Comparison of eight commercial kits for quantitation of antineutrophil cytoplasmic antibodies (ANCA)1

Journal of Immunological Methods 208 Ž1997. 203–211

Comparison of eight commercial kits for quantitation of antineutrophil cytoplasmic antibodies ž ANCA/ 1 Guochun Wang, Elena Csernok ) , Kirsten de Groot, Wolfgang L. Gross Department of Rheumatology, UniÕersity of Lubeck, Lubeckr Rheumaklinik Bad Bramstedt GmbH, P.O. Box 1448, 24576 Bad Bramstedt, ¨ ¨ Germany Received 3 February 1997; revised 21 April 1997; accepted 2 September 1997

Abstract Antineutrophil cytoplasmic antibodies ŽANCA. are used as diagnostic markers for systemic vasculitis. However,the specificity and sensitivity of ANCA detection differs from centre to centre due in large part to variations in methodology. We compared 8 commercial ELISA kits and an in-house method ŽHM. for their specificity and sensitivity in detecting ANCA against proteinase 3 ŽPR3-ANCA, 7 kits. and meyloperoxidse ŽMPO-ANCA, 8 kits.. Sera from 5 patients with systemic lupus erythematosus ŽSLE., 28 with Wegener’s granulomatosis ŽWG., 22 with microscopic polyangiitis ŽMPA., 5 with idiopathic rapidly progressive glomerulonephritis ŽRPGN., and 5 healthy controls were examined by both the indirect immunofluorescence technique ŽIFT. and the ELISA kits. Sera from healthy controls and patients with SLE or cANCAnegative WG were shown to be PR3-ANCA negative by all 7 PR3-ANCA kits. In 25 cANCA-positive sera from WG patients, PR3-ANCA positivity ranged from 44% to 84%. An absolute concordance among the 7 kits was noted in 56% of the cANCA-positive samples. The PR3-ANCA levels in 5 of the 7 kits correlated with the cANCA titers in IFT. Sera from the healthy controls and 4 out of the 5 SLE and pANCA-negative patients were found to be MPO-ANCA negative in all 8 MPO-ANCA kits. In 20 pANCA-positive sera, MPO-ANCA positivity ranged from 25% to 75%. Thirty-five percent of MPO-ANCA-positive sera were confirmed by capture ELISA, immunoblot and inhibition assay. The concordance rate was only 30% among pANCA-positive sera in the 8 MPO-ANCA kits. No significant correlation was observed between pANCA titers and MPO-ANCA levels. The HM showed that 65% of cANCA-positive sera were PR3-ANCA positive, and 45% of pANCA-positive sera were MPO-ANCA positive. Our results indicate that the sensitivities and specificities for ANCA detection differ significantly among the commercial kits tested and underline the necessity of establishing uniform international standards for ANCA ELISA procedures in order to permit more reliable interpretation and comparison of data. q 1997 Elsevier Science B.V. Keywords: ANCA; PR3-ANCA; MPO-ANCA; ELISA; Indirect immunofluorescence

Abbreviations: ELISA, enzyme-linked immunosorbant assay; IFT, indirect immunofluorescence; ANCA, antineutrophil cytoplasmic antibodies; PR3, proteinase 3; MPO, myeloperoxidase; HLE, neutrophil elastase; LF, lactoferrin; WG, Wegener’s granulomatosis; MPA, microscopic polyangiitis; SLE, systemic lupus erythematosus ) Corresponding author. Tel.: q49-4192-902295; fax: q49-4192-902381. 1 Permission for publishing the names of the ELISA kits mentioned in this paper has been obtained from the manufacturers. This study was carried out in May–December 1996; since then modifications andror improvements may have been introduced in some kits. 0022-1759r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 7 . 0 0 1 5 4 - 3

