PR3 immunecomplexes

Journal of Immunological Methods 211 Ž1998. 111–123

Capture-ELISA based on recombinant PR3 is sensitive for PR3–ANCA testing and allows detection of PR3 and PR3–ANCArPR3 immunecomplexes Jiayan Sun a,c , David N. Fass b, Jay A. Hudson a , Margaret A. Viss a , Jorgen Wieslander d , Henry A. Homburger c , Ulrich Specks a,) ¨ a

c

Thoracic Diseases Research Unit, Mayo Clinic and Foundation, Rochester, MN, USA b Hematology Research Unit, Mayo Clinic and Foundation, Rochester, MN, USA DiÕision of Clinical Biochemistry and Immunology, Mayo Clinic and Foundation, Rochester, MN, USA d Wieslab, IDEON, S-223 70 Lund, Sweden Received 15 August 1997; revised 31 October 1997; accepted 14 November 1997

Abstract Proteinase 3 ŽPR3., a constituent of azurophil granules of neutrophils Žpolymorphonuclear cells, PMNs., is the target antigen for most anti-neutrophil cytoplasmic antibodies Žc-ANCA. in Wegener’s granulomatosis ŽWG.. We have recently developed an expression system for recombinant PR3 ŽrPR3. that is recognized by c-ANCA. Here, we report on the development and characterization of two monoclonal antibodies ŽmoABs. and a rabbit polyclonal antiserum generated against this rPR3. Epitope competition analysis indicates that the moABs MCPR3-1 and MCPR3-2 recognize overlapping epitopes on the PR3 molecule that are distinct from the ones recognized by moABs 4A5 and 6A6 developed by others. Since MCPR3-2 does not appear to compete for epitopes recognized by a sizable proportion of PR3–ANCA, we used it to develop a sensitive capture enzyme linked immunosorbent assay ŽELISA. for clinical PR3–ANCA testing. Both purified PMN–PR3 and crude human mast cell line ŽHMC-1.rPR3-S176A cell lysates were used as sources of PR3 target antigen in this assay with equal analytical sensitivity and specificity. Of 109 patients with ANCA-associated disease, 91 Ž83.5%. and 90 Ž82.6%. were PR3–ANCA positive by capture ELISA when PMN–PR3 and HMC-1rPR3-S176A cell lysates were used as antigen, respectively. When HMC-1rPR3 and HMC-1rPR3-S176A cells were used as indirect immunofluorescence ŽIIF. substrate, 88 Ž80.7%. and 92 Ž84.4%. were PR3–ANCA positive, respectively. These differences were not statistically significant. Only 1 of 151 controls without defined ANCA-associated disease tested positive by capture ELISA with either target antigen Žboth negative by PR3–ANCA specific IIF.. The capture ELISA can also be used to detect of PR3–ANCA immunecom-

Abbreviations: a 1-PI, a 1-protease inhibitor; ANCA, anti-neutrophil cytoplasmic antibodies; c-ANCA, cytoplasmic fluorescence pattern ANCA; ELISA, enzyme linked immunosorbent assay; GN, glomerulonephritis; IIF, indirect immunofluorescence; moAB, monoclonal antibody; NGS, normal goat serum; PBS, phosphate-buffered saline; PMN, polymorphonuclear cells; PR3, proteinase 3; rPR3, recombinant proteinase 3; RF, rheumatoid factor; RT, room temperature; WG, Wegener’s granulomatosis ) Corresponding author. Thoracic Diseases Research Unit, Guggenheim Bldg. 642 A, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905, USA. Tel.: q1-507-284-2301; fax: q1-507-284-4521; e-mail: [email protected]. 0022-1759r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 7 . 0 0 2 0 3 - 2

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plexes and, in combination with the rabbit antiserum, for the quantitative measurement of PR3 in biological fluids. q 1998 Elsevier Science B.V. Keywords: ANCA; Capture ELISA; Human; Mast cell; Proteinase 3; Wegener’s granulomatosis

1. Introduction Anti-neutrophil cytoplasmic antibodies ŽANCA. were first described in association with small vessel vasculitis and Wegener’s granulomatosis ŽWG. ŽDavies et al., 1982; Hall et al., 1984; van der Woude et al., 1985.. Based on the fluorescence pattern induced by ANCA on ethanol fixed neutrophil cytospin preparations, they are currently categorized in two broad groups: cytoplasmic staining ANCA Žc-ANCA. and perinuclear staining ANCA Žp-ANCA. ŽFalk and Jennette, 1988; Wiik and van der Woude, 1990.. A multitude of target antigens for ANCA have been identified with various clinical associations ŽKallenberg et al., 1994.. While new target antigens continue to be identified, proteinase 3 ŽPR3. and myeloperoxidase are the most clinically useful because of their strong associations with WG and microscopic polyangitis ŽKallenberg et al., 1994.. The neutrophil serine protease, PR3, has been identified as the principal target antigen for c-ANCA ŽJenne et al., 1990; Ludemann et al., 1990; Gupta et ¨ al., 1990.. Because of its high specificity for WG, the finding of a positive c-ANCA with PR3–ANCA specificity has significant therapeutic implications ŽCohen Tervaert et al., 1989; Nolle ¨ et al., 1989.. The search for simple PR3–ANCA detection systems with high analytical sensitivity and specificity has been hampered by properties of PR3–ANCA as well as by properties of the target antigen: the majority of PR3–ANCA recognize conformational epitopes on PR3 ŽBini et al., 1992. and the purification of PR3 from polymorphonuclear cell ŽPMN. granules for use in solid-phase assays is tedious ŽKao et al., 1988; Zhao and Lockwood, 1996. and prone to result in partial loss of recognizability by at least some PR3–ANCA. The availability of an expression system for recombinant PR3 ŽrPR3. that can be used as substrate for PR3–ANCA detection might offer an alternative solution to some of these problems. We have recently reported on the development of an expression system for conformationally intact, enzy-

