Notes

1. The dialysis of small volumes can be conveniently performed in narrow dialysis tubing by placing a short glass tube, sealed at both ends, in the tubing so that the space available to the sample is reduced. Transfer losses are minimized by conducting the subsequent steps in the same dialysis bag. There are also various microdialysis systems available commercially, for example, the Slide-A-Lyzer units from Pierce Biotechnology (Rockford, IL).

2. The conjugates are stable for several years at 4°C because the NaN3 inhibits microbial growth and the BSA minimizes denaturation and adsorption losses. These conjugates should not be frozen.

3. Purification of the conjugates is usually unnecessary; however, if there is evidence of the presence of free antibody it can be removed by gel filtration in Sepharose CL-6B (Amersham Biosciences, Chalfont St. Giles, UK) or a similar medium with PBS as solvent.

4. The enzyme-labeled antibody can be evaluated by enzyme-linked immunosorbent assay. Immobilize the appropriate antigen on the wells of a microtiter plate or strip (at a concentration of 2-10 |g/ mL), incubate various dilutions of the conjugate for a few hours, wash the wells, add substrate, and measure the amount of product formed (see Chapter 15). This approach may also be used for monitoring conjugate purification in chromatography fractions.

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