Production of recombinant human β2-microglobulin for scintigraphic diagnosis of amyloidosis in uremia and hemodialysis

Abstract

Amyloid of β2-microglobulin (β2m) origin can be diagnosed using 131I-radiolabelled-β2m scintigraphy in patients with uremia and hemodialysis treatment. As the tracer β2m is isolated from another patient, it carries the common risks, including vital infections such as Hepatitis B, C and HIV, which are associated with human plasma products. In order to exclude these risks we have produced recombinant human β2m (rhβ2m) in Escherichia coli. The expression vector pASK40ΔLβ2m(His)5 contains a C- terminal (His)5-tag for purification via immobilized metal ion affinity chromatography (IMAC). Size exclusion chromatography on a Superose 12 column represents the second step of purification. The isolated rhβ2mH5 reacted in an immunochemically identical manner to native human β2m, and showed a single band of ≃ 11.8 kDa in Western blot analysis and revealed a single spot in two-dimensional gel electrophoresis. Mass spectrometry analysis revealed a single peak at the expected molecular mass of 12 415.8 Da. Uniformity was further proven by crystallization and N-terminal amino-acid sequence analysis. The rhβ2mH5 protein was then produced under conditions that allow the intravenous use in humans. Intraveneously applied indium-111- labelled rhβ2mH5 was monitored in hemodialysed patients with and without known β2m-amyloidosis. The tracer was localized specifically to particular areas known to contain amyloid. Thus, this rhβ2mH5 preparation is suitable for detecting amyloid-containing organs of the β2m-class in vivo and fulfills the requirements of a tracer for common use. Finally, the use of indium-111 instead of iodine-131 has reduced the radioactive load and resulted in higher resolution.