Immunoglobulins labeled

Immunoblotting techniques involve the identification of a protein target via antigen-antibody-specific reactions. Proteins are typically separated by electrophoresis in polyacrylamide gels, and then transferred ( blotted ) onto chemically resilient membranes (e.g., nitrocellulose, polyvinylidene difluoride) where they bind in the pattern they took in the gel. The membrane is overlaid with a primary antibody directed to the specific target, then with a secondary antibody (anti-immunoglobulin) labeled with radioisotopes, enzymes or other marker compounds. 

Fig. 4. Testing of a conjugate prepared from purified antibodies to mouse immunoglobulin labeled with alkaline phosphatase. Ordinate conjugate dilution abscissa absorbance at 405 nm of samples diluted 1 4 after 15 min of incubation with substrate. — , Dose response in microtiter wells coated with mouse serum proteins O—O, dose response in uncoated wells.

The azirine derivative (58), when photolysed in the presence of an immunoglobulin, labelled the galactan-binding area.- - Also in the area of carbohydrate-protein binding, novel descriptors have been used in the analysis of the quantitative structure-activity relationship of the binding of p-substituted phenyl glycosides to concanavalin A. " ... 

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. 

Taylor CR, Burns J. The demonstration of plasma cells and other immunoglobulin containing cells in formalin-fixed, paraffin-embedded tissues using peroxidase labelled antibody. J. Clin. Pathol. 1974 27 14-20. 

The following procedure is a suggested method for using NHS-fluorescein to label immunoglobulins. 

Dissolve an immunoglobulin to be labeled in ice-cold, 50mM sodium bicarbonate, pH 8.5, at a concentration of lOmg/ml. 

Dissolve the antibody to be labeled in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5, at a concentration of at least lOmg/ml. The immunoglobulin must be glycosylated to work in this procedure. 

Remove excess fluorophore by dialysis or gel filtration using a desalting resin. Protect the labeled immunoglobulin from light. 

The following protocol is based on the methods recommended by Thermo Fisher for use of the cyanine dye DyLight 649. Antibody reduction is based on the methods of Sun et al. (2005), which results in partially reduced bispecific immunoglobulin containing available thiols in the hinge region for labeling. 

In addition to labeling immunoglobulins with enzymes to provide detectability through their catalytic action on a substrate, antibody molecules also can be labeled or tagged with small... 

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. 

Baranowska-Kortylewicz, J., and Kassis, A.I. (1993b) Labeling of immunoglobulins with bifunctional, sulfhydryl-selective, and photoreactive coumarins. Bioconjugate Chem. 4, 300-304. 

O Shannessy, D.J., Doberson, M.J., and Quarles, R.H. (1984) A novel procedure for labeling immunoglobulins by conjugation to oligosaccharide moieties. Immunol. Lett. 8, 273-277. 

Segal, D.M., and Hurwitz, E. (1976) Dimers and trimers of immunoglobulin G covalently cross-linked with a bivalent affinity label. Biochemistry 15, 5253. 

M. Okochi, H. Ohta, T. Tanaka, and T. Matsunaga, Electrochemical probe for on-chip type flow immunoassay immunoglobulin G labeled with ferrocenecarboaldehyde. Biotechnol. Bioeng. 90, 14-19 (2005). 

Human embryo lung fibroblasts infected with a reference laboratory strain of herpes simplex virus (HS V) type 2 were used to detect antibody to HSV type 2 in serum samples. After treatment of cells with serial dilutions of sera, HRP-labeled immunoglobulins to human IgG (class G immunoglobulins) were added and detected with CL substrate [36], In both cases a sharp detection of the specific antibodies was achieved with chemiluminescent assays, which proved more sensitive than the colorimetric immunoperoxidase assays. 

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