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Dear Madame or Sir, |
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In time for this year's AACC in Chicago (July 25-27, 2006), we are pleased to present our summer newsletter introducing DIARECT's newest product developments.
* We are proud to introduce the dermatomyositis-specific Mi-2 target; this protein is currently our largest full-length recombinant auto-antigen: 220 kDa!
* Very much R&D work has gone into our recombinant Ro/SS-A (60 kDa) antigen. All the more imposing is the final result: a Ro 60 target which combines the high and unrestrained reactivity of the native antigen with all the well-known advantages of an established recombinant manufacturing process: large lot size, superior lot-to-lot consistency and virtually unlimited availability.
* With our next launch we will break new ground: the first commercially available human monoclonal standards. Two human monoclonal dsDNA mAbs will perfectly meet the calibration/standardization needs of ELISA-, FARR- and Crithidia-based dsDNA antibody detection systems.
All new products are now available, and if your order for an evaluation sample is received before July 31st, 2006, we will grant you a 50% AACC rebate off the list price.
Also featured in this newsletter is new information from Surmodics, Inc. First, they present their recent data on StabilZyme® NOBLE BSA-Free Stabilizer, which illustrate the superb stabilizing properties of this unique, BSA-free formulation. The final section is dedicated to a statement from Surmodics, in which they describe their quality system, especially with regard to their proprietary stabilization products.
Pleasant reading, and we are looking forward to seeing you at the AACC.
Your DIARECT team
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Mi-2 Cat.No. 18100 |
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A clinical subgroup of patients suffering from myositis show positive autoantibody test results considered to be highly specific for this disorder. Autoantibodies targeting the Mi-2 nuclear antigen represent one of the serologic hallmarks of idiopathic inflammatory myopathies (IIM). The Mi-2 autoantigen (Mi-2ß) is a roughly 220 kDa protein that is part of the nucleosome remodeling-deacetylase (NuRD) complex (1, 2) apparently involved in transcription regulation.
In IIM Mi-2 antibodies are characterized by diagnostic sensitivity and specificity of approximately 4-18% and 98-100%, respectively. Furthermore, anti-Mi-2 antibodies are strongly associated with dermatomyositis (frequency up to 31%) and have a very high positive predictive value for such a disease subset. Anti-Mi-2 are the only defined myositis-specific autoantibodies clearly directed to a nuclear target. Another rather exceptional feature of Mi-2 antibodies relates to their frequency in children, which is comparable to that in adults.
Mi antibodies were described in 1976 for the first time (complement fixation using calf thymus extract) and the corresponding Mi-2 antigen, immunoaffinity purified in 1985, facilitated the first reliable ELISA analysis of larger serum panels. It took another decade until Seelig and coworkers (3) were able to isolate a full-length cDNA encoding CHD4 (chromodomain helicase DNA-binding protein 4), which they called Mi-2ß. Subsequently different parts of the Mi-2ß autoantigen have been recombinantly expressed and used to systematically study the clinical, laboratory and histological characteristics of an impressively large cohort of anti-Mi-2 positive patients (1).
We now introduce the new, diagnostically superior Mi-2ß target: a human recombinant, full-length autoantigen (MW 221.3 kDa), eukaryotically expressed in baculovirus-infected insect cells.
Ref.
(1) Hengstman, G J D et al. (2006) Clinical characteristics of patients with myositis and autoantibodies to different fragments of the Mi-2ß antigen. Ann Rheum Dis 65, 242-5
(2) Zhang, Y (1998) The dermatomyositis-specific autoantigen Mi-2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities. Cell 95, 279-89
(3) Seelig, H P et al. (1995) The major dermatomyositis-specific Mi-2 autoantigen is a presumed helicase involved in transcriptional activation. Arthritis Rheum 38, 1389-99
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| Dot-blot comparison of three lots of Mi-2 antigen |
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Ro/SS-A (60 kDa; recombinant) Cat.No. 17400 |
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| SDS-PAGE and Western Blot analysis of three amounts of Ro/SS-A (60 kDa) autoantigen. Molecular weight standards (lane 1) and 0.3, 0.6 and 1.2 µg Ro/SS-A (60 kD) (lanes 2-4) were investigated. The proteins were blotted and probed with a patient serum specific for Ro/SS-A. |
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| Comparison of calf-thymus Ro/SS-A and recombinant, human Ro/SS-A in ELISAs. Ro/SS-A antigen was coated at 0.25 µg/mL. Ninety samples were analyzed. |
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Anti-Ro/SS-A antibodies are found with high frequency in everyday clinical practice and are closely associated with Sjogren's syndrome, systemic lupus erythematosus and neonatal lupus. These antibodies belong to the most commonly detected ANA/ENA entities, and most molecular and biochemical details of the target antigens involved have been intensely investigated. Nonetheless, substantial information on the intracellular function of the Ro/La complex - which is composed of small single-stranded RNAs (hYRNAs) and of one or more polypeptides - remained rather sporadic until recently. Its ability to bind nucleic acids and the fact that it shares homologies with gene regulation proteins suggested that it may participate in RNA transcription process.
