Clinica Chimica Acta 442 (2015) 82–83
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Letter to the Editor Autoreactivity to isoforms of glycoprotein 2 in inflammatory bowel disease
We read with great interest the study by Pavlidis et al. reporting the evaluation of a novel ELISA for the detection of autoantibodies (autoAbs) to the major zymogen granule membrane glycoprotein 2 (GP2) in patients with Crohn's disease (CrD) [1]. Glycoprotein 2 has been identified as an autogenic target in Crohn's disease by our group recently [2]. We demonstrated first, that autoreactivity to GP2 is found in up to 30% of patients with CrD and is associated with the clinical phenotype in such patients [3]. Regarding its location in humans, GP2 was initially shown to be synthesized in the exocrine pancreas and to be secreted into the intestine. Later, GP2 has been described as a specific marker of microfold cells (M cells) in the follicle-associated epithelium in the intestine and probably engaged in the interaction with the gut microbiota [4]. Since GP2 modulates innate and adaptive intestinal immune responses, an interplay between secreted and M-cell associated GP2 is proposed regarding the sensing of the microbiota. Pavlidis et al. reported an excellent specificity of 96.0% testing 617 patients with inflammatory bowel disease (IBD) by their new assay [1]. Comparing their results, they refer to methodological issues with regard to the varying sensitivity and specificity data published in previous studies with differing numbers of IBD patients so far. In this perspective, it is interesting to note that different isoforms of GP2 are synthesized in the pancreas. Fukuoka identified a larger and a shorter isoform of GP2 and termed them alpha (GP2a) and beta
(GP2b), respectively [5]. Previous prevalence data of anti-GP2 IgA and IgG were obtained mainly by assays manufactured by GA Generic Assays employing the larger isoform for GP2-specific autoAb detection [6]. In contrast, the recombinant isoform 4 of GP2 used by Pavlidis et al. corresponds to the shorter one (GP2b) [1]. To compare the prevalence of autoAbs to both isoforms in patients with IBD, we expressed them in the Baculovirus system and deployed the purified GP2 isoforms in solid-phase ELISA. Thus IgA and IgG to GP2a and GP2b were determined in 178 patients with CrD, 117 patients with ulcerative colitis (UC), and 154 blood donors. In contrast to anti-GP2a autoAb, antiGP2b IgA, IgG and IgA/IgG demonstrated a higher prevalence in patients with CrD (23/178 vs 17/178, 82/178 vs 69/178, and 23/178 vs 14/178, respectively). Moreover, anti-GP2b IgG revealed a lower prevalence in patients with UC (9/117 vs 18/117), whereas IgA to that isoform was more prevalent in this patient cohort compared to autoAb to GP2a (3/117 vs 1/117). Consistent with Pavlidis et al., there were neither patients with UC nor BD that demonstrated IgA and IgG to the respective isoforms of GP2 simultaneously [1]. Remarkably, IgG to GP2b was significantly less prevalent in BD than anti-GP2a IgG (4/154 vs 17/154, p = 0.005). Thus, in contrast to anti-GP2a IgG, anti-GP2b IgG demonstrated a higher sensitivity and in particular a higher specificity (95.2% vs 87.1%) in our study. Indeed, the area under the curve (AUC) for anti-GP2b IgG was significantly higher than that of anti-GP2a IgG (0.849 vs 0.713, p b 0.001) in accordance with the receiver-operating characteristics curve analysis (Fig. 1). In contrast, the AUC for anti-GP2b IgA was significantly lower compared with that for IgA to GP2a (0.760 vs 0.813, p = 0.008). Thus, we conclude that IgG to GP2b discriminates better between patients with CrD and UC than anti-GP2a autoAbs do. Since anti-GP2 IgG demonstrates a higher sensitivity in patients with CrD compared with IgA to GP2, GP2b should be used as an autoantigenic target for the differential diagnosis of IBD patients. Financial disclosure Dirk Roggenbuck is a shareholder of GA Generic Assays GmbH and Medipan GmbH. Both companies are diagnostic manufacturers. The remaining authors state that they have nothing to declare regarding conflict of interest and funding with respect to this manuscript. References
Fig. 1. Comparison of anti-GP2a and anti-GP2b autoAbs in patients and controls by receiveroperating characteristics (ROC) curve analysis. Recombinant GP2 isoforms alpha (GP2a) and beta (GP2b) were adsorbed onto microtiter plates at equal coating concentrations and used for specific autoAb analysis by ELISA in patients with Crohn's disease, ulcerative colitis, and blood donors as described elsewhere [6]. Thus, IgG and IgA to recombinant GP2a and GP2b were detected and subjected to ROC curve analysis.
http://dx.doi.org/10.1016/j.cca.2015.01.018 0009-8981/© 2015 Elsevier B.V. All rights reserved.