matically active rPR3 expressed in the human mast cell line HMC-1 ŽSpecks et al., 1996. and we have shown that these HMC-1rPR3 cells represent a substrate for PR3–ANCA detection by indirect immunofluorescence ŽIIF. that appears superior to the use of PMN as substrate ŽSpecks et al., 1997.. The studies presented here were performed: Ž1. to characterize two monoclonal antibodies ŽmoABs., MCPR3-1 and MCPR3-2, and a polyclonal rabbit antiserum generated against rPR3 expressed in HMC-1 cells; Ž2. to evaluate their application in a capture enzyme linked immunosorbent assay ŽELISA. for the detection of PR3–ANCA and PR3–ANCArPR3 immune complexes, as well as for the quantitative measurement of PR3; and Ž3. to compare the use of lysates from HMC-1rPR3-S176A cells and purified PR3 from neutrophil ŽPMN. granules as antigens in the capture ELISA for PR3– ANCA detection.

2. Materials and methods 2.1. Materials Unless specified otherwise, all materials were from Sigma, St. Louis, MO. Purified human neutrophil PR3, elastase and cathepsin G were purchased from Athens Research and Technology, Athens, GA. HMC-1rPR3-S176A cells were cultured as described elsewhere ŽSpecks et al., 1996.. These cells express an enzymatically inactive mutant of rPR3 with a substitution of the active site serine at position 176 ŽCampanelli et al., 1990. by alanine Židentical to the designation PR3-S195A which is based on the bovine chymotrypsinogen A numbering ŽJenne et al., 1997... 2.2. Indirect immunofluorescence Ethanol fixed cytospin preparations of human PMN, HMC-1rPR3, HMC-1rVEC and HMC-

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1rPR3-S176A cells were prepared as described elsewhere ŽSpecks et al., 1997.. Cytospin preparations were incubated with primary antibodies for 30 min, washed three times in 1% normal goat serumrphosphate-buffered saline ŽNGSrPBS. for 5 min each, incubated with secondary antibodies for 30 min, washed again three times in 1% NGSrPBS and covered with cover slips in 90% glycerolrPBS. The entire procedure was performed at room temperature ŽRT.. 2.3. Cyto-ELISA A cyto-ELISA was developed to screen for antiPR3 reactivity of hybridoma supernatants using the moAB 4A3 ŽSommarin et al., 1995. as prototype. 1 = 10 6 HMC-1rPR3 or HMC-1rVEC cells were washed and suspended in 20 mM Tris–HCl, 0.5 M NaCl, pH 7.5 Žwash buffer 1. and placed in microtiter wells. After centrifugation of the plates at 900 = g, cells were fixed in 95% ethanol at 48C for 5 min and air dried. Supernatants from clones to be tested for reactivity with rPR3 were incubated in wells containing HMC-1rPR3 cells or HMC-1rVEC control cells for 60 min at RT. After washing three times with wash buffer 1, HRP-conjugated goat anti-mouse IgG ŽBioRad, Hercules, CA, cat a1721011, diluted 1:400 in 20 mM Tris–HCl, 0.5 M NaCl, 0.5% BSA, pH 7.4. was incubated in the wells for 60 min at RT. After washing, 100 m l of 0.1% 5-aminosalicylic acid, 0.01% H 2 O 2 solution were added as substrate for the color reaction which was stopped after 4 min by addition of an equal volume of 1 M NaOH. Absorbance was measured at 490 nm wavelength. 2.4. Generation of antibodies For the generation of the moABs MCPR3-1 and MCPR3-2, mice were immunized with HMC-1rPR3 cell granule extract. HMC-1rPR3 cells were disrupted by nitrogen cavitation Ž350 psi = 20 min. as described for neutrophils ŽBorregaard et al., 1983.. The slow spin supernatant was layered over a discontinuous Percoll gradient and centrifuged at 20,000 = g for 15 min. Of the resulting three bands Žthe

