A comparison of commercial and in-house elisas for antineutrophil cytoplasmic antibodies directed against proteinase 3 and myeloperoxidase

Pathology (1999) 31, pp. 38± 43

A COMPARISON OF COMMERCIAL AND IN-HOUSE ELISAS FOR ANTINEUTROPHIL CYTOPLASMIC ANTIBODIES DIRECTED AGAINST PROTEINASE 3 AND MYELOPEROXIDASE W. P OLLOCK*, K. D UNSTER², J.M. R OLLAND², H. K OH

AND

J. S AVIGE¶

Immunology Department, Gribbles Pathology, South Yarra*, Department of Pathology and Immunology, M onash M edical School, Alfred Hospital, Prahran², Department of Biochemistry, Women’s and Children’s Health Care Network, Carlton, and University Department of Medicine, Austin and Repatriation Medical Centre, Heidelberg¶, Victoria, Australia

Summary This study compares the concordance of results in different ELISAs for antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) or myeloperoxidase (MPO). Sera were considered ªtrue positivesº if they were positive according to the manufacturer’s criteria in a least three of the five PR3-ANCA ELISAs, or in at least four of the six MPO-ANCA ELISAs. Of the 26 sera that demonstrated cytoplasmic fluorescence (C-ANCA), 23 (89%) contained PR3-ANCA and three (11%) had MPO-ANCA. Two sera that were negative by indirect immunofluorescence (IIF) contained PR3-ANCA. Of the 26 sera with perinuclear fluorescence (P-ANCA), 19 (73%) contained MPO-ANCA, and one (4%) had PR3-ANCA. Six sera with P-ANCA did not have PR3- or MPO-ANCA. No serum that was negative by IIF contained MPO-ANCA. For the different PR3-ANCA ELISAs, sensitivities ranged from 88 to 100%, and specificities from 91 to 100%. For the MPO-ANCA ELISAs, sensitivities varied from 59 to 100% and specificities from 83 to 100%. The highest sensitivity and specificity for both the PR3- and MPOANCA ELISAs were obtained with the IBL and Eurodiagnostica assays. The in-house PR3-ANCA ELISA performed slightly less well than the commercial assays, but the performance of the in-house MPO-ANCA assay was comparable or better. Key words: Antineutrophil cytoplasmic antibodies, autoantibodies, ELISA, indirect immunofluorescence, myeloperoxidase, proteinase 3. Abbreviations: ANCA, antineutrophil cytoplasmic antibodies; IIF, indirect immunofluorescence; M PO, myeloperoxidase; PR3, proteinase 3. Accepted 5 August 1998

INTRODUCTION Wegener’s granulomatosis and microscopic polyangiitis are small vessel vasculitides that can result in irreversible renal failure if untreated or if treatm ent is delayed. W hen a systemic vasculitis is suspected, the demonstration of circulating antineutrophil cytoplasmic antibodies (ANCA) is helpful in making this diagnosis 1±4 so that treatment can be instituted immediately. The combination of screening for ANCA on normal peripheral blood neutrophils by indirect im munofluorescence (IIF),5 and testing positive sera in EL ISAs for ANCA

directed against proteinase 3 (PR3) 6 and myeloperoxidase (M PO-ANCA),3 greatly enhances the individual specificities of these tests.7 Sera that produce cytoplasmic fluorescence (C-ANCA) and bind in ELISA s for PR3-ANCA are found in nearly 90% of patients with active generalised Wegener’s granulomatosis 8 and in about 60% of those with active regional or ªlimitedº disease,9 but are uncommon in patients in remission. Sera with perinuclear fluorescence (P-ANCA) and MPO specificity are present in up to 85% of patients with active microscopic polyangiitis.3 However, EL ISAs for PR3- or M PO -ANCA cannot replace IIF because about 10% of patients with Wegener’s granulomatosis or microscopic polyangiitis are negative in these assays.10 ELISA s are useful in interpreting sera that are associated with ªdifficultº IIF patterns such as when coincidental antinuclear antibodies (ANA) are present (these can produce a pattern indistinguishable from P-ANCA), when several antibodies are present simultaneously, when there is high background fluorescence or when the investigator is inexperienced with interpretation. The ELISA s for PR 3- and M PO -ANCA determine the two most clinically important target antigen specificities in patients suspected of having Wegener’s granulomatosis or microscopic polyangiitis. W hile ANCAs occur in rheum atoid arthritis,10 SLE 12 and inflamm atory bowel disease,13,14 PR 3 and M PO specificities are uncom mon, 15 and ANCAs in these diseases probably do not correlate with disease activity or the presence of vasculitis.16 Finally, ELISA s m easure antibody reactivity that correlates well with disease activity in individual patients; levels are usually high at presentation, fall with treatment and antibodies reappear during relapse (but not during the infections which m ay be otherwise confused with relapses).17,18 ANCA reactivity helps to distinguish between patients with a systemic vasculitis, where levels are high at presentation, and those with the non-vasculitic ANCAassociated diseases, where levels are generally low. However, ANCA reactivity determined by ELISA reflects antibody affinity in addition to concentration, making standardisation of results difficult. There are several published methods and comm ercial kits for the detection of PR 3- and M PO -ANCA, and the aim of this study was to determ ine the concordance of results using these different ELISA s.

