Long-term storage of peroxidase-labelled immunoglobulins for use in enzyme immunoassay

Abstract

Optimal conditions for long-term storage of peroxidase-labelled immunoglubulins for use in enzyme immunoassays have been investigated with particular emphasis on the preservation of the immunological and enzyme activity. Immunoglobulin G preparations from different animal species were purified according to conventional methods and labelled with horseradish peroxidase. Conjugates were stored at different temperatures either in liquid form with 50% glycerol, after lyophilisation or as ammonium sulphate precipitates. In addition, the conjugates were also frozen and stated at −40°C. The immunological and enzymatic activity of the conjugates was evaluated at regular intervals throughout storage. The best results ere obtained with conjugates stored as ammonium sulphate precipitates at 4°C. Under these conditions, the complexes retained after 2 years of storage 92 and 91% of their enzymatic and immunological activity respectively and gave excellent reproducibility in ELISA. The conjugates exhibited a very low degree of self-aggregation. The ammonium sulphate did not interfere in the ELISA. Storage at elevated temperatures resulted in a greater decrease in enzymatic than immunological activity but the results obtained with the ammonium sulphate precipitates were clearly superior to those obtained with alternative storage procedures. The estimated half life of the whole enzymatic-immunological activity of conjugates at 4°C was 9 years. The stability of both activities at 25°C was also reasonably good for short periods. Conjugates stored for 40 days at 37°C lost 60% enzyme activity but retained unaltered their immunological activity and gave rise to high blanks in the ELISA, due to auto-aggregation.