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1. Introduction

2. Materials and methods

Antineutrophil cytoplasmic autoantibodies ŽANCA. constitute a class of autoantibodies directed against various cytoplasmic constituents of neutrophils and monocytes. They are of immunodiagnostic significance in Wegener’s granulomatosis ŽWG. and microscopic polyangiitis ŽMPA. ŽGross and Csernok, 1995.. The indirect immunofluorescence technique ŽIFT. using ethanol-fixed neutrophils is a common screening method for ANCA. The two major IFT patterns on ethanol-fixed neutrophils are called cANCA and pANCA. cANCA is strongly associated with antibodies against proteinase 3 ŽPR3-ANCA.. Between 80% and 90% of cANCApositive samples are positive for PR3-ANCA and about 90% of PR3-ANCA-positive samples are positive for cANCA in IFT ŽWieslander, 1991.. pANCA is associated with antibodies against a number of enzymes, namely myeloperoxidase MPO., lactoferrin ŽLF., human leukocyte elastase ŽHLE., cathepsin-G ŽCG., etc. ŽFalk and Jennette, 1988; Coremans et al., 1992; Cohen Tervaert et al., 1993.. ANCA testing should include antigen-specific assays whenever a positive ANCA result is obtained by IFT. This is especially important because the use of standardized IFT methods for a number of years has not resulted in uniform methods for IFT among different laboratories ŽHagen et al., 1993.. In addition, the interpretation of IFT patterns requires a certain level of experience and expertise and can be biased by a high rate of false positivity due to observer error and the variability of substrate cells. Moreover, the diagnostic value of the IFT test for ANCA detection can be greatly increased if supplemented by antigen specific ELISAs ŽHagen et al., in press.. The growing clinical interest in the determination of ANCA has led many manufacturers to develop antigen-specific ELISAs for assessing these autoantibodies in serum and other biological fluids. However, there have been the literature contains no studies comparing different commercial kits in terms from their specificity and sensitivity. For this reason, we investigated 7 PR3-ANCA and 8 MPO-ANCA ELISA kits of different manufacturers as well as an in-house method and compared their results with the levels of IFT-ANCA titers.

2.1. Patients Five healthy controls, 5 patients with SLE, 28 patients with WG, 22 patients with MPA and 5 patients with rapidly progressive glomerulonephritis ŽRPGN. were studied. WG was diagnosed according to the American College of Rheumatology ŽACR. 1990 classification of WG as well as the 1992 Chapel Hill consensus conference definition of WG ŽLeavitt et al., 1990; Jennette et al., 1994.. MPA was diagnosed applying the 1992 Chapel Hill consensus conference definition of MPA ŽJennette et al., 1994.. SLE patients fulfilled the ACR criteria ŽTan et al., 1982.. Serum samples were kept at room temperature ŽRT. for a maximum of 3 h prior to centrifugation Ž1000 = g for 30 min at RT. and stored at y208C until analyzed. The sera were tested in duplicate in all ELISAs. 2.2. Detection of ANCA by IFT ANCA were detected by IFT according to the method described by Nolle ¨ et al. Ž1989. and modified by the European ANCA study group ŽHagen et al., 1993.. Briefly, air-dried, ethanol-fixed cytospin preparations of purified neutrophils were incubated with the test serum at a dilution of 1r2 in phosphate buffer solution ŽPBS.. Antibody binding was detected with fluorescein isothiocyanate-conjugated FŽabX . 2 fragments of rabbit antihuman IgG ŽDako, Copenhagen.. Any positive sample was titered to endpoint. For differentiation of pANCA from antinuclear antibodies ŽANA., samples with perinuclear fluorescence on ethanol-fixed neutrophils were additionally tested on formalin-fixed neutrophils. These were prepared by incubation of cytospin preparations of purified neutrophils with 0.5% formalin at RT for 5 min. Perinuclear fluorescence on the former was taken to indicate the presence of pANCA only if it was associated with exclusively cytoplasmic fluorescence on formalin-fixed cells. If this procedure did not yield a clear-cut differentiation, the sera were further tested on methanol-fixed HEp-2 cells. Results were accepted as pANCA-positive if the neutrophil

G. Wang et al.r Journal of Immunological Methods 208 (1997) 203–211

titer was at least 4-fold greater than that of HEp-2 cells.

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for the detection of PR3-ANCA and MPO-ANCA. The results were graded positive or negative according to the scale provided by each manufacturer. For the purposes of this study, each company was assigned a letter ŽA, B, C, D, E, F, G or H. and its kits are referred to by that letter in the Sections 3 and 4 and tables. The H kit was designed only for MPOANCA detection. The technical data of these kits are presented in Table 1. HM was developed for measurement of PR3ANCA and MPO-ANCA in accordance with standardized European guidelines as described previously ŽHagen et al., 1993. and incorporated some recent modifications. Briefly, highly purified PR3 and MPO diluted to 2 m grml in 0.1 M carbonate buffer ŽpH 9.6. were coated overnight at 48C on microtiter plates ŽNunc InterMed.. After washing with PBS q 0.05% Tween 20, the plates were incubated with blocking buffer Ž2% casein in PBS.. Test sera diluted 1r50 and standard controls were added. Bound antibodies were detected by incubation with AKP-conjugated goat antihuman IgG ŽSigma, St. Louis., followed by development with p-nitrophenyl phosphate. Absorbance values were read at 405 nm. The cut-off value was 20 units.