0031±3025/99/010038± 06  1999 Royal College of Pathologists of Australasia

COM PARIS ON OF ELISAS FOR ANC A

MATERIALS AND METHODS

39

subtracting the absorbance to the HSA-coated plate from that to the antigen-coated plate. The reactivity of a sample was then determined from a standard curve constructed by plotting the mean absorbance of 3 standards against their designated values. A positive serum bound at greater than mean ‰ 3 SD of 35 normal control sera. For all of these assays, the results were described as ªpositiveº (which included borderline results for the Eurodiagnostica assay) or ªnegativeº according to the manufacturer’s criteria.

Sera Seventy-six sera from patients in whom the diagnosis of vasculitis was suspected were tested. These were provided by 3 laboratories that had already examined them for ANCA, and the sera were not selected because of their ability to cause problems in the assays.

IIF IIF was performed by a single experienced scientist. Slides were screened for ANCA by IIF using commercially prepared neutrophil slides (INOVA, San Diego, CA, USA). These were incubated with 1/20 dilution of serum in phosphate-buffered saline (PBS) for 20 min at RT, followed by a wash with PBS, a further incubation with fluorescein-conjugated affinitypurified antihuman IgG (ImmunoConcepts, Sacramento, CA, USA) for 20 min, and another wash. Slides were coverslipped and examined with a standard immunofluorescence microscope at 3 400 magnification. Postive controls (INOVA) were tested in parallel. Patterns were described as: C-ANCA if there were diffuse staining of the granulocyte cytoplasm with central accentuation between nuclear lobes; and P-ANCA when there was fluorescence of the outer rim of the neutrophil nuclear lobes. Titres were not determined.

Sensitivities and specificities of ELISAs Serum ªpositivityº for PR3- or MPO-AN CA was defined as producing a positive reaction in at least 3 ELISAS for PR3-ANC A, or at least 4 ELISAs for MPO-AN CA, respectively. Sensitivity was calculated from the proportion of PR3- or M PO-ANCA positive sera that were identified as positive in a given assay; and specificity was the proportion of PR3- or MPO-AN CA negative sera that were negative in a given assay.

Comparison of binding of individual sera in different ELISAs There are no standardised binding units for PR3- or M PO-ANCA , and most assays (except the INOVA and in-house ELISAs) did not provide criteria for low, medium or strong binding. However, the number of sera that bound at values greater then the maximum provided standard were compared.

ELISA for PR3- and MPO-ANCA Sera were tested for PR3-ANC A using 4 commercial and 1 in-house ELISA; and for M PO-ANCA , using 5 commercial and an in-house ELISA. The commercial ELISAs were performed by the same 2 scientists in the one laboratory; and the in-house ELISAs were performed elsewhere by the scientist who had developed them. The PR 3- and MPO used in the Shield, IBL and ORGentec assays were described as purified or highly purified, but the methods and any verification were not described. The PR3 used in the in-house ELISA (Wieslab) was HPL C-purified and free of contaminants; the M PO (Calbiochem) was not checked for contamination with lactoferrin. The PR3 and M PO in the in-house ELISAs were coated to plastic microtitre plates (Costar) at 0.7 mg/ml in PBS for 1 h at 37 °C, and then blocked with 1% human serum albumin (HSA, Sigma). All assay plates and reagents were stored at 4 °C, and assays were performed according to the manufacturers’ instructions and a protocol that had been developed for the in-house ELISAs (Tables 1 and 2). Positive samples were detected in the in-house ELISAs with alkaline phosphatase-conjugated antihuman IgG (1/500, Silenus), and the net absorbance of each sample was determined by