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lowest band was not detectable consistently., the upper band tested positive for PR3 by dot blot using a rabbit PR3 anti-serum and the moAB 4A3 ŽSommarin et al., 1995.. The PR3 containing granule fraction was collected, the granules were disrupted by repeated freeze–thawing and emulsified with Freund’s adjuvant and used for immunization of the mice. Mouse sera, the fusions, and the clones were tested for reactivity against rPR3 by comparing HMC-1rPR3 cells to sham-transfected HMC1rVEC cells by IIF and by cyto-ELISA. For the generation of the rabbit antiserum, HMC1rPR3-S176A cells were lysed. After batch adsorption of the lysate with the anion exchange resin Accell-QMA ŽWaters, Milford, MA., the supernatant was further purified using cation exchange chromatography on a Mono S column ŽPharmacia, Piscataway, NJ.. The bound proteins were eluted with a NaCl gradient Ž0 to 1 M, 0.1% Triton X-100, 0.05 M acetate, pH 5.5.. The eluted fractions, showing PR3 reactivity by capture ELISA, were pooled and further purified by reverse phase HPLC using a C3 column eluted with acetonitrile. The immunoreactive peak fractions were pooled, lyophilized, resuspended in PBS, emulsified with Freund’s adjuvant, and used as immunogen. Rabbit serum samples were evaluated for reactivity with rPR3 between booster injections by IIF using HMC-1rPR3-S176A cells in comparison to HMC-1rVEC cells. The IgG fraction of the rabbit serum was prepared by adsorption to protein A. Handling and care of the animals required for antibody production were in accordance with institutional guidelines.

2.5. Immunoblotting Five million cells were lysed in 500 m l of lysis buffer Ž50 mM Tris–HCl, 0.15 M NaCl, 0.5% Nonidet-P 40, 0.5 mM EDTA, 75 m grml PMSF, 2 m grml aprotinin, 0.5 m grml leupeptin, pH 7.2.. Proteins were resolved on 12% SDS-PAGE gels under non-reducing conditions, transferred to Immun-Litee membranes, and analyzed using the Immun-Litee chemiluminescent assay according to the manufacturer’s instructions ŽBioRad.. The membranes were incubated overnight at 48C with the

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moAB MCPR3-2 Ž0.7 mgrml. diluted 1:100 in 1% non-fat dry milk, 20 mM Tris, 500 mM NaCl, 0.05% Tween-20, pH 7.5. 2.6. Capture ELISA For PR3–ANCA testing, the following assay procedure was used. Microtiter wells ŽImmulon 1, Dynatech Laboratories, Chantilly, VA. were incubated with 200 m l of MCPR3-2 solution Ž4 m grml in Na bicarbonate buffer, pH 9.5. at 48C overnight. After washing three times with 20 mM Tris–HCl, 0.5 M NaCl, pH 7.5, 0.05% Tween-20 Žwash buffer 2., 200 m l of purified neutrophil PR3 solution Ž0.0625 m grml in 50 mM Tris–HCl, 0.1 M NaCl, 0.1% BSA, pH 7.4. were incubated in the wells for 1 h at RT. Control wells were incubated in parallel with buffer alone. Cell lysates from HMC-1rPR3-S176A cells were used as an alternative antigen source. 5 = 10 6 cells were lysed by incubation in 500 m l of lysis buffer for 30 min on ice. After careful shearing of the DNA, 200-m l aliquots of a 1:8 dilution of the cell lysate in lysis buffer were incubated in MCPR3-2 coated wells for 1 h at RT. Control wells were incubated in parallel with lysis buffer alone. After washing three times with wash buffer 2, 200-m l aliquots of serum dilution were incubated for 1 h at RT, followed by three washes and incubation of alkaline phosphatase conjugated goat anti-human IgG ŽSigma, A-9544. diluted 1:10,000 in 20 mM Tris– HCl, 0.5 M NaCl, 0.5% BSA, pH 7.5 Ždilution buffer 1.. For the color reaction the phosphatase substrate, p-nitrophenyl phosphate was used at a concentration of 1 mgrml in 0.1 M Tris–HCl, 5 mM MgCl 2 , 0.1 M NaCl, pH 9.5. The absorbance was determined after 30 min at a wavelength of 405 nm. For PR3–ANCA testing, serum samples were tested at dilutions of 1:20 Žin dilution buffer 1. in duplicate on wells coated with MCPR3-2 and antigen Žneutrophil PR3 or HMC-1rPR3-S176A cell lysate., and in parallel on control wells coated with moAB alone containing no antigen Žbackground.. Unless specified otherwise, net absorbance values calculated by subtraction of the background value from the value obtained from wells containing captured antigen are reported. Serum samples yielding net absorbance values of 0.100 or greater were considered PR3–ANCA positive.