2.3. PR3-ANCA and MPO-ANCA assays 2.3.1. Assessment of reactiÕity of coating antigens with monoclonal antibodies (MoAbs) The reactivity of the following MoAbs with the coating antigen in each ELISA kit was detected as described below Section 2.3.2: three mouse MoAbs against PR3: WGM2 Žfrom our own laboratory, diluted 1r10 for testing., 4A3 Žthe kind gift of Dr. J. Wieslander, Statens Seruminstitut, Copenhagen, used at a 1r2 dilution., and CLB-ANCA12.8 Žfrom CLB, Amsterdam, diluted 1r100 for testing.; one mouse MoAb anti-MPO ŽDAKO, Glostrup, diluted 1r500 for testing., one mouse MoAb anti-HLE ŽDAKO, Denmark, tested at a 1:500 dilution. and one mouse MoAb anti-LF ŽPAESEL, Hanau, used at a 1r500 dilution.. Bound antibodies were detected by incubation with alkaline phosphatase ŽAKP.-conjugated goat antimouse IgG ŽSigma, St. Louis. for 1 h at RT, followed by development with p-nitrophenyl phosphate. The absorbance ŽOD. was read at 405 nm. 2.3.2. Detection of PR3-ANCA and MPO-ANCA in sera Serum levels of PR3-ANCA and MPO-ANCA were measured according to the manufacturers instructions using the ELISA kits of eight companies. In addition, an in-house method ŽHM. was employed

2.4. Detection of ANCA directed to other target antigens by ELISA ANCAs directed to other target antigens ŽLF, HLE, CG, bactericidalrpermeability increasing pro-

Table 1 Technical data on the commercial ELISA kits for PR3-ANCA and MPO-ANCA as given by the manufacturers Manufacturer

Serum dilution

Calibration range ŽUrml.

Incubation time

Enzymea

Substrate

l reading

Cut-off value ŽUrml.

A B C D E F G H

1r50 1r50 1r100 1r100 1r80 1r100 1r100 1r200

10–80 10–200 0–100 6.3–100 0–320 2–200 0–100 29.4–940

2 h q 1 h q 15 min 30 min q 30 min q 10 min 30 min q 30 min q 10 min 30 min q 15 min q 15 min 1 hq1 hq1 h 30 min q 30 min q 15 min 1 h q 30 min q 30 min 30 min q 30 min q 10 min

HRP HRP HRP HRP AKP HRP AKP HRP

TMB TMB TMB TMB pNPP TMB PLMP TMB

450 450 450 450 405 450 550 450

20 10 15 10 20 20 PR3-ANCA: 2 MPO-ANCA: 6 56.4

a

X

X

HRP: horseradish peroxidase, AKP: alkaline phosphatase, TMB: 3,3 ,5,5 -tetra-methyl-benzidine, pNPP: p-nitrophenyl phosphate, PLMP: phenolphthalein mono-phosphate.

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G. Wang et al.r Journal of Immunological Methods 208 (1997) 203–211

tein wBPIx and lysozyme wLZx. were measured by the in-house method as described in Section 2.3.2. 2.5. Capture ELISA A capture ELISA was developed using the antiMPO MoAb as capture antibody to test the samples for the presence of MPO-ANCA. Briefly, the antiMPO MoAb and irrelevant control IgG were coated onto 96 well microtiter plates ŽNunc InterMed. at a concentration of 2 m grml in 0.1 M carbonate buffer ŽpH 9.6. and incubated overnight at 48C. After the plates were washed, they were incubated with MPO at a concentration of 10 m grml for 1 h at RT. The in-house ELISA procedure described in Section 2.3.2 was followed. 2.6. CompetitiÕe inhibition ELISA assay Competitive inhibition ELISA was performed to determine the specificity of MPO-ANCA activity.

