Immobilized metal ion affinity chromatography for the purification of Fel d I, a cat major allergen, from a house-dust extract'

Abstract

Although the efficient isolation and purification of the major feline allergen have previously been achieved using polyclonal or monoclonal antibody affinity chromatography these methods lead to a relatively low yield of pure allergen. Therefore, attempts were made to establish a process involving ion-exchange chromatography followed by immobilized metal ion affinity chromatography. Although slightly more time consuming than the immunological methods, it gives a higher yield. It consists of four steps. From a crude house-dust exract, precipitation of a Fel d I enriched fraction is obtained by addition of acetone. After dissolution in water, exhaustive dialysis takes place against a citric acid solution to eliminate divalent metal ions .This gives the whole house-dust extract. Then partial isolation and purification are obtained by ammonium sulfate precipitation. The resulting precipitate is dissolved in and dialysed against water to remove ammonium salts. This partially purified house-dust extract can be submitted to chromatography on an anion exchanger and then on a copper ion charged chelating gel. All steps of the isolation and purification were controlled by immunochemical analyses using a crossed immuno-electrophoretic method and enzyme-linked immunoadsorbent assays. The HD75 Q2 Cu2 fraction so obtained can be considered to be a pure cat major allergen Fel d I preparation. Its homogeneity was demonstrated using physico-chemical and immunochemical methods.