Monoclonal antibodies with fluorescein

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... 

This chapter provides protocols for the most convenient methods of labelling mouse IgG monoclonal antibodies with enzymes (alkaline phosphatase and peroxidase), a fluorescent molecule (fluorescein), biotin, and DIG. A general protocol for the evaluation of the labelled monoclonal antibody is also given. 

Figure 7 Immunopotentiating reconstituted influenza virosomes (IRIV) mediated adjuvance in cytotoxic T-cell induction requires CD4+ T cells. CD8+ and CD14+ cells were cultured in the presence of autologous intact or irradiated CD4+ cells. These cultures were stimulated with influenza matrix (IM)58 66 (1 Pg/mL) alone (A) or supplemented with IRIV (1 50) (B). After seven days of incubation both cocultures were restimulated with irradiated IMss-ee pulsed CD14+ cells and cultured for six further days in the presence of interleukin-2 [see Materials and Methods ]. Six days after restimulation, cultures were stained with HLA-A0201 /IM58-66 PE-specilic tetramers and anti-CD8 fluorescein isothiocyanate monoclonal antibodies. Source. From Ref 6.

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. 

BrdU/DNA flow cytometry offers flexibility and diversity in the study of cell kinetics from cells in culture to human tumors in vivo. The essence of the procedure is to pulse label with BrdU by a short-term incubation in vitro or by a single injection in vivo samples are then taken at time intervals thereafter and stained after fixation in ethanol. The cells are then stained with a monoclonal antibody against BrdU that can be either directly conjugated to a fluoro-chrome (usually fluorescein isothiocyanate [FITC]) or, alternatively, bound to a second antibody conjugated with FITC. The cells are then counterstained with propidium iodide (PI) to measure the DNA content and analyzed on the flow cytometer. The results are displayed as linear-red fluorescence on the x-axis vs linear or log-green fluorescence on they-axis. 

The cells and nuclei (0.5-1.0 X 106) are aliquoted into polystyrene tubes. Approximately 10 jjlI of normal horse serum (Vector Laboratories, Burlingame, CA) is added to block any nonspecific binding. The suspension is incubated with biotinylated antiestrogen monoclonal antibody (1D5, Dako Corp., Carpinteria, CA) at 1 25 dilution for 1 hr at 37°C. Aliquots are stained with 10 p,l of fluorescein isothiocyanate (FITC)-conjugated strepta-vidin (Dako buffer containing 0.1% Triton X-100). 

To be of use in microscopy or flow cytometry, this bond needs to be visualized (to the eye or to the photodetector) by the addition of a fluorescent tag. Visualization can be accomplished by one of two different methods. With direct staining, cells are incubated with a monoclonal antibody that has been previously conjugated to a fluorochrome (for example, fluorescein or phycoerythrin or any fluorochrome with appropriate absorption and emission spectra). This procedure is quick and direct it merely involves a half-hour incubation of cells with antibody (at 4°C), followed by several washes to remove weakly or nonspecifically bound antibodies. Cells thus treated are ready for flow analysis (although final fixation with 1% electron microscopic-grade formaldehyde will provide a measure of biological safety and long-term stability). 

The correct control is always an antibody of exactly the same properties as the monoclonal antibody used in the experiment, but with an irrelevant specificity. If we are staining cells with a monoclonal antibody having a specificity for the CD3 protein occurring on the surface of T lymphocytes (and that monoclonal antibody happens to be a mouse immunoglobulin of the IgG2a subclass, conjugated with six fluorescein molecules per molecule of protein and used to stain the cells at a concentration of 10 pg per ml), then an appropri-... 

Direct fluorescent antibody smears have become a more efficient method than Giemsa stains or tissue cultures fiar identifying chlamydia. Commercially prepared kits make specimen collection convenient, and results are available in approximately 24 hours. Good results, however, depend on obtaining an adequate specimen. Fluorescein-labeled monoclonal antibodies in the staining reagent specific for Chlamydia trachomatis outer membrane proteins bind to the C. trachomatis in the smear. Studies that compare direct fluorescein antibody techniques with tissue culture results have found acceptable sensitivity and specificity values. 

Some biodnylated andbodies will not react with some secondary reagents. If this is the case (which can only be established by experiment), it is possible to use one biotinylated primary antibody together with a second, unconjugated, primary antibody and a fluorescein-labeled and-Ig. The cells are incubated with the biotinylated antibody followed by streptavidin-PE together with the second primary monoclonal antibody, followed by an andmouse Ig conjugated to fluorescein. 

Fluorescence resonance energy transfer (FRET) is a technique that has been used to measure distances between pairs of proximal fluorochromes. A suitable pair consists of a donor fluorochrome, which has an emission spectrum that significantly overlaps with the absorption spectrum of an acceptor fluorochrome (2). With the availability of monoclonal antibodies to many cell-surface determinants, intramolecular distances between nearby epitopes and intermolecular distances between adjacent cell-surface macromolecules can be investigated to analyze molecular interactions influencing important cellular events. Such monoclonal antibodies can be conjugated to fluorescein-isothiocyanate (FITC) as the donor, and either tetramethyl-rhodamine-isothiocyanate (TRITC) or phycoerythrin (PE) as the acceptor. 

Immunohistochemical studies of Mn-SOD were done on ovarian cancer tissues. Of four ovarian serous cystadenocarcinoma tissues tested, two stained positively with the antibody used for the ELISA experiments. Antibody localization of Mn-SOD in the tissue is illustrated in Fig. 24. Control sections incubated with a monoclonal antihuman IgG and then stained with fluorescein-conjugated horse antimouse immunoglobulin failed to show uptake of antibody. No reactivity with... 

E. coli OmpA antibodies, was developed. A biotin-labelled anti-TKSNVYGK monoclonal antibody was first incubated with the P40 solution. After addition of streptavidin, the complex formed between the antibody and residual E. coli OmpA was captured by filtration through a biotinylated nitrocellulose membrane. The membrane was further incubated with a rabbit fluorescein-labelled polyclonal anti-OmpA serum, obtained by... 

Immunocapture Immunocapture uses a solid phase coated with avidin or streptavidin to capture nucleic acids containing biotin. Biotin is introduced into the nucleic acids by the use of a biotinylated de-oxyribonucleoside triphosphate (dNTP) or a 5 -bio-tinylated primer during PCR amplification. Haptens such as digoxygenin or fluorescein are also widely used in combination with monoclonal antibodies. 

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