Purified MPO Ž10 m grml. was incubated with equal volumes of various dilutions of MPO-ANCA-positive sera Ž1r50–1r6400. at RT for 2 h and then centrifuged at 10,000 rpm for 20 min. The supernatants were collected and tested by ELISA as described in Section 2.3.2 Žin-house method.. Controls consisted of aliquots of the same sera preincubated with albumin bovine fraction V ŽServa, Heidelberg.. The inhibition was considered significant when there was a reduction of the absorbance values of at least 30% compared to controls. Inhibition% s Absorbance value without inhibition–Absorbance value with inhibition Absorbance value without inhibition

2.7. Immunoblotting Immunoblotting was performed to confirm the positive ELISA results of MPO-ANCA. MPO was electrophoresed Ž1 m grlane. under nonreducing conditions on a 12.5% SDS-PAGE gel. After elec-

Fig. 1. Reactivity of monoclonal antibodies with coating antigen PR3 as detected by the 7 PR3-ANCA ELISA kits. Three MoAbs against PR3 ŽWGM2, 4A3 and CLB-ANCA 12.8. and MoAbs against MPO, HLE and LF were used.

G. Wang et al.r Journal of Immunological Methods 208 (1997) 203–211

trophoresis the samples were transferred to a nitrocellulose membrane ŽSchleicher and Schuell, Germany. and blocked with 5% milk powder in Tris buffered saline ŽTBS., pH 7.4, for 1 h at RT. Parallel to this, total protein was demonstrated with Amido

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black stain for 5 min. The strips were then incubated with sera diluted 1r20 with TBS q 0.05% Tween 20 ŽTBS-T., murine MoAb against MPO diluted 1r100, or murine IgG isotype diluted 1r100 for 1 h at RT. After washing, incubation was performed with sec-

Table 2 Serum levels of cANCA titer in IFT and PR3-ANCA in values obtained using seven commercial ELISA kits ŽA–G. and an in-house assay ŽHM. Serum No.

Diagnosis

cANCA titer

PR3-ANCA Žunitrml. A

B

C

D

E

F

G

HM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

HC HC HC HC HC SLE SLE SLE SLE SLE WG WG WG WG WG WG WG WG WG WG WG WG WG WG WG RPGN WG WG WG WG WG WG WG WG WG WG WG WG RPGN WG

y y y y y y y y y y y y y y y 1:8 1:8 1:16 1:16 1:16 1:32 1:32 1:32 1:64 1:64 1:128 1:128 1:128 1:128 1:256 1:512 1:512 1:512 1:512 1:1024 1:1024 1:1024 1:1024 1:1024 1:2408

1.0 0.5 0.1 0.0 0.1 0.5 0.0 0.1 0.3 0.4 0.4 1.1 4.4 0.8 0.2 2.3 18.0 1.5 50.9 22.8 78.7 24.1 10.5 11.0 12.4 ) 80 ) 80 17.0 72.2 4.3 49.1 8.0 ) 80 ) 80 48.7 12.9 ) 80 ) 80 ) 80 ) 80

0.4 0.5 0.4 0.5 0.2 3.2 0.2 4.3 1.0 0.5 0.4 0.5 1.8 0.7 0.5 0.8 5.2 0.6 1.6 4.6 14.6 1.4 2.1 2.3 0.4 95.6 ) 200 7.9 16.4 2.6 24.6 1.2 28.2 26.2 9.5 1.6 49.3 33.8 23.4 87.7

2.7 0.8 0.0 0.0 0.7 7.2 0.3 0.0 1.7 1.9 0.0 0.4 2.3 0.1 0.0 2.6 21.5 3.5 8.1 9.6 44.0 1.8 3.2 9.4 2.9 ) 100 ) 100 1.3 41.3 0.0 ) 100 27.9 ) 100 98.2 30.5 55.9 ) 100 ) 100 86.3 ) 100

0.14 0.1 0.0 0.0 0.1 0.2 0.1 0.1 1.4 0.2 0.0 0.2 0.6 0.1 0.0 0.4 67.9 0.1 2.5 7.1 31.7 0.5 0.2 5.2 0.3 88.1 ) 100 1.0 17.7 2.0 49.3 3.6 ) 100 ) 100 39.0 2.1 90.8 ) 100 ) 100 ) 100

0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 2.5 0.0 0.0 1.5 48.5 8.5 17.9 27.5 117.0 16.5 8.0 13.0 5.0 209.0 ) 320 45.0 51.5 23.0 61.0 11.0 249.5 121.5 29.5 27.5 ) 320 ) 320 226.0 ) 320