TABLE 1

RESULTS Correlation between IIF results and positivity in ELISAs for PR3- and M PO-ANCA (Table 3) Of the 26 sera with C-ANCA, 23 (89% ) were positive in at least three of the five assays for PR3-ANCA, and three (11%) were positive in at least four of the six assays for M POANCA. A further two sera were negative by IIF but positive in at least three assays for PR3-ANCA. Of the 26 sera with P-ANCA, 19 (73%) were positive in at least four assays for M PO -ANCA and one (4%) in at least three assays for PR 3-ANCA. The remaining six sera were negative for both PR 3 and M PO -ANCA. No serum that was negative by IIF, was positive in four assays for M PO ANCA.

Details of kits for PR3- and MPO-ANC A assays

Kit

Units

Ref. Range

Dynamic Range

Kit format

Tests

Shield Diagnostics (Dundee, Scotland)

12 by 8-well strips

PR 3 and M PO

U/ml

PR 3 neg < 2 MPO neg < 6

0±100

INOVA Diagnostics (San Diego, USA)

12 by 8-well strips plus breakaway

PR 3 and M PO

Units

PR 3 and M PO neg < 20 weak pos 20 ± 39 mod pos 40 ± 80 strong pos < 80

0± 80

IBL (Immuno Biological Laboratories) (Hamburg, Germany)

12 by 8-well strips plus breakaway

PR 3 and M PO

U/ml

PR 3 and M PO neg < 7

0±100

Euro-Diagnostica (M almo, Sweden)

12 by 8-well strips

PR 3 and M PO

EU (ELISA Units)

PR 3 and M PO neg < 10 borderline 10 ±15 pos > 15

ORGentec Diagnostics (M ainz, Germany)

12 by 8-well strips

MPO

U/ml

PR 3 and M PO neg < 25

0± 200

In-house

96-well plate

PR 3 and M PO

%

PR 3 < 22% MPO < 17%

0±100

10 ±1000

40

Pathology (1999), 31, February

POLLOC K et al.

TABLE 2

Assay protocols for individual kits

Serum dilution

Assay vol. ( mL)

Incubation temp ( °C)

Total Incubation (min)

Shield Diagnostics

100

100

18 ± 25

120

INOVA Diagnostics

100

100

Room temp.

90

Kit

Conjugate

Substrate

Stop

Alkaline phosphatase anti-IgG

PM P

NaOH/EDTA

HRP-anti IgG

TM B

H 2SO 4

OD (nm)

Data reduction

550 log/linear (540 ±565) OD sample

450

OD ELISA LOW IBL (Immuno Biological Laboratories) Euro-Dagnostica

ORGentec Diagnostica In-House

100

100

18 ± 24

70

PR 3 50 MPO 75

100 200

20 ± 25

180

100

100

18 ± 28

65

50

100

37

180

HRP-anti IgG

TM B

H 2SO 4

450

log/linear

Alkaline phosphatase anti-IgG

PNP

H 2SO 4*

405

log/linear

HRP-anti IgG

TM B

HCl

450

log/linear

Alkaline phosphatase anti-IgG

PNP

NaOH

405

log/linear

Sensitivities and specificities of PR3 and MPO-ANCA ELISAs (Table 4) The sensitivities of the ELISA s for PR 3-ANCA varied from 88 to 100%, and specificities from 91 to 100%. In the majority of cases the specificity of the PR 3- and M PO ANCA ELISA s could have been im proved without loss of sensitivity by slightly increasing the reference range. However, the converse was not true in the case of the poorer sensitivity of INOVA and ORGentec M PO -ANCA EL ISAs.

3 ELISA LOW

were two sera that bound strongly in three assays, four that bound in two and nine that bound in one only. The number of sera that bound at values greater than the maximum provided standard, varied from one to ten in the four M PO -ANCA ELISA s examined, and there were 16 with strong binding in the in-house ELISA . There was one serum that bound strongly in all four assays, one that bound in three, five that bound in two and seven that bound in one assay only.