To identify the possible causes of high background readings and ways to avoid them, the following measures were evaluated. Blocking of the wells with 2% BSA, 5% milk, or 0.1% Tween-20 for 1 h at RT prior to incubation of the serum sample had no effect on background. Consequently, a blocking step was not made part of the procedure. Preadsorption of serum samples with mouse immunoglobulin or human immunoglobulin also did not result in significant background reduction. For the detection of PR3–ANCArPR3 immune complexes, serum samples were preadsorbed by incubation in MCPR3-2 coated wells for 1 h at RT. The supernatants were then reassayed in duplicate in antigen containing wells as described. For the quantitative determination of PR3, the following modification of the above protocol was used. A total of 50 mM Tris–HCl, 0.1 M NaCl, 0.1% BSA, pH 7.4 was used as wash buffer and for the dilutions of PR3 and primary and secondary antibodies. As primary antibody, the rabbit PR3anti-serum was used at a dilution of 1:50. HRP-conjugated anti-rabbit IgG ŽBioRad, cat a172–1013, diluted 1:400. was used as secondary antibody. The color reaction was obtained and quantified as described for the cyto-ELISA. Each data point was assayed in duplicate. 2.7. Patient serum samples In this study, we used aliquots of the same serum samples from patients with ANCA-associated disease and from control patients which had been previously tested for c-ANCA using PMN cytospin preparations and for PR3–ANCA by IIF using HMC1rPR3 cell cytospin preparations and different commercial ELISA kits ŽTable 1. ŽSpecks et al., 1997.. Since the presence of rheumatoid factor ŽRF. can cause problems in solid phase assays, we have also included 20 serum samples containing RF in this study. In addition, 40 serum samples containing anti-ds-DNA antibodies were evaluated. 2.8. Statistical analysis Pairwise comparisons of the agreement of test results were performed using the McNemar’s test.

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Table 1 Serum samples and PR3–ANCA test results in the various assays Sample group Ž n.

HMC-1rPR3-IIF Žpositivern. a

Capture ELISA PMN–PR3 Žpositivern.

Capture ELISA HMC-1rPR3-S176A lysate Žpositivern.

Healthy volunteers Ž20. Anti-MPO-positive Ž20. ANA-positive Ž20. RF-positive Ž20. Anti-ds-DNA-positive Ž40. Other antibodies Ž20. AMA Ž7. SMA Ž3. LKM-1 Ž3. Jo-1 Ž3. PCA Ž3. Reticulin Ž1. Cytoplasmic staining on commercial PMN slides Ž19. Previously reported as c-ANCA-positive Ž101.

0r20 1r20 0r20 0r20 nd 0r20

0r20 1r20 0r20 0r20 0r40 0r20

1r20 1r20 0r20 0r20 0r40 0r20

1r19

2r19

1r19

88r101

92r101

91r101

a

Except for the RF containing samples the data listed in this column have been previously reported. AMA: anti-mitochondrial antibodies. LKM: anti-liver, kidney microsome antibodies. MPO: myeloperoxidase. PKA: anti-parietal cell antibodies. RF: rheumatoid factor. SMA: anti-smooth muscle antibodies. nd: Not done.

Significance of correlations were analyzed using the F-test for linear regression. In all cases, two-sided p-valuesF 0.05 were considered significant.

1rPR3-S176A cells ŽFig. 1.. No cross-reactivity with purified neutrophil elastase or cathepsin G was detected by immunoblot or direct ELISA Žnot shown.. MCPR3-2 has been used to purify native PMN–PR3

3. Results 3.1. Characterization of antibodies Two moABs designated as MCPR3-1 and MCPR3-2 were selected based on their ability to bind to granule content of HMC-1rPR3 cells but not of cells transfected with the expression vector without insert ŽHMC-1rVEC cells.. IgG was purified from mouse ascites and MCPR3-1 and MCPR3-2 were identified to be of IgG1 subclass. Both antibodies react with PMN–PR3 as determined by IIF generating the characteristic c-ANCA staining pattern on ethanol fixed PMN Žnot shown. and by immunoblots with purified PMN–PR3 and lysates from HMC-

Fig. 1. Western blot of purified PMN–PR3 and HMC-1rPR3-S176 cell lysate. 5=10 6 HMC-1rPR3-S176A cells were lysed, aliquots of the cell lysates Žequivalent to 2.5=10 5 cells per lane, lanes 1 and 3. and purified PMN–PR3 Ž1 m g, lane 2. were separated on a 12% polyacrylamide gel under non-reducing conditions. Proteins were then transferred to filter membranes and probed with the moAB MCPR3-2 Žlanes 1 and 2. and the rabbit polyclonal antiserum Žlane 3..

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from azurophil granule extracts by affinity chromatography. Neither antibody interfered with enzymatic activity of rPR3 against the substrates MeSucc-Ala-AlaPro-Val-pNa or elastin-FITC, or with its complexation with a 1-protease inhibitor Ž a 1-PI. Žnot shown.. Epitope competition analysis was performed as described ŽKwak and Yoon, 1996. using the cytoELISA. We compared our moABs raised against

rPR3 to the moABs 4A5, 6A6 and 4A3 raised against PMN–PR3 ŽSommarin et al., 1995.. The results indicate that MCPR3-1 and MCPR3-2 compete for binding to PR3 ŽFig. 2A., whereas the moABs 4A5 and 6A6 do not compete with MCPR3-1 Žnot shown. or MCPR3-2 ŽFig. 2B and C.. The moAB 4A3 appears to bind to a larger epitope that partially overlaps with those recognized by MCPR3-1 Žnot shown. and MCPR3-2 ŽFig. 2D..

