Reinigung des nierenspezifischen Glykoproteins gp400 mittels Immunaffinitätschromatographie

Abstract

Immunizing of mice with porcine renal brush border membranes by Prof. H. Koepsell and coworkers resulted in the generation of different monoclonal antibodies. These antibodies already have been characterized in detail. One of these monoclonal antibodies, N4A4, recognizes a kidney specific antigen named gp400 and can be used for noninvasive urinary diagnostics as gp400 is excreted into the urine under physiological conditions. As gp400 is expressed in renal cell carcinomas of proximal tubular origin a current study determines whether N4A4 can be used as a diagnostic tool or a therapeutic agent for these tumors.

Since the identity of gp400 is still not known, the goal of the here presented work was the purification of gp400 with biochemical and immunological methods to allow amino acid sequencing.

An approximate tenfold enrichment of gp400 could be initially achieved by vesicle preparation of porcine renal cortex. This vesicular fraction was used as starting material for further purification steps. After comparing different detergents, the anionic detergent cholic acid was used at a concentration of 2 % (w/v) to solubilize gp400.

Lectinaffinity purification with concanavalin A did not result in an enrichment of gp400, probably due to interactions between lectin and detergent. In contrast isoelectric focusing could be used to further concentrate gp400 and to determine its isoelectric point at 6,14, but was not practical for large-scale purification.

Therefore we tried Protein G with covalently coupled monoclonal antibodies as immuno affinity chromatography. GP400 could not be purified using N4A4 most likely due to interference between cholic acid and the antigen-antibody-binding. However, after using L4D6 isolation of gp400 was achieved. As N4A4 showed a positive reaction in western blotting of the protein eluted from the L4D6 column it was proven that the antigen of both monoclonals is identic. A total of 125 pmol of gp400 could be recovered.

After concentration by ultrafiltration and in gel digest an attempt was made to determine the amno acid sequence of gp400. Although some sequence data, which showed 90% homology to a protein encoded by a yet uncharacterized human cDNA clone could be obtained it should be doubted whether the sequence belongs to gp400. Presumably the biggest amount of purified gp400 was already lost during ultrafiltration.