Coupling of antibodies with biotin.

The avidin-biotin bond is the strongest known biological interaction between a ligand and a protein (Kd = 1.3 x 10-15 M at pH 5.0) (1). The affinity is so high that the avidin-biotin complex is extremely resistant to any type of denaturing agent (2). Biotin (see Fig. 1) is a small, hydrophobic molecule that functions as a coenzyme of carboxylases (3). It is present in all living cells. Avidin is a tetrameric glycoprotein of 66,000-68,000 molecular weight, found in egg albumin and in avian tissues. The interaction between avidin and biotin occurs rapidly, and the stability of the complex has prompted its use for in situ attachment of labels in a broad variety of applications, including immunoassays, DNA hybridization (4-6), and localization of antigens in cells and tissues (7). Avidin has an isoelectric point of 10.5. Because of its positively charged residues and its oligosaccharide component, consisting mostly of mannose and glucosamine (8), avidin can interact nonspecifically with negative charges on cell surfaces and nucleic acids, or with membrane sugar receptors. At times, this causes background problems in histochemical and cytochemical applications. Streptavidin, a near-neutral, biotin-binding protein (9) isolated from the culture medium of Streptomyces avidinii, is a tetrameric nonglycosylated analog of avidin with a molecular weight of about 60,000. Like avidin, each molecule of streptavidin binds four molecules of biotin, with a similar dissociation constant. The two proteins have about 33% sequence homology, and tryptophan residues seem to be involved in their biotin-binding sites (10,11). In general, streptavidin gives less background problems than avidin. This protein, however, contains a tripeptide sequence Arg-Tyr-Asp (RYD) that apparently mimics the binding sequence of fibronectin Arg-Gly-Asp (RGD), a universal recognition domain of the extracellular matrix that specifically promotes cell adhesion. Consequently, the streptavidin-cell-surface interaction causes high background in certain applications (12).

Preparation of avidin conjugates.

The high-affinity avidin-biotin system has found applications in different fields of biotechnology, including immunoassays, histochemistry, affinity chromatography, and drug delivery, to name a few. A brief description of avidin and avidin-like molecules, streptavidin, deglycosylated avidin, and NeutraLite avidin is presented in the Chapter 2. With four biotin-binding sites per molecule, the avidin family of proteins is capable of forming tight complexes with one or more biotinylated compounds (1). Typically, the avidin-biotin system is used to prepare signal-amplifying "sandwich" complexes between specificity reagents (e.g., antibodies) and detection reagents (e.g., fluorophores, enzymes, and so on). The specificity and detection reagents are independently conjugated, one with avidin and the other with biotin, or both with biotin, providing synthetic flexibility (2). Avidin conjugates of a wide range of fluorophores, phycobiliproteins, seconday antibodies, microspheres, ferritin, and enzymes commonly used in immunochemistry are available at reasonable prices, making their small-scale preparation impractical and not cost effective (see Note 1). However, conjugations of avidin to specific antibodies, to uncommon enzymes, and to other proteins and peptides are often performed on-site. A general protocol for the conjugation of avidin to enzymes, antibodies, and other proteins is described in this chapter.

Conjugation of antibodies to biotin.

Antibodies can be conjugated to biotin by a number of chemical means. They can then be used in immunochemical procedures in conjunction with secondary reagents coupled to biotin-binding protein proteins such as avidin.

Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.

Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.

Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation.

The high-affinity binding of biotin to avidin, streptavidin, and related proteins has been exploited for decades. However, a disadvantage of the biotin/biotin-binding protein interaction is that it is essentially irreversible under physiological conditions. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin. We synthesized an amine-reactive desthiobiotin derivative for labeling proteins and a desthiobiotin-agarose affinity matrix. Conjugates labeled with desthiobiotin are equivalent to their biotinylated counterparts in cell-staining and antigen-labeling applications. They also bind to streptavidin and other biotin-binding protein-based affinity columns and are recognized by anti-biotin antibodies. Fluorescent streptavidin conjugates saturated with desthiobiotin, but not biotin, bind to a cell-bound biotinylated target without further processing. Streptavidin-based ligands can be gently stripped from desthiobiotin-labeled targets with buffered biotin solutions. Thus, repeated probing with fluorescent streptavidin conjugates followed by enzyme-based detection is possible. In all applications, the desthiobiotin/biotin-binding protein complex is easily dissociated under physiological conditions by either biotin or desthiobiotin. Thus, our desthiobiotin-based reagents and techniques provide some distinct advantages over traditional 2-iminobiotin, monomeric avidin, or other affinity-based techniques.