Dear Madam or Sir,
The DIARECT team
DNA toposisomerase I (EC 126.96.36.199) is a key nuclear enzyme that
interconverts supercoiled DNA to the required topological conformations
for normal DNA replication and transcription. This enzyme is the target
antigen for the so-called Scl-70 autoantibodies, which in turn are a
highly specific marker in scleroderma patients (specificity 98-100%).
Scleroderma is a rare connective tissue disorder characterized by
abnormal thickening of the skin. A number of different autoantibodies
are known to occur in scleroderma patients. The presence of Scl-70
autoantibodies is very often associated with diffuse skin involvement
and pulmonary fibrosis.
Figure: Dot blot analyses of sera from blood donors (1-5) and patients with presumed systemic sclerosis (6-12). Recombinant proteins Scl-70 (full length, lot 24B04) and Scl-70 (truncated, lot 45702) were analyzed and compared to Scl-70 antigen (non recombinant) purified from bovine sources (lots 15D01, 15D02 and 15D03).
Substrate choice is a critical consideration during assay development and can impact factors such as sensitivity, dynamic range and reproducibility. BioFX® TMB Extended Range HRP Microwell Substrate (TMBX) is the most recent addition to SurModics’ family of TMB HRP substrates.
Use of TMBX in a quantitative assay (i) can increase the assays upper limit of quantitation and (ii) maintain the detection limits and analytical sensitivity achieved with other substrates in our TMB family.
Use of TMBX also improves the reproducibility of the assay through lower backgrounds and control of assay timing.
Figure: Standard curve comparison for TMBW and TMBX stopped and read at 450 nm using a model assay system. Rabbit Rabbit anti-mouse IgG was coated on a ELISA plate and stabilized with StabilCoat® Immunoassay Stabilizer. Biotinylated mouse IgG was titrated in StabilZyme® Select Conjugate Stabilizer and incubated for two hours at room temperature. After washing diluted Streptavidin-peroxidase was added and incubated for 20 minutes at room temperature. After a final wash the substrate was added and developed. Results show an extended dynamic range when TMBX is used.
SurModics' choice of substrates with varying detection levels:
AACC and Clinical Lab Expo
Date: July 15 -19, 2012
Location: Los Angeles, CA
AMLI 25th Annual Meeting
Date: August 4 -7, 2012
Location: Minneapolis, MN
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