Fig. 2. Epitope competition analysis of moABs by cyto-ELISA. ŽA. When MCPR3-1 Žsmall filled circles. is bound to its epitope at saturation no signal enhancement is obtained by addition of MCPR3-2 Žlarge open circles. indicating that the two antibodies compete for overlapping binding sites. When MCPR3-2 Žlarge open squares. is bound to its epitope at saturation only a small signal enhancement can be achieved by the addition of MCPR3-1 Žsmall full squares. suggesting that the epitope recognized by MCPR3-1 may be slightly larger than the epitope recognized by MCPR3-2. ŽB. When 4A5 Žlarge open triangles. is bound to its target at saturation a significant enhancement of absorbance can be achieved by addition of MCPR3-2 Žfull small triangles.. If MCPR3-2 is bound at saturation first Žlarge open squares. an equivalent gain in absorbance can be obtained if 4A5 Žsmall full squares. is added, indicating that the epitopes recognized by MCPR3-2 and 4A5 do not overlap. ŽC. When 6A6 is used at saturation Žcrosses. significant signal enhancement can be obtained by addition of MCPR3-2 Žx. and vice versa, indicating that MCPR3-2 and 6A6 also do not compete for the same epitope. ŽD. When MCPR3-2 is used at saturation Žlarge open squares., the addition of 4A3 results in signal enhancement Žsmall full squares.. When 4A3 is used at saturation Žreached at dilution of 1:30; black bars. the addition of MCPR3-2 Žwhite bars. does not result in significant signal enhancement, indicating that 4A3 occupies a larger overlapping epitope. Shown are representative examples of two ŽD. or three repeat experiments ŽA–C..

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3.2. Detection of PR3–ANCA The experiments of this study were designed to address the following questions: Ž1. Can the moAB MCPR3-2 be used as capture antibody for PR3– ANCA testing, or does it compete for epitopes recognized by a sizable proportion of PR3–ANCA? Ž2. Can HMC-1rPR3 cell lysates be used directly as source of antigen in this assay, avoiding the need for the tedious purification procedure of PR3? Because PR3 has been shown to cleave immunoglobulins and ANCA ŽDolman et al., 1995., it is possible that PR3–ANCA-test results obtained by capture ELISA could be affected by the enzymatic activity of PR3, particularly since the moAB MCPR3-2 does not inhibit the enzymatic activity of PR3. Therefore, we first determined whether the inactive mutant PR3-S176A expressed by HMC1rPR3-S176A cells ŽSpecks et al., 1996. is recognized by PR3–ANCA. Ethanol fixed cytospin preparations of HMC-1rPR3, HMC-1rPR3-S176A and HMC-1rVEC control cells were prepared as described ŽSpecks et al., 1997.. Sera from all 109 patients with WG and MPA from our previously reported patient cohort ŽSpecks et al., 1997. were tested by IIF at serum dilutions of 1:4 and 1:16 in parallel on all three substrates. As shown in Table 2, all PR3–ANCA sera that recognized wild type rPR3 expressed in HMC-1rPR3 cells also recognized the inactivated mutant rPR3-S176A. In addition, rPR3S176A was recognized by the remaining three sera from patients with biopsy proven WG and one addi-

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tional serum from a patient with clinically diagnosed WG, indicating that rPR3-S176A represents an equivalent substrate for PR3–ANCA testing to the wild type rPR3 expressed by HMC-1rPR3 cells. While these data might suggest a higher analytical sensitivity of IIF using HMC-1rPR3-S176A cells as substrate, the difference was not statistically significant Ž p s 0.125.. Subsequently, we assayed all 200 serum samples from the reported cohort ŽSpecks et al., 1997., 20 rheumatoid factor containing sera, and 40 anti-dsDNA antibody containing sera in the capture ELISA using both, purified PMN–PR3 and HMC-1rPR3S176A cell lysates, as captured target antigen. Both antigens captured by MCPR3-2 represent sensitive substrates for PR3–ANCA testing ŽTable 2. that is statistically not significantly different from IIF testing using HMC-1 cells expressing rPR3 Žwild type or inactive mutant.. Only one serum sample from a patient with biopsy proven WG was negative in the capture ELISA when HMC-1rPR3-S176A cell lysate was used as antigen Žbackground was low at 0.153.. This sample was positive in the capture ELISA with PMN–PR3 as antigen and by IIF using HMC-1rPR3 and HMC-1rPR3-S176A cells as substrate. One sample from a patient with the clinical diagnosis of WG that had previously been positive by IIF using HMC-1rPR3, HMC-1rPR3-S176A cells and PMN, tested PR3–ANCA negative in the capture ELISA with either substrate. With PMN–PR3 as antigen, one positive result was found in a patient with Q-fever and renal insuf-

Table 2 PR3–ANCA testing by IIF using HMC-1rPR3 and HMC-1rPR3-S176A cell cytospin preparations and by capture ELISA with PMN–PR3 and HMC-1rPR3-S176A cell lysate as captured antigen Diagnosis

n

Biopsy proven WG Clinical WG MPA Total, n % positive p-valuea

66 14 29 109

a

Positive on

Positive by capture ELISA with

HMC-1rPR3

HMC-1rPR3-S176

PMN–PR3

HMC-1rPR3-S176 cell lysate

63 12 13 88 80.7 0.125

66 13 13 92 84.4

66 12 13 91 83.5 1.0

65 12 13 90 82.6 0.5

Calculated using McNemar’s test based on 2 = 2 cross-tabulation of test results. WG: Wegener’s granulomatosis. MPA: microscopic polyangiitis. GN: glomerulonephritis.

