1. Preparations of HRP may vary in their carbohydrate content and this can affect the oxidation reaction. Free carbohydrate can be removed by gel filtration. Increasing the sodium periodate concentration to 0.2 M can also help, but further increases lead to inactivation of the peroxidase.

2. Dialysis of small volumes can be conveniently done 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 carrying out 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).

3. The absorbance at 403 nm is caused by the peroxidase's heme group. The enzyme is often specified in terms of its RZ value; this is the ratio of A403 to A280, and it provides a measure of the heme content and purity of the preparation. Highly purified peroxidase has an RZ of approx 3. Conjugates with an RZ of 0.4 perform satisfactorily.

4. BSA improves the stability of the conjugate and minimizes loses as a result of adsorption and denaturation. NaN3 should not be used with peroxidase conjugates because it inhibits the enzyme. If an antimicrobial agent is required, 0.2% sodium merthiolate (thimerosal) should be used.

5. The performance of the conjugate can be evaluated by enzyme-linked immunosorbent assay as described in Note 4 of Chapter 8 and in Chapter 15.

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