Protein Protocols Protein Protocols

Coupling of Abs with Biotin 359
3. Methods
3.1. Conjugation with Amine-Reactive Biotin
1. Calculate the amount of a 10 mg/mL biotin succinimidyl ester solution (biotin-NHS)
needed to conjugate the desired quantity of antibody at the chosen biotin/antibody molar
ratio, according to the following formula:
(mL of 10 mg/mL biotin-SE) = {[(mg antibody × 0.1)/mol wt
of antibody] × R × mol wt of biotin-SE)} (1)
where R = molar incubation ratio of biotin/protein. For example, using 5 mg of IgG and a
10:1 molar incubation ratio of biotin-XX-SE, Eq. (1) yields:
(mL of 10 mg/mL biotin-XX-SE)
= {[(5 × 0.1)/145,000] × (10 × 568)} = 0.02 mL (2)
2. Dissolve the antibody, if lyophilized, at approx 5–15 mg/mL in either of the two reaction
buffers described in Subheading 2.1. If the antibody to be conjugated is already in solution
in 10–20 mM PBS, without azide, the pH necessary for the reaction can be obtained
by adding 1/10 vol of 1 M sodium bicarbonate. IgM should be conjugated in PBS, pH 7.2
(see Note 3).
3. Weigh 3 mg or more of the biotin-SE of choice, and dissolve it in 0.3 mL or more of DMF or
DMSO to obtain a 10 mg/mL solution. It is essential that this solution be prepared immediately
before starting the reaction, since the succinimidyl esters or any amine-reactive
reagents hydrolyze quickly in solution. Any remaining solution should be discarded.
4. While stirring, slowly add the amount of 10 mg/mL solution, calculated in step 1, to the
antibody prepared in step 2, mixing thoroughly.
5. Incubate this reaction mixture at room temperature for 1 h with gentle stirring or shaking.
6. The antibody conjugate can be purified on a gel-filtration column or by dialysis. When
working with a few milligrams of dilute antibody solution, care should be taken not to
dilute the antibody further. In this case, dialysis is a very simple and effective method to
eliminate unreacted biotin. A few mL of antibody solution can be effectively dialyzed in
the cold against 1 L of buffer with three to four changes. Small amounts of concentrated
antibody can be purified on a prepackaged desalting column equilibrated with the preferred
buffer, following the manufacturer’s directions. Five or more milligrams of antibody can
be purified on a gel-filtration column. The dimensions of the column will have to be proportional
to the volume and concentration of the antibody. For example, for 5–10 mg of
antibody in 1 mL solution, a column with a bed vol of 10 × 300 mm will be adequate. To
avoid denaturation, dilute solutions of biotinylated antibodies should be stabilized by adding
BSA at a final concentration of 0.1–1%.
3.2. Conjugation with Biotin Hydrazide at the Carbohydrate Site
1. It is essential that the entire following procedure be carried out with the sample completely
protected from light (see Note 9).
2. Dissolve antibody (if lyophilized) or dialyze solution of antibody to obtain a 2–10 mg/mL
solution in the reaction buffer described in Subheading 2.1., item 1. Keep at 4°C.
3. Add an equal volume of cold metaperiodate solution. Incubate the reaction mixture at 4°C
for 2 h in the dark.
4. Dialyze overnight against the same buffer protecting from light, or, if the antibody is concentrated,
desalt on a column equilibrated with the same buffer. This step removes the iodate and
formaldehyde produced during oxidation.