9.3 6.7 3.0 2.6 1.1 1.4 0.0 3.6 3.4 1.1 3.4 1.9 1.7 4.5 3.5 1.0 15.0 0.0 1.7 8.4 37.1 2.6 1.9 7.4 1.7 199 ) 200 9.7 46.3 4.5 49.0 5.7 62.5 93.3 10.7 4.8 90.5 43.5 78.6 128

0.9 0.4 0.2 0.4 0.3 1.3 0.2 0.3 0.7 0.7 0.3 0.2 1.5 0.2 0.4 1.4 42.8 0.4 0.8 46.2 74.2 2.5 2.3 3.2 0.9 98.0 ) 100 12.5 49.9 4.8 52.5 3.9 97.9 89.5 38.4 21.6 91.1 80.7 89.9 ) 100

0.9 0.5 0.0 0.0 0.0 0.3 0.1 0.1 0.2 0.2 0.3 0.8 2.3 0.7 0.1 2.0 13.2 1.1 49.2 24.2 79.3 26.7 9.7 9.6 10.5 ) 80 ) 80 15.4 ) 80 6.6 52.9 6.8 ) 80 ) 80 55.3 10.9 ) 80 ) 80 ) 80 ) 80

HC: healthy controls, SLE: systemic lupus erythematosus, WG: Wegener’s granulomatosis, RPGN: rapidly progressive glomerulonephritis, y: negative.

G. Wang et al.r Journal of Immunological Methods 208 (1997) 203–211

208

ond antibody ŽAKP-conjugated antimouse IgG or AKP-conjugated antihuman IgG diluted 1r1000 in TBS-T. for 1 h at RT. After washing with TBS-T,

the assay was developed by incubation with nitroblue tetrasodiumrbromochloroindoly phosphate for 20 min at RT.

Table 3 Serum levels of pANCA titer in IFT and MPO-ANCA values obtained using eight commercial ELISA kits ŽA–H. and an in-house assay ŽHM. Serum No.

Diagnosis

pANCA titer

MPO-ANCA Žunitrml. A

B

C

D

E

F

G

H

HM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

HC HC HC HC HC SLE SLE SLE SLE SLE MPA MPA MPA MPA MPA WG WG WG RPGN RPGN MPA MPA MPA MPA MPA RPGN RPGN MPA MPA RPGN MPA MPA MPA MPA MPA MPA MPA MPA MPA MPA

y y y y y y y y y y y y y y y y y y y y 1:8 1:16 1:32 1:32 1:32 1:32 1:32 1:64 1:64 1:64 1:128 1:128 1:256 1:256 1:256 1:256 1:256 1:256 1:512 1:512

0.2 0.3 0.1 0.1 0.2 1.6 0.2 0.1 0.3 0.6 0.4 1.6 2.2 0.7 0.5 0.4 0.4 0.3 1.3 0.7 1.0 1.1 0.3 9.4 5.1 11.1 44.0 1.8 5.7 4.3 3.6 2.0 1.5 3.8 6.8 ) 80 21.4 50.0 2.4 ) 80

3.6 3.6 0.2 2.2 0.1 12.5 0.0 5.0 0.6 0.8 0.0 3.3 2.1 7.4 0.2 0.7 1.1 0.7 1.2 1.0 0.4 3.4 0.0 9.2 24.0 9.2 179 1.1 17.1 11.3 14.9 19.9 1.5 45.6 24.7 125 47.7 141 11.0 192

3.4 3.1 0.2 0.1 3.0 20.0 0.8 0.4 2.7 2.5 0.0 0.8 2.4 0.7 0.0 1.8 1.4 1.4 2.1 1.3 1.7 5.4 0.0 5.2 35.9 1.4 ) 100 9.6 42.0 9,7 30.1 26.4 6.7 7.4 57.8 ) 100 ) 100 ) 100 16.2 ) 100

0.3 0.3 0.3 0.4 0.3 14.7 0.4 0.4 0.2 0.3 0.2 3.6 0.8 3.1 0.6 0.5 0.6 0.4 0.6 0.5 2.1 3.9 0.3 3.6 78.3 5.8 ) 100 8.6 11.8 9.1 44.9 16.6 8.9 9.0 45.7 60.2 ) 100 ) 100 10.8 ) 100

