Biotinylated Antibodies

Notes The label may also be biotin or any fluorophore. For antibody biotinylation, we use biotin-SP-conjugated AffiniPure Fab Fragment Goat Antimouse IgG (H+L) (Jackson ImmunoResearch Labs, Code Number 115-067-003). 

The procedure given here summarizes the localization of tissue antigens using a primary antibody, biotinylated secondary antibody, avidin-biotin complex, and DAB chromagen on fresh frozen brain... 

If a free amino group forms a portion of the protein that is essential for activity (e.g., the antigen-combining site for antibody), biotinylation with the succinimide ester will lower or destroy the activity of the protein, and other methods of labeling should be tried. Biotin hydrazide has been used to modify the carbohydrate moieties of antibodies (10,11). Other alternatives are the thiol-reactive biotin maleimide (12) or biotin iodoacetamide (13). 

Fig. 5.2. Methods for detection of passenger-ligand interaction. (A) Fluorophore-coupled ligand (B) fluorophore-coupled antibody (C) quaternary complex generated by subsequent rounds of incubation with ligand, primary antibody, biotinylated second antibody, and strep ta-vidin, R-phycoerythrin conjugate (D) quaternary complex generated by subsequent rounds of incubation wi tli ligand, primary antibody, biotinylated second antibody, and streptavidin-coated magnetobeads. L ligand P passenger.

An avidin-biotin system has been used to attach antibodies in the bilayer of DDSs. Xiao et al. developed a three-step strategy to improve the tumor-to-tissue ratio of anticancer agents [184], Two antibodies specific for the CA-125 antigen that is highly expressed on NIH OVCAR-3 cells were used. These cells were prelabeled with biotinylated anti-CA-125 antibody and fluoroscein isothiocyanate (FITC)-labeled streptavidin (SAv) prior to administration of biotinylated liposomes. Both antibodies were specifically bound to the cell surface of OVCAR-3 cells but not to SK-OV-3 cells, which do not express the specific antibody. Antibody biotinylation did not affect its immunoreactivity. 

Competitive immunoassays based on the avidin-biotin approach have also been described. In this case, the antibody is immobilized on microtiter plates. Wells are incubated with a constant concentration of biotinylated antigen in the presence of different concentrations of standards or the sample. After washing, the ABC complex is added. The formed antibody-biotinylated antigen-ABC sandwich is then detected by the addition of enzyme substrate. The immobilized enzyme-activity is inversely proportional to the concentration of analyte in the sample, resulting in a typical sigmoidal calibration curve. 

It is essential (Kendall et al., 1983 Yolken et al., 1983) to biotinylate correctly the antibody or enzyme, since there is a pronounced optimum (Section 3.2.2). However, current avidin/biotin methods may have to be changed with respect to biotinylation procedures to attain their full potential. The optimal dilutions of the biotinylated antibody, biotinylated enzyme and avidin solutions have to be determined and the test performed as detailed in Table 14.4. It is essential to use incubation periods of about 20 min (pronounced optimum) for the reaction between the biotinylated proteins and avidin (Kendall et al., 1983). 

BSA Anti-BSA antibodies Biotinylated antibodies streptavidin Cy-BtoBn Fig. 8.5 Principle of a solid-phase streptavidin-cymantrenyl-biotin competitive immunoassay [65. ... 

It is possible to perform an enzyme-linked immunosorbent assay (ELISA) using a streptavidin-coated 96-well plate the primary antibody, biotinylated-rabbit-anti-streptavidin, specifically recognizes the avidin linked to the helicate as the bioprobe. The detection limit is better than that with commercially available organic dyes such as antistreptavidin-FICT (FiB3). 

Secondary antibody biotinylated horse anti-mouse antibody (Vector laboratories)... 

Secondary antibody (biotinylated goat anti-mouse IgG). 

Fig. 35 TEM image of peptide fibres coated with streptavidin-gold nanoparticles, (a, b) Peptides with biotin directly attached (using SAF-pl-biotin) particles are 10 nm, (c) Peptide fibre with biotinylated anti-FLAG antibody attached that was then bound to gold-labelled streptavidin particles are 5 nm. Reprinted with permission from Ryadnov and Woolfson [76]. Copyright 2004 American Chemical Society...

A more recently introduced format is the AlphaScreen assay. The assay principal behind this technology has previously been described above. In the kinase format a biotinylated peptide is bound to a streptavidin donor bead, and a phopshospecific antibody is bound to the acceptor bead. When the substrate is phosphorylated, the beads come in close proximity and a signal is generated. An example using the assay for the detection of inhibitors of serine kinases is presented by Von Leo-prechting [26]. 

The LANCE cAMP assay is a competitive assay in which cAMP produced by the cells competes with fluorescent-labeled acceptor cAMP for a cryptate tagged donor antibody. The principal of the assay is shown in Fig. 6. On the left strepta-vidin conjugated Europium binds to biotinylated cAMP. An antibody labeled with the fluorescent dye Alexa binds to the cAMP, bringing the donor and acceptor into close proximity, and energy transfer occurs. When the cell releases cAMP, it competes with the biotin-labeled cAMP for the antibody, and a signal decrease is observed. In the TR-FRET assay the antibody is directly labeled with either Eu or Tb. In this format an increase in cAMP also causes a decrease in signal. 

Valdivieso-Garcia, A. Riche, E. Abubakar, O. Waddell, T. E. Brooks, B. W. A double antibody sandwich enzyme-linked immunosorbent assay for the detection of Salmonella using biotinylated monoclonal antibodies. J. Food Prot. 2001, 64,1166-1171. 

Recently, SETA BioMedicals has developed a new near-infrared squaraine-based label Seta-633, which can be used to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter [19]. This label exhibits lower quantum yields and shorter fluorescence lifetimes when free in solution, but these values substantially increase upon interaction with proteins, which is contrary to tracers like Cy5 or Alexa 647. It was demonstrated in a model assay that a biotinylated Seta-633 binds to anti-biotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 2.76 fold upon binding to a specific antibody (anti-biotin, MW =160 kDa), while the titration with BSA or nonspecific antibody does not result in a noticeable change in lifetime (Fig. 13). The label is compatible with readily available light sources (635 nm or 640 nm lasers) and filter sets (as for Cy5 or Alexa 647) and its... 

Figure 8.1 The results of IHC of two experiments using Dynabeads (Dynal, New York, NY) coated with biotinylated anti-mouse IgG (first experiment) and protein S-100 (second experiment), (a) Positive control showing red color (S-100) localized in the melanoma cells, (b) Strong positive red color circles all beads coated with biotinylated anti-mouse antibody after the heating AR treatment (first experiment), (c) Using the heating AR treatment, S-100-coated polymer beads show positive red color around the beads as circles (second experiment), (d) Negative control of the first experiment. No red color could be seen for polymer beads (arrows) that had been treated with exactly the same protocol as that of slide (b), but omitting the avidin-biotin-peroxidase (label). Bar = 50pm. Reproduced with permission from Shi et al., J. Histochem. Cytochem. 2005 53 1167-1170. See color insert.

Using the same versatile modular synthetic strategy, the same group developed biotinylated bi- (438) and tetra-antennary (439) mannosylated glycoconjugates to capture and detect E. coli cells, and compared the relative capturing ability of these molecules to commercial polyclonal antibodies (Fig. 47).318 Instead of aliphatic spacers, tetraethylene glycol linkers were used to diminish nonspecific binding and to impart flexibility for a better fit in the active sites. 

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