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DIARECT Newsletter No. 1/2007
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Dear Madame or Sir, |
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Whereas most if not all SLE-associated autoantigens are localized in
the cell nucleus, a number of myositis-specific antigens are
cytoplasmic and play a major role in protein biosynthesis. For example,
the myositis-related aminoacyl-tRNA synthetases (Jo-1, PL-7, PL-12) are
cytoplasmic targets that catalyze the linking of aminoacyl residues to
their corresponding tRNAs. Another cytoplasmic antigen complex which
plays a key role in myositis diagnosis is the signal recognition
particle (SRP), an RNA-protein complex. In terms of diagnostic
relevance, SRP54 has been shown to be the main target, and we are
pleased to present this new recombinant antigen, which can now be
included into your in-vitro diagnostic tool box.
In this newsletter we also report on recently performed studies
clarifying the importance of PM/Scl 100 and PM/Scl 75 antigens for
improving polymyositis, scleroderma, and overlap syndrome testing.
And our partners from SurModics, Inc., have put together a highly
informative overview table to help you in deciding which of the famous
stabilization product family is most suitable for your application.
Pleasant reading!
Your DIARECT team
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SRP54 and Polymyositis in-vitro Diagnostics |
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| SDS-PAGE analysis of five independent lots of SRP54 |
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Cell Biology of SRP
The signal recognition particle (SRP) is a cytoplasmic
ribonucleoprotein complex that directs the translocation of newly
synthesized protein from the polysome to the endoplasmic reticulum. SRP
is composed of six polypeptides and a tRNA-like molecule known as 7SL
RNA. The 54-kDa subunit of SRP (SRP54) is a GTP-binding protein and has
been shown to interact with the signal sequences of nascent secretory
and membrane proteins. The 54-kDa subunit of SRP contains three
domains: an N-terminal helical bundle domain, a GTPase domain, and the
M-domain that binds the 7SL RNA as well as the signal sequence.
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| Dot-blot analysis of three SRP54 lots. One microliter of each sample was applied. |
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Autoantibodies and Disease
Human autoantibodies often recognize epitopes that are conserved during
evolution and are essential for the cellular function of the
autoantigen. Anti-SRP autoantibodies occur almost exclusively in
patients with polymyositis, an autoimmune syndrome characterized by
chronic muscle inflammation of unknown cause (1). These auto-antibodies
immunoprecipitate several of the SRP subunits (plus RNA) but
predominantly recognize the SRP54 subunit (2). Anti-SRP autoantibodies
are rare: about 5% of myositis patients are positive, however 18% of
those with cytoplasmic antibodies other than Jo-1. The classic
'anti-SRP syndrome' is a severe form of polymyositis in which the
myositic inflammation is acute and aggressive in onset, and myalgias
and cardiac involvement are common. Normally poor response to therapy
is observed with a 5-year survival rate in the range of 25%.
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Recombinant Human Antigen
DIARECT is now introducing the full-length, human SRP54 produced in the
baculovirus/Sf9 expression system. Excellent test results with this
antigen were obtained in line assays (immuno-dot) as well as EIA
systems.
Ref. (1) Targoff, I.N. et al. (1990) Antibody to signal recognition particle in polymyositis. Arthritis Rheum 33, 1361-70.
(2) Römisch, K. et al. (2006) Human autoantibodies against the 54 kDa
protein of the signal recognition particle block function at multiple
stages. Arthritis Research & Therapy 8:R39.
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| Analysis of different myositis patient sera in western blots (Data courtesy of Prof. Humbel, Luxembourg) |
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New insights into PM/Scl Antigens |
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Of the numerous nucleolar targets, the clinical significance and
diagnostic relevance of PM/Scl 100 antibodies for premium in-vitro
testing of myositides, scleroderma and overlap syndromes is undisputed.