1.4 0.7 0.3 1.0 0.6 69.3 0.8 0.7 0.7 2.3 0.5 5.2 4.7 9.3 2.7 2.8 8.4 3.5 4.1 2.3 11.1 44.6 1.1 8.5 131.5 8.9 ) 320 13.9 30.4 23.6 154.9 43.8 17.9 20.3 58.5 127.4 258.0 229.6 16.7 251.9

12.1 0.4 0.0 3.2 5.2 117.4 2.9 7.5 5.9 0.0 4.6 4.8 0.7 5.8 3.4 0.0 1.3 5.5 1.6 2.0 4.0 ) 200 3.2 22.4 140.4 20.9 ) 200 12.8 108.1 16.0 124.8 70.1 18.4 29.1 81.0 ) 200 ) 200 ) 200 39.5 ) 200

0.2 1.1 0.5 0.2 0.7 17.8 0.6 1.9 1.9 1.3 0.0 1.6 3.0 4.0 2.1 1.2 0.6 0.0 3.5 1.9 1.6 5.6 1.3 6.8 48.8 4.0 ) 100 3.8 23.8 2.5 18.2 12.0 4.7 4.8 22.9 28.8 76.2 ) 100 9.0 ) 100

35.7 0.0 15.5 30.8 11.0 77.8 23.7 15.0 34.5 33.2 22.4 50.1 21.1 32.1 30.5 10.3 21.0 27.5 50.0 34.4 29.9 110.8 16.3 42.5 317.5 0.0 ) 940 61.2 154.6 137.3 148.1 236.9 79.3 45.8 275.1 331.2 791.0 ) 940 96.0 ) 940

1.2 0.9 0.8 0.8 2.4 3.3 1.8 0.4 0.9 1.2 2.6 2.5 3.0 1.1 1.2 0.9 0.7 0.6 2.2 1.8 4.5 3.8 1.5 9.2 8.7 10.9 45.7 2.2 9.8 17.6 55.7 4.4 33.6 10.5 33.4 77.8 32.6 66.7 24.8 90.2

HC: healthy controls, SLE: systemic lupus erythematosus, WG: Wegener’s granulomatosis, RPGN: rapidly progressive glomerulonephritis, MPA: microscopic polyangiitis, y: negative.

G. Wang et al.r Journal of Immunological Methods 208 (1997) 203–211

3. Results 3.1. PR3-ANCA ELISA kits 3.1.1. ReactiÕity of MoAbs with coating antigen PR3 The reactivities of the MoAbs WGM2, 4A3 and CLB-ANCA12.8 with coating antigens were observed in each PR3-ANCA kit. The strongest reactivities of the three MoAbs with coating antigens of the PR3-ANCA kits were all obtained using the E kit, all of the lowest reactivities with the C kit. No reactivity was found in these wells by adding the anti-MPO, anti-HLE and anti-LF MoAbs ŽFig. 1.. 3.1.2. The specificity and sensitiÕity of PR3-ANCA detection Five healthy controls, 5 SLE patients with high levels of ANA Žall G 1:128. as disease control, 5 cANCA negative WG patients, 25 cANCA-positive patients Ž23 WG and 2 RPGN. were selected for PR3-ANCA ELISAs. The individual cANCA titers and PR3-ANCA values obtained from each kit are shown in Table 2. Sera from healthy controls and SLE and cANCA-negative patients were shown to be PR3ANCA-negative by all 7 PR3-ANCA kits and HM ŽTable 2.. Of the 25 cANCA-positive sera, 11 Ž44%. were found to be PR3-ANCA positive by the B and F kits, 13 Ž52%. positive by D, 15 Ž60%. positive by A, C and HM, 17 Ž68%. positive by E, and 21 Ž84%. positive by G kit ŽTable 2.. 3.1.3. Concordance among the different kits Absolute concordance among the seven PR3ANCA kits was noted in 14 out of 25 cANCA-positive samples Ž56%; 11 positive and 3 negative.. 3.1.4. Correlation between PR3-ANCA ELISA Õalues and IFT titers of cANCA Serum levels of cANCA titers in IFT correlated significantly with the PR3-ANCA levels as determined by A Ž r s 0.44, p - 0.05., C Ž r s 0.56, p 0.01., D Ž r s 0.41, p - 0.05., E Ž r s 0.44, p 0.05., G Ž r s 0.45, p - 0.05. and the in-house method Žr s 0.44, p - 0.05., but not with the levels

