DIARECT Newsletter No. 1/2008

The emergence of sophisticated anti-neutrophilic cytoplasmic antibodies (ANCA) diagnostics was first described in the 1985 Lancet article of van der Woude and coworkers showing that the detection of ANCA was indeed related to Wegener's granulomatosis (ref. 1).
Two ANCA patterns may be seen with indirect immunofluorescence of ethanol-fixed neutrophils: a cytoplasmic pattern (c-ANCA) and the artifactual perinuclear pattern (p-ANCA). The major antigen for c-ANCA is a 29-kDa serine protease (ref. 2), proteinase 3 (PR3). PR3 and the major antigen of p-ANCA, myeloperoxidase (MPO), are present within the azurophilic granules of neutrophilic granulocytes. Some clinical overlap has been seen, but the two patterns have different disease associations. The c-ANCA pattern has been predominantly associated with Wegener's granulomatosis, whereas p-ANCA has been associated with microscopic polyarteritis, other vasculitides, idiopathic necrotizing and crescentic glomerulonephritis (c.f. DIARECT newsletter 2/2007).
Over the last two decades ANCA have become the established tool for the diagnosis of systemic vasculitides. The major role for ANCA testing is in diagnosing renal insufficiency of unknown origin, where a positive test indicates whether the patient will benefit from immunosuppressive treatment or not (ref. 3). A negative test result almost completely rules out the presence of systemic vasculitis.
In this clinical setting the major autoantigens are proteinase 3 and myeloperoxidase. For a long time the best test for antibodies to these antigens has been ELISA. Nevertheless, in recent years several methods have been published as an alternative to or optimization of conventional direct ELISA methods.
PR3 and MPO ANCAs recognize conformational epitopes that may be hidden or even destroyed during the coating process. One of the alternative approaches to avoid this problem has been the development of capture and so-called anchor techniques in order to increase assay sensitivity without loss of specificity (ref. 4, 5). Furthermore, a novel ELISA format based on a mixture of human native and recombinant PR3 has been claimed to significantly improve the diagnostic potential for ANCA-associated vasculitis (ref. 6). It remains to be seen which antigen or antigen mixtures and which assay design (capture, anchor etc.) will stand the test of time. Presently, however, there is no indication that the global diagnostic market requires recombinant human PR3 antigen.
Therefore, we at DIARECT have focused on establishing a manufacturing process for native PR3 from human peripheral blood polymorphonuclear cells. In collaboration with our partner in Luxembourg, Prof. R.-L. Humbel (LLIP, Luxembourg), this new PR3 antigen has been stringently tested with respect to specificity, sensitivity, and signal-to-noise ratio.
When sera classified according to their immunofluorescence pattern are tested in ELISAs employing PR3 and MPO antigens, there is virtually no overlap (Figure 1).One remarkable finding - the MPO abs positive cANCA serum - documents a type of diagnostic anomaly which is characteristic for sera from Churg-Strauss-syndrome patients (R.-L. Humbel, personal communication). Also present are sera from Morbus Wegener patients under treatment, in which the antiPR3 titer has been markedly reduced.
We wish to thank Prof. R.-L. Humbel for carrying out these tests and for supplying us with interesting serum samples.

1. van der Woude, F.J., Rasmussen, N., Lobatto, S. et al. (1985) Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener's granulomatosis. Lancet 1/425-9


2. Niles, J.L., McCluskey, R.T., Ahmad, M.F., Arnaout, M.A. (1989) Wegener's granulomatosis autoantigen is a novel neutrophil serine proteinase. Blood 74, 1888-93


3. Segelmark, M., Westman, K. Wieslander, J. (2000) How and why should we detect ANCA? Clin. Exp. Rheumatol. 18, 629-35


4. Westman, K.W.A., Selga, D., Bygren, P., et al. (1998) Clinical evaluation of a capture ELISA for detection of proteinase-3 antineutrophil cytoplasmic antibody Technical Note. Kidney International 53, 1230-6


5. Hellmich, B., Csernok, E., Fredenhagen, G., Gross, W.L. (2007) A novel high sensitivity ELISA for detection of antineutrophil cytoplasm antibodies against proteinase-3. Clin. Exp. Rheumatol. 25 (Suppl. 44), 6-10


6. Damoiseaux, J., Dähnrich, C., Rosemann, A., et al. (2008) A novel ELISA using a mixture of human native and recombinant proteinase-3 significantly improves the diagnostic potential for ANCA-associated vasculitis. Ann. Rheum. Dis. Online First, publ. March 28, 2008 as 10.1136/ard.2007.086579