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Table 3 Absorbance readings obtained from antigen containing wells, wells lacking antigen Žbackground. and net absorbance values Antigen PR3–ANCA status Ž n. PMN–PR3 positive Ž95. negative Ž125. HMC-1r PR3-S176A lysate positive Ž94. negative Ž126.

Wells with antigen

Background median Žrange.

Net absorbance

0.752 Ž0.264–1.413. 0.257 Ž0.082–1.740.

0.221 Ž0.086–0.851. 0.317 Ž0.090–1.764.

0.424 Ž0.123–1.179. y0.022 Žy0.409–0.094.

0.802 Ž0.269–1.673. 0.185 Ž0.101–1.170.

0.214 Ž0.099–0.910. 0.159 Ž0.094–1.151.

0.550 Ž0.148–1.425. 0.008 Ž –0.148–0.094.

ficiency of indeterminate etiology, whereas another serum from a healthy volunteer tested positive with HMC-1rPR3-S176A cell lysate as antigen. Both of these samples were ANCA negative by IIF on all substrates. The medians and ranges of absolute, background and net absorbance values obtained with the two antigen preparations for PR3–ANCA positive and negative serum samples are listed in Table 3. Background absorbance of ) 0.500 occurred significantly more frequently with PMN–PR3 than with HMC1rPR3-S176A lysates as antigen: 42% vs. 8.4% of PR3–ANCA negative samples and 8.4% vs. 6.4% of PR3–ANCA positive samples yielded such values. While high background in PR3–ANCA positive samples was mostly caused by the presence of PR3–ANCArPR3 immunecomplexes, the cause of high backgrounds in PR3–ANCA negative samples remained unclear.

The net absorbance values obtained with purified PMN–PR3 showed a significant correlation with those obtained with HMC-1rPR3-S176A cell lysate as antigen ŽFig. 3; r s 0.694, p - 0.001.. It has been previously shown that c-ANCA IIF titers do not always correlate well with PR3–ANCA solid phase assay readings ŽNolle ¨ et al., 1989.. We compared the capture ELISA absorbance data presented here with the c-ANCA titer readings obtained previously on ethanol fixed PMN and HMC-1rPR3 cell slides reported elsewhere ŽSpecks et al., 1997.. The absorbance readings obtained with purified PR3 from PMN did not correlate with c-ANCA titers obtained using PMN slides Ž r s 0.149, p s 0.15.. There was a statistically significant, yet weak correlation with titers obtained using HMC-1rPR3 cell slides Ž r s 0.231, p - 0.05.. In contrast, there was a better correlation between c-ANCA titers obtained on PMN slides and the capture ELISA absorbance readings

Fig. 3. Correlation of absorbance readings of PR3–ANCA positive sera obtained by capture ELISA using PMN–PR3 or HMC-1rPR3-S176A cell lysates as antigen.

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obtained when HMC-1rPR3-S176A cell lysates were used as antigen Ž r s 0.472, p - 0.001.. The strongest correlation was observed between PR3–ANCA titers obtained by IIF on HMC-1rPR3 cells and the capture ELISA absorbance readings obtained with HMC-1rPR3-S176A cell lysates Ž r s 0.576, p 0.001.. 3.3. Reproducibility of PR3–ANCA determination To evaluate the interassay variation of PR3– ANCA test results obtained with the capture ELISA, we tested three individual c-ANCA positive serum samples with titers of 1:8, 1:64 and 1:512 Žas deter-

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mined by routine c-ANCA determination on PMN cytospin preparations., respectively and three normal control sera on 5 consecutive days using both PMN– PR3 and HMC-1rPR3-S176A cell lysate as antigen. The entire coating procedure as well as the preparation of fresh cell lysate was performed separately for each experiment. As shown in Fig. 4, the day-to-day variation is higher with HMC-1rPR3-S176A cell lysates as antigen than with purified PMN–PR3. The apparent day-to day variation can be reduced if the data are normalized for a defined control sample ŽFig. 4C and D.. To determine the coefficient of variation ŽCV., 20 serum samples containing PR3–ANCA as deter-

Fig. 4. Reproducibility of PR3–ANCA determination. Three c-ANCA negative serum samples Žpatients 1 to 3., one sample each with a c-ANCA titer of 1:512 Žpatient 4., of 1:64 Žpatient 6. and of 1:8 Žpatient 5. were tested by capture ELISA using PMN–PR3 Žpanels A and C. and HMC-1rPR3-S176A cell lysate Žpanels B and D. as target antigen. Panels A and B show the means" SEM of the net absorbance values obtained in five consecutive experiments. The complete cell lysis and plate coating procedure was repeated for each individual experiment. Panels C and D show the data expressed as percentage of a defined control sample Žpatient 4., indicating that the variability can be reduced by normalizing the data for a defined control sample.