[1] Pavlidis P, Shums Z, Koutsoumpas AL, Milo J, Papp M, Uemurea T, et al. Diagnostic and clinical significance of Crohn's disease-specific anti-MZGP2 pancreatic antibodies by a novel ELISA. Clin Chim Acta 2015 [in press]. [2] Roggenbuck D, Hausdorf G, Martinez-Gamboa L, Reinhold D, Büttner T, Jungblut PR, et al. Identification of GP2, the major zymogen granule membrane glycoprotein, as the autoantigen of pancreatic antibodies in Crohn's disease. Gut 2009;58:1620–8. [3] Roggenbuck D, Reinhold D, Werner L, Schierack P, Bogdanos DP, Conrad K. Glycoprotein 2 in Crohn's disease. Adv Clin Chem 2013;60:187–208. [4] Schierack P, Rödiger S, Kolenda R, Hiemann R, Berger E, Grzymajlo K, et al. Speciesspecific and pathotype-specific binding of bacteria to zymogen granule membrane glycoprotein 2 (GP2). Gut 2014 [in press]. [5] Fukuoka S. Molecular cloning and sequences of cDNAs encoding alpha (large) and beta (small) isoforms of human pancreatic zymogen granule membrane-associated protein GP2. Biochim Biophys Acta 2000;1491:376–80.
Letter to the Editor [6] Roggenbuck D, Reinhold D, Wex T, Goihl A, von Arnim U, Malfertheiner P, et al. Autoantibodies to GP2, the major zymogen granule membrane glycoprotein, are new markers in Crohn's disease. Clin Chim Acta 2011;412:718–24.
Dirk Roggenbuck Faculty of Science, Brandenburg University of Technology, Senftenberg, Germany GA Generic Assays GmbH, Dahlewitz, Berlin, Germany Corresponding author at: Faculty of Science, Brandenburg University of Technology, Großenhainer Str. 57, 01968 Senftenberg, Germany. Tel.: +49 33708 441716; fax: +49 33708 441725. E-mail address: [email protected]. Nadja Röber Institute of Immunology, Medical Faculty of the Technical University Dresden, Dresden, Germany Dimitrios P. Bogdanos Institute of Liver Studies, Division of Transplantation Immunology and Mucosal Biology, King's College London School of Medicine at King's College Hospital, London, UK
83
Alexander Goihl Dirk Reinhold Institute of Molecular and Clinical Immunology, Otto-von-Guericke-University, Magdeburg, Germany Karsten Conrad Institute of Immunology, Medical Faculty of the Technical University Dresden, Dresden, Germany Martin W. Laass Department of Pediatrics, Medical Faculty of the Technical University of Dresden, Dresden, Germany 11 January 2015
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Letter to the Editor Autoreactivity to isoforms of glycoprotein 2 in inflammatory bowel disease
We read with great interest the study by Pavlidis et al. reporting the evaluation of a novel ELISA for the detection of autoantibodies (autoAbs) to the major zymogen granule membrane glycoprotein 2 (GP2) in patients with Crohn's disease (CrD) [1]. Glycoprotein 2 has been identified as an autogenic target in Crohn's disease by our group recently [2]. We demonstrated first, that autoreactivity to GP2 is found in up to 30% of patients with CrD and is associated with the clinical phenotype in such patients [3]. Regarding its location in humans, GP2 was initially shown to be synthesized in the exocrine pancreas and to be secreted into the intestine. Later, GP2 has been described as a specific marker of microfold cells (M cells) in the follicle-associated epithelium in the intestine and probably engaged in the interaction with the gut microbiota [4]. Since GP2 modulates innate and adaptive intestinal immune responses, an interplay between secreted and M-cell associated GP2 is proposed regarding the sensing of the microbiota. Pavlidis et al. reported an excellent specificity of 96.0% testing 617 patients with inflammatory bowel disease (IBD) by their new assay [1]. Comparing their results, they refer to methodological issues with regard to the varying sensitivity and specificity data published in previous studies with differing numbers of IBD patients so far. In this perspective, it is interesting to note that different isoforms of GP2 are synthesized in the pancreas. Fukuoka identified a larger and a shorter isoform of GP2 and termed them alpha (GP2a) and beta
