proteins to the pattern. Different color antibodies can be used to create multicomponent patterns...

Another popular assay format for kinase assays is the Lanthascreen. This format is a variation on the LANCE assay, but employs Tb as the cryptate. In this format N-terminally fluorescently tagged peptide substrate (acceptor) is phosphorylated by the kinase. Next, a phophospecific antibody which is labeled with terbium binds specifically to the phosphorylated product, placing the donor and acceptor in close proximity, generating a signal [25]. 

Figure 7.1. Protein expression mapping using an antibody array. The antibody array consists of monoclonal antibodies specific for a set of proteins in the organism of interest gridded onto a filter. To determine if a protein is expressed under the conditions being tested, a crude lysate is obtained and the proteins within the lysate are labeled with a fluorescent tag. The lysate is applied to the filter and the proteins are allowed to bind to the relevant antibody. Bound proteins are visualized via the fluorescent tag.

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

A spectrophotometric assessment of the F/P ratio should be done after purification of the tagged antibody. The measurement of absorbance at 495 nm (for fluorescein) divided by the absorbance at 280 nm should be between 0.3 and 1.0 to obtain a good fluorescent derivative of acceptable activity and low background. This usually translates into a ratio of about 4-7 fluorescein molecules per protein molecule. 

The level of TRITC modification in a macromolecule can be determined by measuring its absorbance at or near its characteristic absorption maximum ( 575nm). The number of fluor-ochrome molecules per molecule of protein is known as the F/P ratio. This value should be measured for all derivatives prepared with fluorescent tags. The ratio is especially important in predicting the behavior of antibodies labeled with TRITC. For a TRITC-labeled protein, the ratio of its absorbance at 575-280 nm should be between 0.3 and 0.7. 

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. 

It is well known that a very important feature of many biological systems is specific recognition at the molecular level. Antibodies as a group are widely used for molecular recognition, e.g., affinity assays. This feature can be used by labeling an anti-human growth hormone antibody fraction with a fluorescent tag... 

FIA Fluorescence immunoassay uses a fluorescent tag on the antibody or antigen. Fluorescent labels absorb light of one wavelength and reemit it at another wavelength. The label is excited by UV and emits visible light. Common fluorescent labels are fluorescein, Texas red, and GFP (green fluorescent protein). 

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