Recent studies have now implicated Ro in two distinct cellular processes: small RNA quality control and the enhancement of cell survival following exposure to ultraviolet irradiation (1). The findings that Ro binds 5S rRNA precursors in Xenopus oocytes and variant, apparently misfolded U2 snRNAs in mouse ES cells strongly suggest that Ro may participate in a general quality control pathway for defective mRNAs as well as misfolded and mutant proteins. A conserved role for the Ro 60 kDa protein in facilitating cell survival after ultraviolet irradiation has recently emerged from studies of Ro in mammalian cells and bacteria. In one strain of mice lacking the Ro 60 protein, the number of apoptotic keratinocytes increases two-fold following UV irradiation. Secondly, mouse embryonic stem cells lacking Ro have a lower survival rate after UV exposure. Since Ro binds misfolded pre-5S and U2 RNAs, one possibility is that, following irradiation, Ro sequesters nascent RNAs that misfold or fail to assemble into RNPs. More work is clearly needed to elucidate the mechanism(s) by which the Ro protein enhances the survival of irradiated cells and to determine whether the Y RNAs contribute to this function.
For diagnostic purposes preparations of natural Ro 60 kDa protein purified from calf thymus have been available for a long time. However, because of varying lot-to-lot consistency with these products, it has been our goal to produce the recombinant human target. The development of a recombinant Ro/SS-A 60 kDa antigen, which reacts with patient's sera to the same extent as the calf thymus-derived protein has been an immense challenge.
The DIARECT human recombinant Ro/SS-A 60 kDa antigen fulfils those requirements and finally bridges a long lasting gap. Now our customers have the reactivity of the calf-thymus antigen and the lot-to-lot consistency of recombinant production.
Ref.
(1) Chen X, Wolin, S L (2004) The Ro 60 kDa autoantigen: insights into cellular function and role in autoimmunity. J Mol Med 82, 232-9
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Human monoclonal dsDNA autoantibodies dsDNA-mAb32 Cat.No. 33200; dsDNA-mAb33 Cat.No. 33300 |
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The successful advances in recombinant expression of a great variety of human autoantigen targets have led to rapid progress in the field of in vitro autoimmune diagnostics over the last decade. Presently recombinant production of the majority of human autoantigens is possible with highest purity and large lot sizes (several 100 mgs), therefore fulfilling all quality requirements of diagnostic kit manufacturers whatever the diagnostic device format may be.
In sharp contrast to these improvements in autoantigen availability no comparable advances in the standardization of autoantibody tests have evolved. Even today there is no real alternative to the traditional use of sera from autoimmune patients as standard as well as calibration materials, a situation that still causes problems:
* serious supply problem because of the bad health status of affected patients
* complex polyclonal autoantibody mixtures in individual patient sera with variations in specificities, affinities and titres of autoantibodies
* sera exhibiting multiple reactivities
As an alternative to patient-derived autoantibodies, human monoclonal antibodies could be used but are difficult to generate and produce routinely. One of the exceptions, in which a panel of stable human hybridomas has been obtained by cell fusion of peripheral B lymphocytes from SLE patients with a heteromyeloma fusion partner (1), are human monoclonal autoantibodies that bind dsDNA with high affinity (2).
This panel of anti-dsDNA monoclonal antibodies has been investigated in great detail (3) and two out of this panel are now available:
* one of these mAbs (dsDNA-mAb33) shows an extremely good correlation in ELISA-based devices when compared with the international WHO standard preparation (Wo/80; 4)
* the second mAb (dsDNA-mAb32) has shown excellent applicability in FARR and Crithidia type assays.
