Use of Anti‐(β2 Microglobulin) mAb to Study Formation of Amyloid Fibrils

Abstract

Three mAbs, IgG,k 1F11, 7B6 and 14H3, were raised against in vitro-self-aggregated &Z-microglobulin. They recognize the native and unfolded forms of the protein and bind its fibrillar form that is present in amyloid tissue. When assayed in fibrillogenesis tests in vitro, mAb 14H3 inhibited fibril formation from m-microglobulin. This mAb recognizes a sequential epitope corresponding to the C-terminal octapeptide, residues 92-99, of /32-microglobulin. By using synthetic peptides it has been found that the integrity of the sequence is essential for the formation of the immunocomplex : the binding affinity is lowered by one order of magnitude (Kd from lo-' M to M) by removal of Met99 and completely abolished when both Asp98 and Met99 are lost or Arg98 is substituted with Lys. The other two mAbs, IF11 and 7B6, which bind sequences 20-41 and 63-75, respectively, are without effect on P;?-microglobulin fibrillogenesis. These two mAbs recognize p-microglobulin bound to the heavy chain in the major histocompatibility complex of type I located in the cell membrane, a property which is not shared by mAb 14H3.

Lysine 58‐cleaved β2‐microglobulin is not detectable by 2D electrophoresis in ex vivo amyloid fibrils of two patients affected by dialysis‐related amyloidosis

“…After 2D-PAGE, the proteins in the gel were transferred onto a PVDF membrane as previously described (Stoppini et al 1997). The b2m isoforms were identified with an anti-human b2m polyclonal antibody (Dako) using a chemiluminescent procedure (ECL plus Western blotting detection reagent Amersham Bioscience).…”

A Circulating β₂-Microglobulin Intermediate in Hemodialysis Patients

“…In 1998, Bellotti et al [3] first demonstrated the presence of a partially unfolded β 2 m that could refold reversibly into a N-β 2 m in an amyloid deposit from hemodialysis (HD) patients. Prior to this report, Stoppini et al [4] reported that a monoclonal antibody specific for C-terminal peptides, which are folded inward in a N-β 2 m molecule, could inhibit fibril formation in vitro. In addition, a conformational variant of β 2 m with an unfolded C-terminus was found in amyloid deposits from HD patients using a monoclonal antibody against C-terminal octapeptide of β 2 m [5].…”

Conformational dynamics of the β2-microglobulin C terminal in the cell-membrane-anchored major histocompatibility complex type I

“…The following materials were obtained from the commercial source given in parentheses: reagents used for immunofluorescence analysis (Tecnogenetics, Becton Dickinson, Pharmingen, Clontech and Dako); mouse isotyping kit and reagents for electrophoresis (Bio-Rad); reagents for cell culture and enzyme immunoassay (ICN, Gibco BRL, Hyclon). mAb 14H3 was prepared as previously described [5]. The recombinant MHCI complex, produced as described by Springer et al [4] was kindly supplied by K. Doering and recombinant human i2-m was prepared as described by Reid et al [6].…”

“…Immunological typing of the antibody was accomplished with peroxidase-conjugated rabbit anti-mouse immunoglobulins. The purification of mAb 14H3 from cell culture medium was carried out as previously described [5] by affinity chromatography on a protein G-conjugated Sepharose column. Scatchard analysis.…”

“…In this type of amyloidosis, the MHCI can be considered the storage component of the amyloid fibril precursor and its synthesis and dissociation kinetics are most likely associated with progression of the disease. We have recently described a monoclonal antibody (mAb 14H3) produced against the C-terminal end of i2-m (sequence 92-99) that recognizes i2-m as a monomer or as a fibrillar polymer, but which is apparently unable to detect folded i2-m in the MHCI anchored to the intact cell membrane [5]. In this study, we show that following in vitro cell damage of both the Jurkat T cell line and peripheral blood cells from normal subjects, the 92 -99 epitope of i2-m becomes accessible to the 14H3 mAb, possibly as a consequence of conformational changes in the MHCI.…”