However, the debate on the particular diagnostic importance of the
100-kDa antigen vs. its 75-kDa counterpart has been increasing over the
last decade. Recently Raijmakers et al. (1) described a new,
N-terminally elongated PM/Scl 75 polypeptide and were able to determine
its diagnostic value for analysing sera from patients with
polymyositis/scleroderma overlap syndrome. The data of this
investigation indicated that the use of the 'long' PM/Scl 75 isoform in
addition to PM/Scl 100 in ELISA significantly increases the number of
patients in which anti-PM/Scl autoantibodies can be detected.
To establish comparable conditions for testing full-length constructs
of PM/Scl 100 and PM/Scl 75 antigens in parallel, DIARECT has expressed
both polypeptides in Baculovirus-infected insect cells and purified
them by IMAC.
We made these recombinant human antigens available to a number of
internationally distinguished research and reference labs (Prof.
Humbel, Luxembourg; Prof. Pruijn, Nijmegen; Dr. Mierau, Aachen). The
results were characterized by a rather high degree of uniformity in
that most of the PM/Scl-positive sera were positive for the PM/Scl 100
target with a somewhat smaller percentage of sera also positive for
PM/Scl 75. Surprisingly a rather small number of sera clearly negative
for PM/Scl 100 were borderline and low to moderately positive for
PM/Scl 75. Therefore, the addition of PM/Scl 75 (to complement PM/Scl
100) has the potential to increase sensitivity without any loss of
specificity.
If you are interested in testing your valuable serum bank specimens
with both PM/Scl targets (100-kDa and 75-kDa ), DIARECT is now offering
evaluation samples of both antigens for the price of one.
Ref. (1) Raijmakers, R. et al. (2004) PM-Scl-75 is the main autoantigen
in patients with the polymyositis/scleroderma overlap syndrome.
Arthritis Rheum. 50:565-9.
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Autoantigens for Myositis Diagnostics |
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Over the last couple of years DIARECT has introduced a number
myositis-related autoantigens some of which were until then
commercially very limited, if not completely inaccessible . We now
offer the most complete panel of recombinant targets for polymyositis
and dermatomyositis autoantibody testing:
| 12900 | Histidyl-tRNA Synthetase (Jo-1) | Poly/Dermatomyositis | | 15600 | Threonyl-tRNA Synthetase (PL-7) | Poly/Dermatomyositis | | 15700 | Alanyl-tRNA Synthetase (PL-12) | Poly/Dermatomyositis | | 18400 | SRP54 | Poly/Dermatomyositis | | 18100 | Mi-2 | Dermatomyositis | | 16000 | PM/Scl 100 | Poly/Dermatomyositis | | 17000 | PM/Scl 75 (on request) | Poly/Dermatomyositis |
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SurModics Stabilizers |
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SurModics offers a full line of high-performance protein stabilization
reagents for the in vitro diagnostics market. These proprietary
stabilizers are formulated for a variety of applications, including the
preservation of antibodies and antigens used in immunoassays, HRP and
AP conjugates, and microarray proteins immobilized to beads in
suspension or dried in array formats. Through its expertise in surface
modification and protein stabilization, SurModics is bringing
innovation together ™ with customers around the world.
Click here for the pdf file
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North American Distributor |
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North American customers please contact our distributor:
SurModics, Inc.
9924 West 74th Street
Eden Pairie, MN 55344
Tel: 952-829-2709
Fax: 952-829-2743
read more
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Calendar 2007, where you can meet the DIARECT and SurModics management |
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107th ASM General Meeting
Toronto, Ontario, Canada, May 21 - 25, 2007
2007 AACC Annual Meeting
San Diego, CA, USA, July 15 - 19, 2007
2007 AMLI Annual Meeting
The Westin
Michigan Avenue
Chicago, IL, USA, August 9 - 12, 2007
8th Dresden Symposium on Autoantibodies
Dresden, Germany, September 12 - 15, 2007
Medica
Düsseldorf, Germany, November 14 - 17, 2007
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