209

as determined by B Ž r s 0.28, p ) 0.05. or F Ž r s 0.31, p ) 0.05.. 3.2. MPO-ANCA ELISA kits 3.2.1. ReactiÕity of MoAbs with coating antigen MPO The coating antigens of all MPO-ANCA ELISA kits reacted with the anti-MPO MoAb, but did not react with the anti-PR3 ŽWGM2, 4A3 and CLBANCA12.8., anti-HLE or anti-LF MoAbs. The highest reactivity of the anti-MPO MoAbs with coating antigens of the commercial MPO-ANCA kits was exhibited by G Ž A 405 s 0.819., followed in decreasing order by F Ž A 405 s 0.709., C Ž A 405 s 0.595., E Ž A 405 s 0.503., H Ž A 405 s 0.418., D Ž A 405 s 0.410. and A Ž A 405 s 0.316.. The lowest absorbance was 0.289 obtained from B. 3.2.2. The specificity and sensitiÕity of MPO-ANCA detection Five healthy controls, 5 SLE and 10 pANCAnegative patients Ž5 MPA, 3 WG and 2 RPGN., 20 pANCA-positive patients Ž17 MPA and 3 RPGN. were selected for MPO-ANCA ELISAs. Table 3 shows the individual data on pANCA titers and the MPO-ANCA values obtained by each kit. Sera from 5 healthy controls, 10 pANCA-negative patients and 4 out of 5 SLE patients were shown to be MPO-ANCA negative by all eight MPO-ANCA ELISA kits and HM ŽTable 3.. One serum from an SLE patient with ANA Ž1:640., anti-LF Ž27.3 Urml. and antihistone Ž29 Urml, normal value - 20 Urml. reactivities was shown to be MPO-ANCA positive by all of the kits except A and HM. However, this serum was found to be MPO-ANCA negative in capture ELISA and by immunoblot. In the patients with pANCA positivity Ž n s 20., five sera Ž25%. were found to be MPO-ANCA positive by A, 9 Ž45%. by HM, 11 each Ž55%. by C and D, 12 Ž60%. by G, 13 each Ž65%. by B and E, and 15 each Ž75%. by F and H. However, 7 of these 20 sera Ž35%. were MPO-ANCA positive in capture ELISA, immunoblot and inhibition assay Ždata not shown.. 3.2.3. Concordance among the different kits Absolute concordance among the eight MPOANCA kits occurred with 6 out of the 20 pANCApositive samples Ž30%; 5 positive and 1 negative..

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3.2.4. Correlation between MPO-ANCA ELISA and pANCA titers Serum levels of MPO-ANCA obtained from each kit were also examined by IFT for possible correlations with the pANCA titers. However, no significant correlation was found between pANCA titers and MPO-ANCA levels Ždata not shown..

4. Discussion In recent years a growing number of reports have dealt with diseases related to the presence of PR3ANCA and MPO-ANCA in serum. One puzzling fact that emerges from the literature is the variability of the prevalence of these autoantibodies in diseases known to be associated with ANCA ŽDavenport et al., 1994; Rao et al., 1995.. Early studies showed that the sensitivities and specificities of cANCA ŽPR3-ANCA. for active WG were greater than 90% ŽVan der Woude et al., 1985; Nolle ¨ et al., 1989; Venning et al., 1990.. The sensitivity of MPO-ANCA for MPA was about 45% ŽKallenberg, 1996., whereas a recent European multicenter study reported the sensitivity of PR3-ANCA for WG to be 69% with a specificity of 87%; the sensitivity of MPO-ANCA for MPA was 58% ŽHagen et al., in press.. Differences in the patient and control samples account for some of the discrepancies, as do differences in methodology. Although many commercial ELISA kits are now available for the detection of PR3rMPO-ANCA, there is no uniform international methodology. The present study confirms that marked variations in the sensitivity for ANCA detection exist among the commercial ELISA kits tested. For PR3-ANCA detection, our results show that the specificities of the 7 commercial kits as well as our HM do not differ. With regard to sensitivity, between 44% and 84% of the 25 cANCA-positive WG patients were found to be PR3-ANCA positive using the commercial kits and 60% positive by the HM. A comparison of the results of the 7 commercial kits showed that the concordance was only 56% in cANCA positive samples. Additionally, the serum levels of PR3-ANCA obtained from only 5 of the 7 commercial PR3-ANCA kits correlated with cANCA titers. These data suggest that the sensitivity of PR3ANCA differed among the ELISA kits studied.