Ref.
(1) Grunow, R. et al. (1988) The high efficiency human B cell immortalizing heteromyeloma CB-F7. J Immunol Meth 106, 257-65
(2) Winkler, T H et al. (1991) IgG human monoclonal anti-DNA autoantibodies from patients with systemic lupus erythematosus. Clin Exp Immunol 85, 379-85
(3) Simon, T et al. (1996) Standardizing the diagnosis of autoantibodies: an evaluation of human monoclonal anti-dsDNA autoantibodies for standardization of a commercial ELISA assay system. Presentation "Antibody Engineering" Meeting, GBF, Braunschweig, September 1996
(4) Feltkamp, T E et al. (1988) The first international standard for antibodies to double stranded DNA. Ann Rheum Dis 47, 740-6
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| Comparison of Wo80 and dsDNA mAb33. Identical dilutions of both antibodies were compared using a commercially available dsDNA-antibody ELISA kit. |
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StabilZyme® NOBLE Stabilizer |
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StabilZyme NOBLE BSA-Free (bovine serum albumin-free) Stabilizer, Product Number SZ04, is a stabilizer and diluent for biologically active components in solution. This stabilizer incorporates a unique technology without the use of BSA to protect the activity of conjugated proteins and unmodified proteins in solution--those commonly used in diagnostic immunoassays, such as competitive, direct, and sandwich ELISAs. StabilZyme NOBLE Stabilizer is also recommended as a stabilizer for protein and serum controls in solution.
StabilZyme NOBLE Stabilizer allows for storage at ready-to-use concentrations to ensure accurate interpretation of results, eliminating potential dilution errors by end users. Compared to conventional immunoassay diluents, StabilZyme NOBLE Stabilizer often reduces background and increases binding, resulting in an increased signal-to-noise ratio.
StabilZyme NOBLE Stabilizer is sterile-filtered and contains a mercury-free, azide-free preservative. It should be used at 100% concentration to dilute your reagent at its optimum concentration. This diluted reagent can be used in your assay as normal. It should be stored at 4ºC and protected from the direct exposure to light.
Please Note: StabilZyme NOBLE Stabilizer has limited buffering capacity. In competitive ELISAs, add buffer if the test samples have extreme pH variability. Ensure that the pH is between 6.8 and 7.0. Avoid phosphate buffers at concentrations greater than 2mM concentration in the final volume, for this may cause precipitation.
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To demonstrate the stabilizing efficacy of StabilZyme NOBLE stabilizer, four different species of HRP conjugates were stored in StabilZyme NOBLE stabilizer in their respective use concentrations. The HRP conjugates included rabbit (IgG) anti-mouse IgG-HRP, mouse (IgG) anti-rabbit IgG-HRP, goat (IgG) anti-rabbit IgG-HRP and chicken (IgY) anti-mouse IgG-HRP. The enzyme activity was tested by ELISA, and the percent retained activity was determined by comparing the activity of the StabilZyme NOBLE stabilizer stored at 4ºC to that of the StabilZyme NOBLE stabilizer stored at 37ºC.
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SurModics is in the business of developing unique chemical technologies and products to enhance or enable their customers' devices, products and/or systems. SurModics' Stabilization Products have been implemented into hundreds of diagnostic test kits as critical components; therefore, SurModics understands that Quality is of the utmost importance and that customer satisfaction is essential to its success. SurModics continually strives to maintain the highest standards in meeting its customer requirements from product development through distribution.
SurModics has both ISO 9001:2000 and 13485:2003 certifications. All SurModics' Stabilization Products are manufactured per ISO 9001 standards under stringent controls and established procedures to ensure lot-to-lot consistency and complete lot traceability. The raw material components of the Stabilization Products are proprietary; however, each component is fully qualified before it is released to manufacturing. All components are quarantined until required testing and inspection have been performed and acceptance criteria have been met. Each batch of product is given a unique identification (lot number), while each individual product is clearly recorded and identified through receiving, inspection, processing, product inspection, release, shipment, and distribution. Final Quality control testing confirms that the lot of final product meets pre-defined product specifications for proprietary components, physical characteristics, and performance.
SurModics is proud of the standard of excellence its employees strive for every day to deliver the Stabilization Products with the Quality and consistency customers require. Through the combination of these products and diagnostic test systems, SurModics and its partners bring innovation together around the world.
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