The biotin–avidin system

4.7 The biotin–avidin system

Biotin is synthesized by plants and many microorganisms; in particular, large amounts are formed by the intestinal flora. It is essential for warm-blooded animals and acts as a coenzyme in

CO 2 fixation and transcarboxylation reactions. Avidin, found in egg white, has an extremely high affinity (> 10 15 /mol) for biotin, and bind- ing is so strong that for most purposes the binding can be treated as though it is covalent. There are four sites on the avidin molecule at which biotin may be bound. Biotin is a small molecule that can be easily coupled to proteins, including antibodies, enzymes and many antigens, with- out significant loss of the protein’s biological activity. The loss of activity is much less than the damage done by coupling a large enzyme directly or following exposure to oxidizing agents, as in radiolabelling. Sandwich assays may then be performed in which antibodies bound to solid- phase antigen may be revealed by adding, in sequence, biotinylated anti-immunoglobulin, avidin and then biotinylated enzyme.

Avidin may be conjugated with enzyme, thus eliminating one step in the assay. Considerable amplification of the resultant signal can be obtained by use of preformed complexes of avidin and biotinylated enzyme aformed such that a few biotin-binding sites are still free on the avidin molecules. An advantage of the avidin–biotin system is that avidin may be labelled with enzyme, fluorochromes, radiolabels, ferritin, etc., so that the same biotinylated antibody may be used with the different avidin conjugates in enzyme immunoassay, radioimmunoassay, immunofluores- cence and electron microscopy, without the need for preparing separate labelled antibodies.

Streptavidin from Streptomyces avidinii is also used. Although there are no problems with non- specific binding, the biotin-binding site is thought to be less accessible which sometimes neces- sitates coupling the biotin via a spacer arm. The technique described below may be used with proteins and carbohydrates. The biotinylated proteins tend to retain more of their activity after conjugation. Many different biotin-linked antibodies are commercially available. Similarly, avidin may be purchased already substituted with a wide variety of fluorochrome, enzyme or radioisotope labels.

4.7.1 Biotinylation

MATERIALS Biotin-O-succinimide ester

IgG fraction of antiserum (see Section 1.3) Enzyme; for example, horseradish peroxidase Sephadex G-25 buffer-exchange column (see Appendix B.1) or dialysis tubing

0.1 M bicarbonate buffer, pH 8.4 Dimethyl sulphoxide (DMSO)

0.1 M tris(hydroxymethyl)-aminomethane (Tris)–HCl buffer, pH 8.4

4.7THE BIOTIN–AVIDIN SYSTEM

METHOD

1 Equilibrate IgG and enzyme separately in bicarbonate buffer using the Sephadex G-25 column or by dialysis.

2 Adjust each protein concentration to 1 mg/ml in bicarbonate buffer (see Appendix B.5.1–B.5.4).

3 Dissolve 1 mg biotin ester in 1 ml DMSO just before use.

4 Add 75 µl biotin solution to each 1 ml of protein solution.

5 Mix immediately and rotate for 4 h at room temperature.

6 Re-equilibrate the column with 0.1 M Tris–HCl buffer, pH 7.4, and buffer exchange the two samples on this column (or dialyse against this buffer).

TECHNICAL NOTE The coupling reaction must be performed in a buffer lacking Tris or other primary amines as these will compete with amino groups on the protein. Hence the reason for switching to Tris after coupling.