Production of recombinant human beta(2)-microglobulin for scintigraphic diagnosis of amyloidosis in uremia and hemodialysis

Amyloid of beta(2)-microglobulin (beta(2)m) origin can be diagnosed using I -131-radiolabelled-beta(2)m scintigraphy in patients with uremia and hemodi alysis treatment. As the tracer beta(2)m is isolated from another patient, it carries the common risks, including viral 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 beta(2)m (rh beta(2)m) in Escherichia coli. The expression vector pASK40 Delta L be ta(2)m(His)(5) contains a C-terminal (His)(5)-tag for purification via immo bilized metal ion affinity chromatography (IMAC). Size exclusion chromatogr aphy on a Superose 12 column represents the second step of purification. The isolated rh beta(2)mH(5) reacted in an immunochemically identical manne r to native human beta(2)m, and showed a single band of approximate to 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 b y crystallization and N-terminal amino-acid sequence analysis. The rh beta( 2)mH(5) protein was then produced under conditions that allow the intraveno us use in humans. Intraveneously applied indium-111-labelled rh beta(2)mH(5 ) was monitored in hemodialysed patients with and without known beta(2)m-am yloidosis. The tracer was localized specifically to particular areas known to contain amyloid. Thus, this rh beta(2)mH(5) preparation is suitable for detecting amyloid-co ntaining organs of the beta(2)m-class in vivo and fulfils the requirements of a tracer for common use. Finally, the use of indium-111 instead of iodin e-131 has reduced the radioactive load and resulted in higher resolution.