Rhodamine labeling antibody with

Labelled antibodies with a fluorescent derivative obtained from fluorescein, rhodamine or luminarin, are well suited for the measurement of a number of human and animal immunoglobulins. However, the transposition of this principle to immunochemical testing for simple organic molecules remains, as yet, non-existent. There may be discrimination between the labelled and non-labelled species at the isolation stage for small organic molecules. This explains that only a few examples of analysis followed by fluorescence measurement. 

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. 

The detection of flu viruses via a fluorescent sandwich immunoassay was reported by Bucher.(10) However, the method sensitivity was too low for direct detection of the virus. A novel sandwich immunoassay was described by Ogcr((lff7 for the detection of Botulinum Toxin A. Antibodies specific for Clostridium botulinum were covalently attached to the surface of a tapered fiber. After the capture of the antigen, a sandwich was formed with a rhodamine-labeled anti-toxin IgG, and the evanescent wave was measured. The assay was highly specific with detection limits near 5 ppb. 

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. 

Figure 7,13. AChR dusters on a rat rayotube in culture, visualized with (A) rhodamine anti-43K protein (B) fluorescein-labeled a-bungarotoxin (corresponding field) (C) rhodamine-labeled anti-vinculin and (D) fluorescein-labeled g-bungarotoxin (corresponding field). The antibodies were gifts from R. Bloch. University of Maryland Medical School. Note that AChR and 43K tend to aggregate in the same regions, whereas AChR and vinculin appear to exdude each other. Additional photographs are in Ref. 107.

Diluted human serum (1 pL) was incubated with the peptide microarray and bound antibodies were detected using a rhodamine-labeled anti-human IgG. Signal was detected using a slide scanner (Affymetrix model 418) with data collection in the Cy3 channel. A reported eightfold gain in sensitivify at 100% specificity over standard ELISA was achieved using the peptide microarray. 

In addition to the wide range of commercial probes obtainable, 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 fluorescein, rhodamine, Texas red, aminomethylcoumarin (AMCA), and phycoerythrin. Figure 311 shows the reaction of fluorescein isothiocyanate, one of the most common fluorescent probes, with an antibody molecule. 

The isothiocyanate derivatives of fluorescein and rhodamine are widely used to label antibodies. They react with the amino groups of the immunoglobin G (IgG) molecule under alkaline conditions and a molar excess of about 20 is usually optimal. More recently, N-... 

Fluorescently labelled antibodies can be used to visualise cellular or subcellular structures. This is done by incubating antibodies against specific cellular antigens with frozen or fixed tissues sections, or even permeabilised cells (Javois 1994). Unbound antibodies are removed by washing, and then a second anti-immunoglobulin antibody coupled to a fluorescent group, such as fluorescein or rhodamine, is added to the preparation. The sample is washed free of excess fluorescent antibody and visualised using a fluorescence microscope. 

Apply rhodamine-labeled mouse monoclonal anti-K antibody Wash with PBS... 

Finally, a double labelling with antibodies and a viability substrate can be performed. De Vos and Nelis (2003, 2006) combined ChemChrome V6 with tetram-ethyl rhodamin isothiocyanate (TRITC) labelled antibodies for the detection of Aspergillus fumigatus. In these approaches, the ChemChrome reagent, yielding green fluorescence, ensures the primary detection by the ChemScan, whereas the TRITC label results in red fluorescence, to be observed microscopically. 

The first report of this type was by Zavala and Brandon [48] who sectioned root tips at low temperature in order to try and prevent the redistribution of hormones. Their tissue sections were then probed with dihydrozeatin antibodies labelled with rhodamine. For electron microscopy, samples were freeze-substituted in ethanol or acetone and then embedded in polymers prior to sectioning and probing with colloidal gold-labelled antibodies. Although apparent specific labelling was reported in this work, it has been criticised [147] because dihydrozeatin is a relatively rare cytokinin, that had not been... 

Fluorescence detection relies on the visualization of a secondary antibody that has been labeled with a fluorophore such as fluorescein (FITC), Texas Red, Tetramethyl rhodamine (TRITC), or R-phycoerythrin. Although this method of detection has a reduced sensitivity of twofold to fourfold compared to chemiluminescence detection, it presents a tenfold greater linear dynamic range, thus providing better linearity and better quantiflcation within the detection limits. Since secondary antibodies can be labeled with fluor-ophores of distinct colors, multiplexing (simultaneous detection of several antigens) of the same blot is feasible. 

Incubate with Rhodamine and/or FITC labeled secondary antibody for 1 hr at RT. 

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. 

Antibodies labeled with fluorescent molecules have several applications, particularly in cytochemistry and cell sorting. There are many fluorochromes used in labeling (1), such as coumarin derivatives, phycobiliproteins, and rare earth chelates however, fluorescein and rhodamine (Table 1) are the most commonly used. 

Direct labeling of a biomolecule involves the introduction of a covalently linked fluorophore in the nucleic acid sequence or in the amino acid sequence of a protein or antibody. Fluorescein, rhodamine derivatives, the Alexa, and BODIPY dyes (Molecular Probes [92]) as well as the cyanine dyes (Amersham Biosciences [134]) are widely used labels. These probe families show different absorption and emission wavelengths and span the whole visible spectrum (e.g., Alexa Fluor dyes show UV excitation at 350 nm to far red excitation at 633 nm). Furthermore, for differential expression analysis, probe families with similar chemical structures but different spectroscopic properties are desirable, for example the cyanine dyes Cy3 and Cy5 (excitation at 548 and 646 nm, respectively). The design of fluorescent labels is still an active area of research, and various new dyes have been reported that differ in terms of decay times, wavelength, conjugatibility, and quantum yields before and after conjugation [135]. New ruthenium markers have been reported as well [136]. 

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