Fluorescent labels antibody labeling with

Recently, polyclonal mouse antihuman IgG antibodies fluorescently labeled with Cy5 were used to detect the binding of human myeloma proteins to biotinylated monoclonal antibodies. Again, Cy5-labeled antibodies were used to determine bacterial, viral, and protein antigens bound to biotinylated IgGs. 

In preparation for the TNT assay, the analog trinitrobenzene (TNB) was fluorescently-labelled with the cyanine dye Cy5 (ex 650, em 670) to form Cy5-TNB. A monoclonal antibody, 11B3 was immobilized on the surface of the probe as the recognition molecule. All buffers, except for the regeneration buffer, contained phosphate buffered saline pH 7.4 with 10% ethanol and 2 mg/ml bovine serum albumin. The regeneration buffer contained 50 50 PBSrethanol... 

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the soHd phase. The analyte-indicator on the soHd phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

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]. 

The LANCE cAMP assay is a competitive assay in which cAMP produced by the cells competes with fluorescent-labeled acceptor cAMP for a cryptate tagged donor antibody. The principal of the assay is shown in Fig. 6. On the left strepta-vidin conjugated Europium binds to biotinylated cAMP. An antibody labeled with the fluorescent dye Alexa binds to the cAMP, bringing the donor and acceptor into close proximity, and energy transfer occurs. When the cell releases cAMP, it competes with the biotin-labeled cAMP for the antibody, and a signal decrease is observed. In the TR-FRET assay the antibody is directly labeled with either Eu or Tb. In this format an increase in cAMP also causes a decrease in signal. 

Indirect immunofluorescence assay (IFA) A laboratory test used to detect antibodies in serum or other body fluid. The specific antibodies are labeled with a compound that will make them glow a fluorescent green color when observed microscopically under ultraviolet light. 

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... 

FIGURE 1.2 In situ localization of the OCP-green fluorescence protein (GFP) fusion protein Immunogold labeling of a thin section of OCP-GFP transformed Synechocystis PCC6803 OCP-GFP cells were labeled with a polyclonal antibody against the GFP coupled to 10 nm gold particles. Bar = 0.5 pm. 

Use of sulfo-NHS-LC-SPDP or other heterobifunctional crosslinkers to modify PAMAM dendrimers may be done along with the use of a secondary conjugation reaction to couple a detectable label or another protein to the dendrimer surface. Patri et al. (2004) used the SPDP activation method along with amine-reactive fluorescent labels (FITC or 6-carboxytetramethylrhodamine succinimidyl ester) to create an antibody conjugate, which also was detectable by fluorescent imaging. Thomas et al. (2004) used a similar procedure and the same crosslinker to thiolate dendrimers for conjugation with sulfo-SMCC-activated antibodies. In this case, the dendrimers were labeled with FITC at a level of 5 fluorescent molecules per G-5 PAMAM molecule. 

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... 

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. 

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