DISCUSSION Comparison of binding of individual sera in different ELISAs (Figs. 1 and 2) The num ber of sera that bound at values greater than the maximum provided standard, varied from two to 15 in the three PR 3-ANCA ELISA s examined in this way, with 11 in the strong positive category in the in-house ELISA . There

TABLE 3 IIF pattern

We have compared the performance of various comm ercial and in-house ELISA s for PR3- and M PO -ANCA, and found that there was m ore concordance for different PR3ANCA assays than for the M PO -ANCA ELISA s. The highest sensitivity and specificity for both the PR3- and M PO-ANCA ELISA s were obtained with the IBL and

Correlation between IIF and ELISA for detection of ANCA n

Shield

INOVA

IBL

Euro

ORGentec

in-House

PR 3-ANCA positive by ELISA

+ in > /3 ELISAs

C-ANCA

26

22 (85% )

23 (89% )

23 (89% )

22 (85% )

18/24 (75% )

23 (89% )

P-ANCA

26

3 (12% )

0 (0% )

1 (4% )

1 (4% )

3/25 (12% )

1 (4% )

negative

23

3 (13% )

11 (4% )

2 (9% )

2 (9% )

4/20 (20% )

2 (9% )

MPO-AN CA positive by ELISA

+ in > /4 ELISAs

C-ANCA

26

6 (23% )

1 (4% )

4 (15% )

3 (12% )

1 (4% )

3/25 (12% )

3 (12% )

P-ANCA

26

23 (89% )

15 (58% )

21 (81% )

22 (85% )

12 (46% )

20/25 (80% )

19 (73% )

negative

23

2 (9% )

0 (0% )

2 (9% )

2 (9% )

0 (0% )

2/20 (10% )

0 (0% )

COM PARIS ON OF ELISAS FOR ANC A

TABLE 4

41

Sensitivities and specificities of ELISAs for detection of PR3- and M PO-ANCA Shield

INOVA

IBL

Euro-Diagnostics

ORGentec

In-House

Sensitivity (% )

25/26 (96%)

24/26 (92%)

26/26 (100%)

25/26 (96%)

21/24 (88%)

Specificity (%)

47/50 (94%)

50/50 (100%)

50/50 (100%)

50/50 (100%)

41/45 (91%)

Sensitivity (% )

22/22 (100%)

16/22 (73%)

22/22 (100%)

22/22 (100%)

13/22 (59%)

21/21 (100%)

Specificity (%)

45/54 (83%)

54/54 (100%)

48/54 (89%)

49/54 (91%)

51/51 (100%)

45/49 (92%)

PR 3-ANCA ELISA

MPO-AN CA ELISA

Eurodiagnostica assays. The in-house PR 3-ANCA ELISA performed slightly less well than the commercial assays, but the performance of the in-house MPO-ANCA assay was comparable or better. Diagnostic laboratories m ay prefer to establish the less expensive in-house assays. There are further savings possible since we have observed that there is no reduction in the magnitude of binding between two plates coated s ntially with the same PR3 or M PO . One of the concerns about EL ISAs for PR 3- and M PO ANCA has been the purity of the target antigens. The best preparations of PR 3 are isolated chromatographically using the method of Kao;19 and while M PO is comm ercially available, it may be contaminated by lactoferrin which is also occasionally recognised by ANCA.20 Details of the antigen purification methods or purity as demonstrated by SD S ± PA GE were available only for the PR3 that was used

in the in-house EL ISA. In other assays PR 3 and M PO were described as ªpurifiedº or ªhighly purifiedº but no details were given. Cloned PR3 and M PO have been used in EL ISAs,21±24 but these proteins lack some conformational epitopes and do not detect all of the corresponding autoantibodies.21,23 The origin of the PR 3 and M PO used in the commercial EL ISAs was not indicated, and it is possible that several assays have used m aterial from a comm on source, such as M PO from Calbiochem. This would make standardisation of the binding results easier. The sera examined in this study were all from patients suspected to have a systemic vasculitis, but there were insufficient clinical data to verify that ªANCA-positiveº sera were from patients with an active vasculitis. Furtherm ore, the sera were not chosen because they were ªdifficultº and presented problems in IIF or the EL ISAs. Thus the results described here m ay be superior to those in

Fig. 1 Comparison of binding in PR3-AN CA ELISAs. PR3, proteinase 3. Sera binding above maximum provided standard are indicated. *Sera not compared in these assays with maximum standard provided in kit. Values for in-house ELISA given only as negative or low, medium or high positives.

42

POLLOC K et al.