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mined by IIF using HMC-1rPR3-S176A cells were pooled. These sera had titers ranging from 1:8 to 1: 512 as previously determined by standard IIF using PMN cytospin preparations. This pooled sample was assayed in 10 consecutive experiments including the entire coating and cell lysis procedure. For PMN– PR3 as target antigen the mean net absorbance value " SEM was 1.36 " 0.05, for HMC-1rPR3-S176A cell lysates it was 0.69 " 0.07. The interassay CV was 11.99% when PMN–PR3 and 31.01% when HMC-1rPR3-S176A cell lysate was used. The intra-assay CV calculated based on experiments performed in 10 repeat wells within the same assay was 5.61% for PMN–PR3 and 13.03% for HMC-1rPR3S176A cell lysate as antigen. 3.4. Detection of PR3–ANCAr PR3 immunecomplexes Sera from patients with WG may contain free PR3–ANCA as well as PR3–ANCArPR3 immunecomplexes ŽBaslund et al., 1994.. The clinical significance of the presence of such immunecomplexes remains unclear. In capture ELISA systems, such immunecomplexes may be the source of significant background that, rarely, may obscure the presence of free PR3–ANCA. Fig. 5 shows two PR3– ANCA positive sera Žnot from the series of 200 patients. that generated background values in the capture ELISA that were as high as the values generated in wells containing the antigen. Removal of free PR3 and PR3–ANCArPR3 immunecomplexes from the samples by preadsorption of these sera with MCPR3-2 resulted in substantial background reduction. The presence of free PR3–ANCA in these samples was confirmed by incubating the MCPR3-2-preadsorbed sample with added exogenous purified PMN–PR3. The addition of this mixture to MCPR3-2 coated wells resulted in doubling of the signal, thereby proving the presence of free PR3–ANCA in these samples which would have been overlooked otherwise. While these data confirm that the capture ELISA results can be confounded by the presence of PR3–ANCArPR3 immunecomplexes, they also show that, when combined with preadsorption of the serum sample with MCPR3-2, this capture ELISA can be used for the detection of such immunecomplexes.

Fig. 5. The presence of circulating PR3–ANCArPR3-complexes can mask the presence of free PR3–ANCA. Shown are two serum samples that yielded background absorbance values on MCPR3-2 coated wells not containing antigen ŽB. that were as high as the absorbance obtained in antigen containing wells ŽA.. Subtraction of background would have falsely resulted in a negative PR3– ANCA result. After solid phase preadsorption with MCPR3-2 Žremoving PR3 and PR3–ANCArPR3 immunecomplexes from the serum samples. the supernatant was retested in wells without antigen, resulting in reduction of background absorbance ŽC.. When free PR3 was added to the supernatant and the mixture was assayed in parallel in wells without antigen ŽD., the absorbance almost doubled, clearly confirming the presence of free PR3ANCA in these serum samples.

3.5. QuantitatiÕe determination of PR3 Microtiter wells were coated with the moAB MCPR3-2 as described in Section 2. To generate a standard curve for PR3, serial dilutions of purified enzymatically active PMN–PR3 were prepared and

Fig. 6. Application of the capture ELISA for quantitative PR3 measurements. Shown is the dose response curve for concentrations of purified PMN–PR3 ranging from 1.5 to 200 ngrml. Each data point represents the mean"SEM of 15 consecutive experiments performed by three different operators. The dilutions of PR3 were prepared freshly for each of the experiments.

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incubated in the wells for 1 h at RT. The assay was performed as described in Section 2. Fig. 6 shows the resulting standard curve which indicates that the detection limit is about 2 ngrml and that it is almost linear over a 20-fold concentration range. The curve shown resulted from 15 experiments performed on consecutive days by three different operators. Additional experiments indicated that the absorbance readings are not affected by the presence of PR3ra 1-PI complexes Žnot shown..

4. Discussion Solid phase assays for PR3–ANCA testing have been developed in order to allow antigen-specific, reader-independent, semiquantitative PR3–ANCA testing. Various methods of antigen-preparation have been reported and the thorough standardization and clinical evaluation of several of these methods is still in progress ŽHagen et al., 1996.. Previous studies have suggested that the sensitivity of most PR3– ANCA ELISA assays based on the direct coating of the purified antigen to the plastic plate is inferior to the standard IIF method of c-ANCA detection using neutrophil cytospin preparations ŽSpecks et al., 1997; Wieslander, 1991.. One possible explanation is that immobilization may result in partial denaturation of the antigen with alteration of conformational epitopes. This problem might be circumventable by immobilizing the antigen via capturing moABs. Indeed, a recently described capture ELISA method, using a combination of three different moABs against PR3 to capture the antigen, promises a sensitivity equivalent to the standard c-ANCA detection method by IIF ŽBaslund et al., 1995.. Detailed data about the analytical sensitivity of another capture ELISA used for PR3–ANCA detection are currently not available ŽMerkel et al., 1997.. The moABs MCPR3-1 and MCPR3-2 described here were raised using granule extracts of HMC1rPR3 cells as immunogen and were shown to react specifically with rPR3 and native PMN–PR3. Our epitope competition analysis would suggest that they recognize overlapping epitopes on PR3. They do not compete for the epitopes recognized by the moABs 4A5 and 6A6 developed by Sommarin et al. Ž1995., but their moAB 4A3 seems to recognize a larger