For MPO-ANCA detection, the data obtained from the 8 commercial kits varied widely. For this reason, capture ELISA, immunoblots and inhibition assays were performed to confirm the ELISA results. Twenty-five percent to 75% of the 20 pANCA-positive serum samples were found to be MPO-ANCA positive by the 8 commercial kits, and 45% by HM. However, only 35% of the sera were confirmed to be MPO-ANCA positive by capture ELISA, immunoblot, and inhibition assay. These 35% were also shown to be MPO-ANCA positive by all of the commercial kits and HM except for the A kit. The concordance rate for the results on the pANCA-positive sera was very low Ž30%.. In addition, the serum levels of MPO-ANCA obtained in each of the 8 commercial kits showed no significant correlations with the pANCA titers. These results demonstrate that the specificity and sensitivity of MPO-ANCA detection differed markedly among the ELISA kits tested. Various factors may influence the sensitivity and specificity of ELISAs. One important factor is the heterogeneity of antigen preparations. In this study, the purity of these antigens was assessed by reactivity with a panel of MoAbs against the main granule proteins of the neutrophil ŽPR3, MPO, HLE and LF.. All the coating antigens in the PR3-ANCA kits only reacted with MoAbs against PR3, and all coating antigen of the MPO-ANCA kits only reacted with MoAb anti-MPO. However, the read-out absorbance values of the MoAbs in these ELISAs differed from kit to kit. This may be because the coating antigens of these kits have different epitopes or structures recognized by ANCA, a difference that could lead to disparities in the sensitivity and specificity of ANCA detection by ELISAs. Other factors, including differences in sample dilutions, incubation time, substrate, and the choice of cut-off point, could have a marked affect on the ELISA results as well. As shown in Table 1, there are substantial methodology differences between the kits. These can contribute to the variations in sensitivity and specificity of ANCA detection in the kits tested. The different sensitivities and low concordances of ANCA detection in the ELISA kits could partly explain the differences reported in the literature. In conclusion, this study has shown that there are sig-

G. Wang et al.r Journal of Immunological Methods 208 (1997) 203–211

nificant differences in sensitivity among the ELISA kits tested for ANCA detection. Our data emphasize the necessity for establishing uniform international standards for ANCA ELISA procedures in order to permit more reliable interpretation and comparison of biological and medical data. Acknowledgements We are grateful to the following companies: The Binding Site ŽBirmingham, UK., DLD Diagnostika ŽHamburg., DPC Biermann ŽBad Nauheim., Elias ., Medizintechnik ŽFreiburg., Euroimmun ŽLubeck ¨ Ž . Ž . Genesis UK , Shield Diagnostics Dundee, UK and Wieslab AB ŽLund. for providing us with the ELISA kits used in this study. References Cohen Tervaert, J.W., Mulder, L., Stegeman, C., Elema, J., Huitema, M., The, H., 1993. Occurrence of autoantibodies to human leukocyte elastase in Wegener’s granulomatosis and other inflammatory disorders. Ann. Rheum. Dis. 52, 115. Coremans, I.E.M., Hagen, E.C., Daha, M.R., Van der Woude, F.J., Van der Voot, E.A.M., Kleijburg van der Keur, C., 1992. Antilactoferrin antibodies in patients with rheumatoid arthritis are associated with vasculitis. Arthritis Rheum. 35, 1466. Davenport, A., Lock, R.J., Wallington, T.B., Feest, T.G., 1994. Clinical significance of antineutrophil cytoplasm antibodies detected by a standardized indirect immunofluorescence assay. Q. J. Med. 87, 291. Falk, R.J., Jennette, J.C., 1988. Antineutrophil cytoplasmic autoantibodies with specificity from myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N. Engl. J. Med. 318, 1651. Gross, W.L., Csernok, E., 1995. Antineutrophil cytoplasmic autoantibodies ŽANCA.: Immunodiagnostic and pathophysiological aspects in vasculitis. Curr. Opin. Rheumatol. 7, 11. EECrBCR Group for ANCA Assay Standardization, Hagen, E.C., Andrassy, K., Csernok, E., Daha, M.R., Gaskin, G., Gross, W.L., Lesaver, P., Luedemann, J., Pusey, C.D., Rasmussen, N., 1993. The value of indirect immunofluorescence and solid phase techniques for ANCA detection: A report on the first

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