High concentration labeling method

A number of modifications have been introduced into the original coupling method, all aimed at finding a better compromise between labeling and inactivation. Avrameas and Ternynck (1971) introduced the two-step procedure which reduced polymerization of IgG and activated more POase. They observed that, even at high concentrations of GA, POase was not polymerized. Following treatment of POase with an excess of GA, free GA is removed and activated... 

In the second step, the purified, transaminated DNA is brought to a higher pH (8.5-10) in order to decrease the protonation of the reactive amino group. Viscidi et al. (1986) labeled the N -substituted cytosine residue with biotin using the NHS-biotin ester, whereas, e.g., Hurskainen et al. (1991) labeled the same intermediate with a europium chelate (for time-resolved fluorescence). The Cq/qj increases about 1 log but this is compensated by the high probe concentration. This method yields probes with a similar detectability (10 molecules) as enzymatically labeled biotin probes, but the reagents are inexpensive and easily available. 

---