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epitope that partially overlaps with the binding sites of MCPR3-1 and MCPR3-2. These findings are consistent with the data indicating that MCPR3-2 does not compete for a sizable number of PR3–ANCA binding sites. This is in contrast to 4A5 and 6A6 which inhibit the binding of 61% and 11% of PR3– ANCA sera, respectively ŽSommarin et al., 1995.. The antibody 4A3 only partially inhibited the binding of about 22% of PR3–ANCA sera, which were all also inhibited by 4A5. One of our goals was to identify an easily accessible abundant source of antigen. The analytical specificity of PR3–ANCA ELISA systems for PR3– ANCA depends on the purity of the immobilized antigen. Described purification procedures for PR3 are tedious, require large numbers of neutrophils as starting material and expose personnel to the risk of infection. The principal of the capture ELISA circumvents the antigen purity issue, provided that the capturing antibodies used have no cross-reactivity with other relevant antigens. Unfortunately, PR3 has been shown to cleave immunoglobulins including ANCA ŽDolman et al., 1995.. Consequently, the proteolytic activity of PR3 might interfere with the stability of the capturing antibody, particularly since MCPR3-2 does not inhibit the enzymatic activity of PR3. This reasoning contributed to the design of the enzymatically inactive rPR3-S176A mutant expressed in HMC-1rPR3-S176A cells ŽSpecks et al., 1996.. The direct comparison of HMC-1rPR3S176A cell lysates to purified enzymatically active PMN–PR3 showing equivalent analytical sensitivity for PR3–ANCA indicates: Ž1. proteolytic degradation of immobilized MCPR3-2 by active PR3 does not represent a technical problem, and Ž2. HMC1rPR3-S176A cell lysates represent a convenient source for specific target antigen, obviating the tedious purification of PR3. The PR3–ANCA values obtained for a pooled control serum with the two antigen preparations were more variable with the HMC-1rPR3-S176A cell lysate. This was expected as the coating procedure contains more variables when cell lysates are used. Both the cell counting procedure as well as the preparation of a homogeneous lysate solution are operator-dependent procedures at this stage of assay development. As suggested by the lower intra-assay CV, the inter-assay CV is likely to be significantly

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improved when large batches of plates are prepared and the assay is performed on plates from the same batch. Our data also indicate that expression of the sample data as percentage of a standardized control serum will further reduce the variability of the results. Some patients with PR3–ANCA also may have circulating PR3–ANCArPR3 immunecomplexes ŽBaslund et al., 1994.. Rarely, this may cause high backgrounds in the capture ELISA to the extent of masking the presence of free PR3–ANCA. It was not a problem among 101 consecutive c-ANCA positive serum samples and we had to search for such samples by screening a large serum bank. Nevertheless, serum samples yielding high background readings should be further tested for the presence of such immunecomplexes by repeating the assay after preadsorption of the serum sample with MCPR3-2 and after addition of free exogenous PR3 to the sample as described above. Future studies will have to determine the clinical relevance of the relationship of free PR3–ANCA to PR3–ANCArPR3 immunecomplexes. This assay may represent a convenient tool for such studies. Finally, this assay allows the quantitative determination of PR3. Since the assay results are not affected by the presence of PR3– a 1-PI complexes, the primary reading does not allow a distinction between free PR3 and PR3– a 1-PI complexes. However, in combination with antibodies against a 1-PI this assay can be used for the detection of such complexes and preadsorption of the sample with antibodies against a 1-PI allows the measurement of free PR3. The availability of sensitive assays that allow the specific distinction between PR3 and elastase are of crucial importance for the study of neutrophil serine protease mediated inflammatory conditions ŽVender, 1996., particularly since there are significant differences in substrate and inhibitor spectrum between elastase and PR3 despite their structural homology and functional similarities ŽPadrines et al., 1994; Rao et al., 1993; Robache-Gallea et al., 1995; Spector et al., 1995.. Acknowledgements The authors want to thank Mr. Robert D. Litwiller, Mr. Randall S. Miller and Ms. Elaine M. Wiegert for

excellent technical assistance with the antigen purification, antibody production and immunofluorescence assays, respectively; Ms. Amber M. Hummel for maintenance of the cell lines; Dr. Steven V. Bittorf and Ms. Hua Tang for performing some of the experiments resulting in Fig. 2d and Fig. 6; Mr. Darrell Schroeder from the Department of Biostatistics for help with the statistical analysis of the data; and Ms. Kathryn L. Stanke for help with the preparation of the manuscript. The authors are also indebted to Dr. Joseph H. Butterfield for the kind gift of the HMC-1 parent cell line. This work was supported by a grant-in-aid ŽAHA96008260. from the American Heart Association, a Research Grant from the American Lung Association of Minnesota Žboth to US. and funds from the Mayo Foundation. It was performed during the tenure of a Clinician-Scientist Award ŽAHA94004360. from the American Heart Association to US.

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