[Immunoenzymatic assay of dyes used in affinity chromatography of proteins]

Immobilized reactive dyes are frequently used in pseudoaffinity chromatography since it has been evidenced that they could specifically interact with some biologicals of interest, and, as a consequence, be separated and purified by this way. This specificity was so high in some cases, that a single step purification process was possible. In addition to these features, the wide variety of dye molecules available associated to their chemical stability and low cost make possible their use as ligands for industrial applications. Nevertheless, one drawback of the use of dye sorbents for protein purification is the possible leakage of dye molecules, with a risk of biological properties alteration of the purified product. We describe a new quantitative assay method for dyes that could contaminate biologicals separated through special affinity columns. This method based on the specific recognition of dyes by antibodies showed a high specificity and a much better sensitivity than the classical spectrophotometric approaches. It has been applied to the quantification of dyes used in affinity chromatography (Cibacron Blue F3-GA, Basilen Bleue E3-G and Procion Red HE-3B) in the presence of proteins having a special affinity for these dyes. This immunoenzymatic assay is also easily applicable to the detection of leachables from dye affinity columns submitted to drastic regeneration treatments.