(GP2b), respectively [5]. Previous prevalence data of anti-GP2 IgA and IgG were obtained mainly by assays manufactured by GA Generic Assays employing the larger isoform for GP2-specific autoAb detection [6]. In contrast, the recombinant isoform 4 of GP2 used by Pavlidis et al. corresponds to the shorter one (GP2b) [1]. To compare the prevalence of autoAbs to both isoforms in patients with IBD, we expressed them in the Baculovirus system and deployed the purified GP2 isoforms in solid-phase ELISA. Thus IgA and IgG to GP2a and GP2b were determined in 178 patients with CrD, 117 patients with ulcerative colitis (UC), and 154 blood donors. In contrast to anti-GP2a autoAb, antiGP2b IgA, IgG and IgA/IgG demonstrated a higher prevalence in patients with CrD (23/178 vs 17/178, 82/178 vs 69/178, and 23/178 vs 14/178, respectively). Moreover, anti-GP2b IgG revealed a lower prevalence in patients with UC (9/117 vs 18/117), whereas IgA to that isoform was more prevalent in this patient cohort compared to autoAb to GP2a (3/117 vs 1/117). Consistent with Pavlidis et al., there were neither patients with UC nor BD that demonstrated IgA and IgG to the respective isoforms of GP2 simultaneously [1]. Remarkably, IgG to GP2b was significantly less prevalent in BD than anti-GP2a IgG (4/154 vs 17/154, p = 0.005). Thus, in contrast to anti-GP2a IgG, anti-GP2b IgG demonstrated a higher sensitivity and in particular a higher specificity (95.2% vs 87.1%) in our study. Indeed, the area under the curve (AUC) for anti-GP2b IgG was significantly higher than that of anti-GP2a IgG (0.849 vs 0.713, p b 0.001) in accordance with the receiver-operating characteristics curve analysis (Fig. 1). In contrast, the AUC for anti-GP2b IgA was significantly lower compared with that for IgA to GP2a (0.760 vs 0.813, p = 0.008). Thus, we conclude that IgG to GP2b discriminates better between patients with CrD and UC than anti-GP2a autoAbs do. Since anti-GP2 IgG demonstrates a higher sensitivity in patients with CrD compared with IgA to GP2, GP2b should be used as an autoantigenic target for the differential diagnosis of IBD patients. Financial disclosure Dirk Roggenbuck is a shareholder of GA Generic Assays GmbH and Medipan GmbH. Both companies are diagnostic manufacturers. The remaining authors state that they have nothing to declare regarding conflict of interest and funding with respect to this manuscript. References
Fig. 1. Comparison of anti-GP2a and anti-GP2b autoAbs in patients and controls by receiveroperating characteristics (ROC) curve analysis. Recombinant GP2 isoforms alpha (GP2a) and beta (GP2b) were adsorbed onto microtiter plates at equal coating concentrations and used for specific autoAb analysis by ELISA in patients with Crohn's disease, ulcerative colitis, and blood donors as described elsewhere [6]. Thus, IgG and IgA to recombinant GP2a and GP2b were detected and subjected to ROC curve analysis.
http://dx.doi.org/10.1016/j.cca.2015.01.018 0009-8981/© 2015 Elsevier B.V. All rights reserved.
[1] Pavlidis P, Shums Z, Koutsoumpas AL, Milo J, Papp M, Uemurea T, et al. Diagnostic and clinical significance of Crohn's disease-specific anti-MZGP2 pancreatic antibodies by a novel ELISA. Clin Chim Acta 2015 [in press]. [2] Roggenbuck D, Hausdorf G, Martinez-Gamboa L, Reinhold D, Büttner T, Jungblut PR, et al. Identification of GP2, the major zymogen granule membrane glycoprotein, as the autoantigen of pancreatic antibodies in Crohn's disease. Gut 2009;58:1620–8. [3] Roggenbuck D, Reinhold D, Werner L, Schierack P, Bogdanos DP, Conrad K. Glycoprotein 2 in Crohn's disease. Adv Clin Chem 2013;60:187–208. [4] Schierack P, Rödiger S, Kolenda R, Hiemann R, Berger E, Grzymajlo K, et al. Speciesspecific and pathotype-specific binding of bacteria to zymogen granule membrane glycoprotein 2 (GP2). Gut 2014 [in press]. [5] Fukuoka S. Molecular cloning and sequences of cDNAs encoding alpha (large) and beta (small) isoforms of human pancreatic zymogen granule membrane-associated protein GP2. Biochim Biophys Acta 2000;1491:376–80.
Letter to the Editor [6] Roggenbuck D, Reinhold D, Wex T, Goihl A, von Arnim U, Malfertheiner P, et al. Autoantibodies to GP2, the major zymogen granule membrane glycoprotein, are new markers in Crohn's disease. Clin Chim Acta 2011;412:718–24.
Dirk Roggenbuck Faculty of Science, Brandenburg University of Technology, Senftenberg, Germany GA Generic Assays GmbH, Dahlewitz, Berlin, Germany Corresponding author at: Faculty of Science, Brandenburg University of Technology, Großenhainer Str. 57, 01968 Senftenberg, Germany. Tel.: +49 33708 441716; fax: +49 33708 441725. E-mail address: [email protected]. Nadja Röber Institute of Immunology, Medical Faculty of the Technical University Dresden, Dresden, Germany Dimitrios P. Bogdanos Institute of Liver Studies, Division of Transplantation Immunology and Mucosal Biology, King's College London School of Medicine at King's College Hospital, London, UK
83
Alexander Goihl Dirk Reinhold Institute of Molecular and Clinical Immunology, Otto-von-Guericke-University, Magdeburg, Germany Karsten Conrad Institute of Immunology, Medical Faculty of the Technical University Dresden, Dresden, Germany Martin W. Laass Department of Pediatrics, Medical Faculty of the Technical University of Dresden, Dresden, Germany 11 January 2015