PBC is a chronic cholestatic liver disease that predominantly occurs in middle-aged women of various ethnic and racial backgrounds. The disease slowly progresses over decades and is supposed to be caused by immune reactions against host antigens. Histologically, it is characterized by inflammatory destruction of intrahepatic small bile ducts, subsequent fibrosis, and finally, liver cirrhosis. It is more frequently diagnosed now than in the past, probably because of a greater awareness of the disease and better as well as more sensitive diagnostic methods (1, 2). Routine testing of the occurence of autoantibodies specific for PBC such as so-called anti-mitochondrial antibodies (E2 subunits of pyruvate and other oxoacid dehydrogenases) and antibodies against distinct components of the nucleus such as SP100 or components of the nuclear pore complexes (GP210 and P62 nucleoporins; 3, 4).
Nuclear autoantibodies specific for the nuclear membrane are found in a subset of patients with PBC, including a few who are M2-negative. The ANA pattern in PBC include 1) multiple nuclear dots (MND-ANA), seen in 10-15% of PBC and mainly associated with sicca syndrome and 2) nuclear membrane associated ANA (NM-ANA), seen in 25-50% of PBC and not associated with lamin autoantibodies. In a majority of cases, the anti-MND target is SP100, whereas anti-NM target is GP210.
SP100, the main autoantigenic target of MND-ANA reactivity, is a 53-kDa nuclear protein with transcription stimulating activity (5). One of the main diagnostic relevancies of these nuclear targets is that even PBC patients negative for M2 antigens may require essential clinical attention. Nevertheless, an anti-MND diagnostic result per se is neither specific for liver disease nor for PBC, since it is also present in a significant number of patients with different rheumatologic disorders (6, 7). Upon comparison with anti-MND determined by indirect immunofluorescence, anti-SP100 appears to be a more sensitive serological marker of liver disease, and it is more PBC-specific (7). IIF pictures may complicate these findings: even if two pictures clearly show anti-MND structures, only one type may be closely associated with the clinical diagnosis of PBC and therefore positive for SP100 (Figure 2).
We would like to thank Prof. R.-L. Humbel for critically evaluating this antigen.
Full-length SP100 expressed using the baculovirus system now available. Another PBC-relevant autoantigen, GP210 is in the last R&D phase and will be available for trial soon.

1. Worman, H.J. (1997) Molecular biological methods in diagnosis and treatment of liver diseases. Clin. Chem. 43, 1476-86


2. Jones, D.E.J. (2000) Autoantigens in primary biliary cirrhosis. J. Clin. Pathol. 53, 813-21


3. Wesierska-Gadek, A., Hohenuer, H., Hitchman, E., Penner, E. (1996) Autoantibodies against nucleoporin p62 constitute a novel marker of primary biliary cirrhosis. Gastroenterol. 110, 840-7


4. Courvalin, J.C., Lassoued, K., Bartnik, E. et al. (1990) The 210-kD nuclear envelope polypeptide recognized by human autoantibodies in primary biliary cirrhosis is the major glycoprotein of the nuclear pore. J. Clin. Invest. 86, 279-85


5. Guldner, H.H., Szostecki, C., Grotzinger, T., Will, H. (1992) IFN enhance expression of Sp100, an autoantigen in primary biliary cirrhosis. J. Immunol. 149, 4067-73


6. Pawlotsky, J.M., Andre, C., Metreau, J.M. et al. (1992) Multiple nuclear dots antinuclear antibodies are not specific for primary biliary cirrhosis. Hepatology 16, 127-31


7. Muratori, P., Muratori, L., Cassani, F. et al. (2002) Anti-multiple nuclear dots (anti-MND) and anti-SP100 antibodies in hepatic and rheumatological disorders. Clin. Exp. Immunol. 127, 172-5

AACC Annual Meeting 2008

July 29-31, 2008 - Washington, DC, USA

(SurModics' booth number 246-248)

AMLI Annual Meeting 2008

August 9-12, 2008 - Seattle, WA, USA

(Booth number 12, exhibitor's venue, The Grand Hyatt, Seattle, WA)

6th International Congress on Autoimmunity

September 10-14, 2008 - Porto, Portugal

Biotechnica 2008

October 7-9, 2008 - Hannover, Germany

6th Immundiagnostisches Meeting

October, 7-9, 2008 - Dresden, Germany

ACR / ARHP Scientific Meeting

October 24-29, 2008 - San Francisco, CA, USA

Medica 2008

November 19-22, 2008 - Düsseldorf, Germany

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