Fluorescent antisera

In recent years, serological techniques have been developed for identifying and enumerating wild yeasts in the presence of culture yeast (see Chapter 16) [92, 93]. It is now possible by fluorescent antisera to detect under an ultraviolet microscope certain wild yeasts (including those in the genus Saccharomyces) at levels as low as one cell per 10 culture yeasts. 

A switch in biosynthesis, within a clone of cells, from one class of immunoglobulin to another class having the same antigen-binding specificity would be consistent with the two genes-one polypeptide hypothesis. Evidence for such a switch has been derived from experiments at the cellular level. Individual plasma cells from the bone marrow of patient Til were shown, with class-specific fluorescent antisera, to synthesize either IgG or IgM, but no individual cells producing both classes were identified (38). In a subsequent study it was found that 100% of plasma cells that were stained by fluorescent antiidiotypic antibodies directed to Til IgG were also stained by antiidiotypic antibodies pre-... 

The authors have furthermore examined the sensitivity of the assay. They found that if fluorescent antisera were diluted eightfold, with bleached fluorescent antisera, the percentage of fluorescent cells remained unchanged. The sensitivity of detection can be further increased with an additional layer of G anti-R-Ig Fl, but minute amounts of immunoglobulin (below 3%), would however escape detection. 

In chicken tissues, fluorescent antiserum against connectin stained only skeletal and cardiac muscles. Chicken gizzard and aorta were negatively stained (Ikeya et al., 1983). Examinations by SDS-gel electrophoresis confirmed that connectin-like proteins are not present in nonmuscle cells (D. H. Hu, unpublished observations, 1984). 

Where possible, the antibodies used for labeling should be pure (see Chapters 2-5). Fluorochrome-labeled, affinity-purified antibodies produce less background and lower non-specific fluorescence than are obtained with fluorescent antiserum or... 

Inoculation of cell cultures with virus-containing material produces characteristic changes in the cells. The replication of many types of viruses produces the cytopathic effect (CPE) in which cells degenerate. This effect is seen as the shrinkage or sometimes ballooning of cells and the disruption of the monolayer by death and detachment of the cells (Fig. 3.6). The replicating virus can then be identified by inoculating a series of cell cultures with mixtures of the virus and different known viral antisera. If the virus is the same as one of the types used to prepare the various antisera, then its activity will be neutralized by that particular antiserum and CPE will not be apparent in that tube. Alternatively viral antisera labelled with a fluorescent dye can be used to identify the virus in the cell culture. 

Figure 6.1. Fluorescence polarization immunoassay for theophylline. (A) Effect of theophylline rabbit antiserum ( ) and normal rabbit serum (A) on the fluorescence polarization of the theophylline-umbelliferone conjugate. (B) Fluorescence polarization of the theophylline-umbelliferone conjugate in the presence of varying concentrations of theophylline. (Reprinted from Ref. 1, with permission from Academic Press.)...

Both IgG and Csb were found to stimulate the respiratory burst separately and independently in one study but in another was active only in the presence of IgG. The divergent findings may be attributable to differing methods. In one study serum was mixed with agarose beads which fix complement with the binding of Csb- One set of beads was then heated at 50° for 30 min to remove 35 and the other was boiled in 2 M NaCl to remove IgG. The completeness of the removal of each component was verified by the loss of reactivity of the beads with a fluorescent monospecific antibody. Beads prepared in this way with either IgG or with Cjb attached elicited the formation of O by PMNs whereas the release of lysosomal contents occurred only with beads containing both IgG and Cab. The effects of the IgG-agarose were blocked by Ffab lj and those of Cjb were blocked by antiserum to C,. 

Figure 11. Microscope photo of human RBC sensitized with rabbit antihuman antiserum labeled with fluorescent microspheres (340 nm in diameter) bonded to goat antirabbit IgQ...

Indirect Fluorescence. This method is normally used for antibody quantitation or screening. Antigen is immobilized onto a solid support, and antiserum is added. If specific antibodies are present, these antibodies will be immobilized on the support. Following a rinse step, fluorophore-labeled antibodies to the particular antibody class are added (e.g., fluorescein-anti-IgG). These labeled antibodies are immobilized only if primary antibodies to the immobilized antigen are present on the solid support. Fluorescence intensity thus increases with antibody concentration. 

Typically 1 adduct out 10 nucleotides. Depends on the method used for the detection of antiserum bound in microtiter plates, i.e., colorimetry, fluorescence, or chemiluminescence. 

Some assays or applications do not require a purified antibody. In this case the polyclonal antibodies can be used as antiserum and the monoclonals as either ascitic fluid or supernatants. Other assays require that the antibodies present in the serum, ascites or supernatants be in purified form some examples include the following (1) when the antibodies are used after chemical modifications such as labeling with fluorescent probes or isotopes ... 

We perceived the need for sensitive assays that do not rely on the use of radioisotopes or extensive analytical methodology and that could accurately detect protein-bound acetaminophen in biological fluids in the presence of unbound acetaminophen. To this end, we recently developed sensitive avidin biotin-amplified ELISA (A-B ELISA) and particle concentration fluorescence immunoassays (PCFIA) which use antiserum specific for the major acetaminophen-protein adduct associated with toxicity (13-16). These assays are new tools to study the relation between formation of the 3-Cys-A protein adduct and acetaminophen-induced toxicity. In this report we review how these assays were developed, validated in laboratory animals, and used to quantify 3-Cys-A protein adduct formation in human acetaminophen overdose patients. 

To confirm that the cell bodies analyzed were from PM4 neurons, Lucifer yellow (a fluorescent dye) was injected (using 5 nA hyperpolanzing pulses of 100 ms duration at 5 Hz for 5-10 min) into the cell bodies prior to their removal. After removal of the cell bodies, the brain was fixed for 1 h in 4% (w/v) paraformaldehyde in PBS before processing with anti-Lucifer yellow antiserum (Molecular Probes, Leiden, The Netherlands). In all such preparations, processes in the protocerebrum and optic lobe, with the characteristic anatomical features of PM4, were stained (Fig. 1). We find that the presence of Lucifer yellow m the cell body... 

The role of (1 3)-)3-D-glucanase in cell wall biosynthesis and its distribution in the mycelium of Sclerotium rolfsii have been studied. Specific zones of immunofluorescence appeared in the hyphal tips, clamp connections, new septa, and lateral branching when a specific antiserum was used with the direct method of fluorescent antibody staining. Enzyme activity in the cell wall preparation was inactivated by diethylpyrocarbonate but a majority of the activity was in a latent form which was unaffected by the ester. 

One of the most careful investigations of this question is that by Haber and Richards (13), who utilized purified rabbit antibodies specific for the 2,4-dinitrophenyl (Dnp) and 2,4,6-trinitrophenyl (Tnp) groups. In contrast to many studies of this type, precautions were taken to ensure that the separated H chains were free of L chains, and analyses for purity were carried out. The H chains were recycled through Sephadex G-lOO until they were essentially free of L chains by two criteria failure to react with antiserum to L chains and the absence of the N-terminal alanine characteristic of rabbit L chains, as measured by a sensitive isotope-dilution assay. Light chains of antibody, either alone or combined with nonspecific H chains, possessed no activity detectable by the method of fluorescence quenching. By contrast, a large percentage of the H chains, polyalanylated or in combination with nonspecific L chains, were able to bind hapten specifically, but with reduced affinity. Some of... 

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