Fluorescein labeling antibodies with

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.

In a particle concentration fluoroimmunoassay system that is based on microtiter plates with 0.22-/ filters on the bottoms, microparticles are used with a flowthrough wash system. Either fluorescein-labeled antibodies or cells serve as labels.(41) Car-boxyfluorescein diacetate, which crosses the cell membrane, is hydrolyzed by cytoplasmic esterases, thereby trapping carboxyfluorescein within the cell. The method is also useful for screening and isotyping antibodies. 

The high molar absorptivities and quantum yields of the large protein fluorophore phycoerythrin (240,000 Da) have been exploited in energy transfer assays. Phyco-erythrin has been used as both donor and acceptor, with several bound antigen molecules per phycoerythrin molecule/86,94) The usefulness of BPE is indicated in competitive assays for human IgG that use fluorescein-labeled antibody as donor to... 

Refining the standardized measurement referred to above to provide a calibrated scale is a more difficult task, although calibration beads of various types now available facilitate the process. Beads with manufacturer s reported equivalent fluorescein content have been available for some time (Flow Cytometry Standards Corporation, San Juan, Puerto Rico), and these are useful for calibration of measurements using fluorescein-labeled antibodies, although... 

This method was modified by Lisi et al. (L8) using antibody-coated microspheres [Ab— -]. Added unlabeled antigen [Ag] and fluorescein-labeled antibody [F-Ab] bind to the antibody-coated microspheres as in the conventional sandwich immunoassay [F-Ab Ag Ab - -]. After the reaction is complete the suspension is introduced into a flow cytometer with a laser light source. By gating fluorescence light accumulation on scattered light pulses,... 

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. 

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. 

Rao et al.20 demonstrated a fluorescence polarization immunoassay for evaluating serum concentrations of tricyclic antidepressants (amitriptyline, imipramine, clomipramine, and doxepin) with respect to nonresponse, compliance, therapeutic window, and influences of age, sex, substance abuse, and toxicity. Abbott Laboratories TDx/TDxFLx Toxicology Tricyclic Assay FPIA (fluorescence polarization immunoassay) was used. This assay of 50 /uL samples contained tricyclic antidepressant antibodies raised in rabbits and fluorescein-labeled tricyclic antidepressant as a tracer. The assay was calibrated with imipramine in the range of 75 to 1000 fig/L (268 to 3571 nmol/L). Intra-assay and inter-assay coefficients of variation for internal quality control samples from the manufacturer were 4.2 and 4.7%, respectively. The limits of detection were 72,71,64, and 72 nmol/L for amitriptyline, imipramine, clomipramine, and doxepin, respectively. This high-throughput immunoassay was easy to use although amitriptyline, dosulepine, desipramine, and nortriptyline showed cross-reactivities ranging from 74 to 100%. 

Phase-separation immunoassays have been reported, in which the solid phase particles are formed after the immunoreaction is completed.(42) Phase-separation immunoassays are advantageous since the unstirred layer of solution near a solid surface alters diffusion and binding kinetics at the surface in comparison with the properties of the bulk solution. In phase-separation assays for IgG and IgM, capture antibodies are bound with monomers suitable for styrene or acrylamide polymerization.(42) Monomer-labeled capture antibodies are reacted with analyte and with fluorescein- and/or phycoerythrin-labeled antibodies in a sandwich assay, followed by polymerization of the monomers. Fluorescence of the resulting particles is quantitated in a FACS IV flow microfluorometer, and is directly proportional to analyte concentration. 

A report of a particle-based FPIA<56) details improvement in the limit of detection by two orders of magnitude over a conventional FPIA format for rabbit IgG, to 10 ° M. Goat anti-rabbit capture antibody (binder) is immobilized on polystyrene microspheres or colloidal gold particles, and the sample competes with a fluorescein-labeled rabbit IgG probe. The observed increase in sensitivity is apparently due to the large increase in effective mass of the binding complex. The polarization range is rather limited, though, reportedly due to low concentrations of immobilized antibodies. 

An example of an evanescent wave fiber optic immunoassay and the associated optics has been described in detail for measurement of anti-rabbit IgG.(130) Rabbit antibody is immobilized on the distal face of an optical fiber. Unlabeled anti-rabbit IgG competes with fluorescein-labeled anti-rabbit IgG for rabbit antibody binding sites... 

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.

It is entirely possible that surface staining cannot be accomplished before fixation. Some antibody-antigen complexes cannot withstand chemical fixation and/or permeabilization. An empirical evaluation must be made. In this example, cells are first stained with a monoclonal antibody against a cell-surface receptor, fixed with ethanol, and then the DNA is stained with propidium iodide. The cells are analyzed for two-color fluorescence, the green of the fluorescein-labeled surface marker and the red of the labeled DNA intercalator. This approach works for antibody-antigens that are unaffected by fixation. 

The dissociation constant (Kd) of a monoclonal antibody with fluorescein isothiocyanate- (FITC)-labeled insulin and unlabeled insulins from several species were measured using CE with laser-induced fluorescence detection (CE-LIF) (9). Kd determinations were made by separating free FITC-labeled insulin and its complex with the antibody in equilibrated solutions in 6 s or less (Fig. 3). Dissociation and association rates for insulin, FITC-insulin, and the antibody are fast enough to reach equilibria in less... 

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