Biotin biotinylated antibody

Using the characteristic of a high-affinity complex between avidin and biotin, biotinylated antibodies have wide applications in various immunochemical assays, especially where signal amplification is required. A method is described here for the biotinylation of immunoglobulins. The procedure utilizes water-soluble succinimidyl ester of biotin that reacts with primary amines of the lysine residues or the amino terminus on the antibody to form amide bonds. The method is simple and specific and results in stable conjugates retaining full immunologic activity. 

Biotin-hydrazide has been used to biotinylate antibodies at their oxidized carbohydrate residues (O Shanessy et al., 1984, 1987 O Shanessy and Quarles, 1985 Hoffman and O Shannessy, 1988), to modify the low-density lipoprotein (LDL) receptor (Wade et al., 1985), to biotinylate nerve growth factor (NGF) (Rosenberg et al., 1986), and to modify cytosine groups in oligonucleotides to produce probes suitable for hybridization assays (Reisfeld et al., 1987) (Chapter 27, Section 2.3). 

Figure 18.14 NHS-SS-PEG4-biotin can be used to label a primary antibody molecule that has specificity for a protein or interest. Incubation of the biotinylated antibody with a sample, such as a cell lysate, allows the antibody to bind to its target. Capture of the antibody-antigen complex on an immobilized streptavidin reagent effectively isolates the targeted protein from the other proteins in the sample. The disulfide linkage in the spacer arm of the biotin tag permits elution of the immune complex from the streptavidin support using DTT and without using the strong denaturing condition typically required to break the streptavidin-biotin interaction.

Figure 20.19 Biotinylated antibodies can be formed by reacting NHS-LC-biotin with available amine groups to create amide bonds.

Figure 22.21 Antibodies may be conjugated to liposomes using an indirect approach incorporating a (strept)avidin-biotin system. Biotinylated liposomes may be complexed with biotinylated antibodies using (strept)avidin as a bridging molecule or may be complexed with an antibody-(strept)avidin conjugate.

Other substances that exhibit specific binding may be used to separate the free and the bound fractions when attached to a solid phase the ability of staphylococcal protein A to bind to the FC fragment of certain isotypes of IgG can be utilized the strong binding of the vitamin biotin to tetravalent avidin may also be employed. Biotin may be readily incorporated into antibody molecules and these molecules may be subsequently captured by an avidin solid phase. Alternatively avidin may be used to provide a link between a biotinylated antibody and a biotinylated solid phase. 

The above represent the past and present of the most common enzyme-mediated methods of antigen detection. There are alternate procedures available, involving such methods as antibiotin antibody steps that combine the avidin-biotin systems with a further antibiotin/antienzyme sandwich for still greater sensitivity. Also, there are methods that follow a PAP procedure with a biotinylated antibody to the PAP immunoglobulin followed by ABC detection (15). The obvious problem created with this approach is the tremendous... 

The technique described here is for use with monoclonal primary antibodies of mouse origin, but can easily be adapted for use with polyclonal antibodies from other species (i.e., rabbit). This method uses a secondary biotin-labeled antibody and a detection system that employs a biotin-avidin horseradish peroxidase complex linker step, the so-called ABC (avidin-biotin complex) detection system (5) (see Chapter 25). In this detection system, avidin acts as a bridge between the biotinylated secondary antibody and a biotin-labeled peroxidase enzyme. The anchored enzyme, in the presence of H2O2 can then convert the substrate, diaminobenzidine, to a brown or black reaction product that is easily identifiable in the tissue section. 

High-quality reagents needed for biotin-avidin immunostaining are all available commercially. Kits are also available commercially for biotinylating antibodies (see Chapter 7). The major consideration in biotinylating antibodies is the use of biotin with a carbon spacer arm at least 1 nm long, since the binding site of biotin on avidin and probably streptavidin is in a deep depression (4). 

The use of biotinylated antibodies provides perhaps the greatest versatility and sensitivity of all methods The affinity of biotin for avidin or the more usually used streptavidm is very high, and the latter can be conjugated to radioisotope, fluorescent moiety, or enzyme Again, the basic procedures are the same as outlined for l25I-labeled antibodies with the additional steps required for streptavidin binding and subsequent incubation with enzyme substrate (see also Chapters 17 and 18)... 

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