Pathology (1999), 31, February

Fig. 2 Comparison of binding in M PO-ANCA ELISAs. MPO, myeloperoxidase. Sera binding above maximum provided standard are indicated. *Sera not compared in these assays with maximum standard provided in kit. Values for in-house ELISA given only as negative or low, medium or high positives.

a routine laboratory where patients are less likely to have systemic vasculitis. All the EL ISAs tested in this study used reagents that detected only IgG ANCA, and not the occasional ANCA that are predominantly IgM .25,26 The presence of IgM ANCA may contribute to the disparity between IIF and EL ISA results that is sometimes noted when a polyspecific reagent is used in IIF but not in the ELISA s. None of the com mercial ELISA s determined non-specific ªstickinessº by subtracting the binding of individual sera to a plate coated with another antigen such as HSA . However this technique was used in both of the in-house ELISA s. The methods used to calculate the results for individual sera differed for each of the com mercial and in-house assays, and the units of positivity were arbitrary. There are no international standards for quantitating PR 3- or M PO ANCA antibody levels, partly because binding depends on the antibody avidity as well as concentration. Thus each comm ercial PR 3- and M PO-ANCA assay kit provided a maximum standard that varied from 80 (INOVA) to 1000 binding units (Eurodiagnostica). For these reasons it was not possible to compare the amount of binding of individual sera in different assays. However some sera that bound very strongly in one assay produced low levels of binding in another. The number of sera binding at levels greater than the m axim um standard varied too in different assays. This is surprising because the m axim um standard usually defines the upper limit of expected values. The variation in binding in different assays is important to note because the clinician probably expects uniformly strong binding in all patients with systemic vasculitis at presentation, and thereafter to be able to correlate the amount of binding with the response to treatment even with different assays. It should be noted that the degree of binding was more consistent with the PR3ANCA ELISA s than with the M PO -ANCA assays.

All of the assays examined in this study were easy to perform and used similar methodology. Except for the Eurodiagnostica kits, the EL ISAs for PR3- and M PO ANCA produced by the one company had interchangeable reagents and identical incubation times, so that both assays could be performed in parallel. Incubation times for each step and total assay times were similar for all assays, although screening EL ISAs now available can detect positivity within minutes. Eight well strips were available for all of the commercial assays, and breakaway single wells were available for the INOVA and IBL kits. Reproducibility, in general, within and between assays for PR 3- and MPO -ANCA antibodies was not a problem. Intra- and inter-assay coefficients of variation provided with the Shield, IBL and in-house ELISA s were all described as less than 15%. The Eurodiagnostica assays were also of ªacceptable reproducibilityº. M any laboratories perform an ELISA for PR3-ANCA on sera that produce a C-ANCA pattern; and M PO -ANCA EL ISA on sera with a P-ANCA pattern. However, we have confirmed here that sera associated with C-ANCA can still be directed against MPO, 27 and those that produce a P-ANCA pattern can be directed against PR3. In addition, a confounding factor in ANCA IIF interpretation is the presence of additional autoantibodies reacting with other cytoplasmic or nuclear com ponents. For exam ple, in this study the three C-ANCA positive sera that bound to M PO but not to PR3 all produced cytoplasmic fluorescence of HEp2 cells. Since there are further ANCA specificities such as bactericidal/permeability-increasing protein (BPI) 28 and others yet to be identified, sera with ANCA demonstrated by IIF but non-reactive in the ELISA s for PR3- and M PO ANCA should probably still be reported as ANCA positive. Some patients with active Wegener’s granulomatosis have

COM PARIS ON OF ELISAS FOR ANC A

shown such reactivity. Furthermore, some sera that are negative by IIF can still have PR 3-ANCA by antigenspecific EL ISA (and probably likewise for M PO -ANCA). These argum ents suggest that all sera from patients suspected of having Wegener’s granulomatosis or m icroscopic polyangiitis should be tested in both PR 3- and M PO ANCA ELISA s in addition to IIF. Footnotes i. Eurodiagnostica now indicate in the package insert that the inter- and intra-assay coefficients of their ELISAs are < 15%. ii. ORGentec has now modified and improved their assays.

A CKNOWLEDGEMENTS The authors would like to thank the following suppliers of microscope slides and ELISA kits that were used in the study; M urex Diagnostics Australia Pty Ltd; Immunodiagnostics; ALS Pty Ltd; Sanofi Diagnostics Pasteur Pty Ltd; and Carter-Wallace Australia Pty Ltd. Address for correspondence: J.S., University Department of M edicine, Austin and Repatriation M edical Centre, Austin Campus, Heidelberg, Vic 3084, Australia.

