Methods

1. Prepare 1 mg of the IgG antibody in 1 mL of 0.1 M sodium carbonate buffer, pH 9.0.

2. Prepare the solution of the isothiocyanate immediately prior to use.

3. Add 50 ^L of the isothiocyanate solution to the antibody slowly with stirring at room temperature and incubate the mixture at 4°C for 8 h in the dark.

4. Terminate the reaction by adding 0.1 mL of 0.1 M ethanolamine, pH 8.5, and incubate at room temperature for 15 min.

5. Remove the excess fluorochrome derivative by gel filtration in Sephadex G-25 equilibrated with PBS (see Note 2). Measure the A280.

6. Pool the fractions in the first (colored) peak, store the conjugate in aliquots at -20°C or in the dark at 4°C with BSA (0.1% final concentration) and NaN3 (0.05% final concentration; see Notes 3 and 4).

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