Interaction with biotinylated antibodies

Useofavidin-biotin technology in immunoaffinity chromatography can lead to improved performance of the affinity column, which translates both into yields of the target material and stability of the column (1,2). The presence of free biotin-binding sites of the avidin-4 esin allows secondary interaction with biotinylated antibody or its complexes with avi-din that may have leaked from the affinity resin. 

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 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.

Gold labeling may be used in the recently introduced catalyzed reporter deposition-immunogold technique where biotinylated tyramide molecules are attached the antibody-antigen complex site the biotinylated sites are visualized by interaction with streptavidin-gold (46,47). 

The avidin-biotin interaction has also been used to immobilize antibodies and proteins, especially in commercial systems based on surface plasmon resonance (SPR) measurements (e.g., the BIAcore). The extraordinary affinity (Kl 10-15 M) of avidin (or its bacterial relative, streptavidin) for the vitamin biotin is the basis of this immobilization procedure. A solid support (e.g., glass beads, sensor chip, optical fiber) covered with avidin can be used as an activated carrier for a very sturdy immobilization of previously biotinylated antibodies. In spite of the many methods for biotinylating proteins described in the literature, the use of biotinyl N-hydroxysuccinimide ester (BNHS) and similar derivatives, remains the most useful [65]. 

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