DIARECT Newsletter No. 2/2007

In time for this year's AACC in San Diego (July 15-19, 2007) we are glad to present to you a number of high-grade clinical, scientific and technical articles about some of our older and new products.
Professor Humbel from Luxembourg has kindly provided us his most recent essay on SRP auto-antibodies.
Although DIARECT's classical domain is recombinant protein engineering, we have consistently been prodded by many of our customers also to develop certain "native" antigens with the well-known DIARECT quality features. We are now pleased to introduce myeloperoxidase (MPO), one of the main antigen targets of ANCA associated diseases.
Time and again one of the most frequent requests of many of our customers using the famous StabilZyme® product line is the behavior of these stabilizers upon dilution with PBS. Here our technical bulletin gives you all of the answers.
Looking forward to seeing you in sunny California at the AACC.

Autoantibodies reactive with the signal recognition particle (SRP) were first discovered in serum of a patient with polymyositis by Reeves et al. in 1986. Other examples followed in the years thereafter. Targoff et al. could show that these autoantibodies correlated to a distinct subgroup of myositis patients. In 2002 Miller et al defined the special character of the disease and found that muscle biopsies showed active myopathy with pathological changes in endomysial capillaries, but little inflammation. Myopathy appears to be a more accurate description than myositis, and a number of studies have confirmed that anti-SRP autoantibodies are associated with a syndrome of a necrotizing myopathy within the spectrum of immune-mediated myopathies that differs from "typical" polymyositis.

Antineutrophil cytoplasmic antibodies (ANCA) have become an established tool for the diagnosis of autoimmune systemic vasculitis and inflammatory disorders. In this clinical setting the major antigens are proteinase 3 and myeloperoxidase, and autoantibodies to these antigens from the azurophilic granules of neutrophils can best be tested by antigen-specific diagnostic devices like ELISA, line assays or in multiplex systems.
We have focused on establishing a manufacturing process for myeloperoxidase (MPO), which can be isolated from human peripheral blood polymorphonuclear cells.
MPO is the product of a single gene of 11 kb in size. Its initial translation product is an 80-kD protein, which following proteolytic removal of the 41 amino acid signal peptide, undergoes N-linked glycosylation with the incorporation of mannose-rich side-chains to generate an 89- to 90-kD enzymatically inactive apoproMPO. With the insertion of a heme, apoproMPO is converted to the enzymatically active proMPO. The removal of the N-terminal 125 amino acid proregion by proteolytic cleavage results in the production of a 72- to 75-kD protein, which undergoes a second proteolytic cleavage to generate the 467 amino acid heavy subunit (57 kD) and the 112 amino acid light subunit (12 kD) of MPO, which associate as a heavy-light protomer. Mature MPO has a molecular mass of approx. 150 kD and consists of a pair of heavy-light protomers whose heavy subunits are linked by a disulfide bond along their long axis. The mannose-rich carbohydrate and the two hemes are covalently bound to the heavy subunit.
MPO is involved in the oxygen-dependent microbicidal system of peripheral blood polymorphonuclear cells. It catalyzes the peroxidation of chloride into hypochlorite and its functional significance is twofold: (i) the generation of hypochlorite is important for the intracellular killing of phagocytosed microorganisms, and (ii) hypochlorite inactivates protease inhibitors and, as such, allows lytic enzymes released from neutrophils to degrade cells and other foreign material in the vicinity of neutrophils.
As the major target of p-ANCA immunofluorescence pattern MPO autoantibodies show high prevalences as well as clinical association with microscopic polyangiitis, idiopathic crescentic glomerulonephritis, Churg-Strauss syndrome, and classic panarteriitis nodosa.
We have now established a validated production process for MPO with a respective R&D project for PR3 to be finalized by the end of the year.
MPO autoantigen has been vigorously tested for assay performance parameters such as sensitivity and specificity. On the basis of convincing signal-to-noise ratio as well as superb lot-to-lot consistency we now make this new antigen available to you for critical examination..

A study was performed to evaluate the use of 1X Phosphate Buffered Saline (PBS) prepared from a 10X concentrate as a diluent for StabilZyme® HRP, SELECT®, NOBLE and AP Conjugate Stabilizers.
The final concentrations of PBS in the StabilZyme stabilizers were 50%, 25% and 10% (corresponding to 50%, 75% and 90% final product concentrations).
Each solution was stored at room temperature and visually observed daily for precipitation. If precipitation occurred, the date of observation was documented.
The results showed that after 13 days at room temperature, precipitation occurs at PBS concentrations greater than or equal to 25%. This study is ongoing with PBS concentrations of 10% and 5%. Results for these concentrations will be available at a later date.