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11. Scott DGI, Sharrack BR, Webb FWS. Antineutrophil cytoplasmic antibodies and rheumatoid arthritis. Clin Exp Rheumatol 1990; 8: F24 (Suppl. 4). 12. Nassberger L, Sjohlm AG, Jonsson H, Sturfelt G, Akesson A. Autoantibodies against neutrophil cytoplasm components in systemic lupus erythematosus and in hydralazine-induced lupus. Clin Exp Immunol 1990; 81: 380 ±3. 13. Saxon A, Shanahan F, Landers C, Ganz T, Targan S. A distinct subset of antineutrophil cytoplasmic antibodies is associated with inflammatory bowel disease. J Allergy Clin Immunol 1990; 86: 202 ±10. 14. Snook JA, Chapman RW, Fleming K, Jewell DP. Anti-neutrophil nuclear antibody in ulcerative colitis, Crohn’s disease and primary sclerosing cholangitis. C lin Exp Immunol 1989; 76: 30 ± 3. 15. Roozendaal C, Broekroelofs J, Horst G, Kleibeuker JH, Nelis GF, Limburg PC, et al. Prevalence and characterisation of ANCA in inflammatory bowel disease. Clin Exp Immunol 1995; 101 (Suppl): 70A. 16. Schellong SM , Boker KHW, Haubitz M , Manns M, Alexander K. Prospective study of pANCA in inflammatory bowel disease. Clin Exp Immunol 1995; 101 (Suppl): 45A. 17. Nolle B , Specks U, Ludemann J, Rohrbach M S, De Remee R A, Gross WL. Anticytoplasmic autoantibodies: their immunodiagnostic value in Wegener’s granulomatosis. Ann Intern M ed 1989; 111: 28 ±40. 18. Cohen-Tervaert JW, Huitema M G, Hene RJ, Sluiter WJ, The TH, van der Hem GK, et al. Prevention of relapses in Wegener’s granulomatosis by treatment based on antineutrophil cytoplasm ic antibody titre. Lancet 1990; 336: 709 ±11. 19. Kao RC, Wehner NG, Skubitz KM , Gray BH, Hoidal JR. Proteinase 3. A distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J Clin Invest 1988; 82: 1963 ±73. 20. Audrain M AP, Beranger TAR, Lockwood CM, Esnault VLM . High immunoreactivity of lactoferrin contaminating commercially purified myeloperoxidase. J Immunol Methods 1994; 176: 23 ±31. 21. Bini P, Gabay JE, Yeitel A, Malchior M , Zhou J-L , Elkon KB. Antineutrophil cytoplasmic autoantibodies in Wegener’s granulomatosis recognise conformational epitope(s) on proteinase 3. J Immunol 1992; 149: 1409 ±15. 22. Campanelli D, M elchior M, Fu Y, Nakata M , Shuman H, Nathan C, et al. Cloning of cDNA for proteinase 3: a serine protease, antibiotic, and autoantigen from human neutrophils. J Exp Med 1990; 172: 1709 ±15. 23. Short AK, Lockwood C M, Bollen A, Moguilevsky N. Neutrophil and recombinant myeloperoxidase as antigens in ANCA positive systemic vasculitis. Clin Exp Immunol 1995; 102: 106 ±11. 24. Falk RJ, Becker M , Terrell R, Jennette JC. Antimyeloperoxidase autoantibodies react with native but not with denatured myeloperoxidase. C lin Exp Immunol 1992; 89: 274 ± 8. 25. Jayne DRW, Jones SJ, Severn A, Shaunak S, M urphy J, Lockwood CM . Severe pulmonary haemorrhage and systemic vasculitis associated with circulating antineutrophil cytoplasm ic antibodies of IgM class only. Clin Nephrol 1989; 32: 101± 6. 26. Esnault VLM , Solimani B, Keogan M T, Brownlee AA, Jayne DRW, Lockwood CM , Association of IgM with IgG ANCA in patients presenting with pulmonary haemorrhage. Kidney Int 1992; 41: 1304 ±10. 27. Segelmark M , Baslund B, Wieslander J. Some patients with antimyeloperoxidase antibodies have a cANC A pattern. Clin Exp Immunol 1994; 96: 458 ±65. 28. Zhao M H, Jones SJ, Lockwood CM . Bactericidal/permeabilityincreasing protein (BPI) is an important antigen for antineutrophil cytoplasmic autoantibodies (ANCA) in vasculitis. Clin Exp Immunol 1995; 99: 49 ± 56.