Development of NC1 and NC2 domains of Type VII collagen ELISA for the diagnosis and analysis of the time course of epidermolysis bullosa acquisita patients

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Abstract

Background

Epidermolysis bullosa acquisita (EBA) is an acquired autoimmune mechanobullous disease. EBA patients possess autoantibodies against type VII collagen which is composed of a collagenous domain flanked by non-collagenous NC1 and NC2 domains. It was reported that major epitopes reside within the NC1 domain and minor epitopes reside within NC2 domain.

Objective

The aim of this study is to develop a sensitive and specific ELISA to facilitate the diagnosis of EBA.

Methods

We developed ELISAs using recombinant NC1 domain produced by mammalian expression system and recombinant NC2 domain produced by mammalian or bacterial expression system to characterize autoantibodies in EBA. Next, we developed an ELISA using a combination of the NC1 (mammalian expression) and NC2 (bacterial expression). We tested the ELISAs with 49 EBA sera, 55 normal control sera, 20 pemphigus vulgaris and 20 bullous pemphigoid sera.

Results

When we evaluated the 49 EBA sera using the NC1 and NC2 ELISAs, 38 (77.5%) reacted with NC1 domain only, 7 sera (14.2%) reacted with both NC1 and NC2 domains, and one serum (2%) reacted with NC2 domain only. Therefore, to increase the sensitivity of the assay, we developed an ELISA coated with a mixture of recombinant NC1 and NC2 domains, resulting in 93.8% sensitivity and 98.1% specificity. By analyzing the time course of two EBA patients, ELISA scores fluctuated in parallel with their disease activity.

Conclusion

We conclude that the NC1 + NC2 ELISA can be a practical assay for the diagnosis and follow up of the antibody titers of EBA patients.

Introduction

Epidermolysis bullosa acquisita (EBA) is an acquired mechanobullous disease characterized by the presence of autoantibodies against type VII collagen. The etiology of EBA is unknown, however an autoimmune pathogenesis is suggested by the demonstration of IgG deposits at the dermo-epidermal junction of EBA patients by immunofluorescence [1], [2], [3], [4].

The diagnostic clinical criteria for EBA are trauma-induced blisters that heal with milia and scarring in the absence of family history of epidermolysis bullosa. The immunopathological criteria are subepidermal blisters and linear deposits of IgG in the basement membrane zone (BMZ). The demonstration of IgG autoantibodies labeling the dermal side of salt-split skin is the most specific diagnostic assay [5].

Type VII collagen is a structural component of anchoring fibrils which stabilize the dermo-epidermal adherence. Type VII collagen is composed of central collagenous three identical alpha helical chains. Each alpha chain is flanked on one side by a 145 kDa amino-terminal non-collagenous domain (NC1) and on the other side by a 34 kDa carboxyl-terminal noncollagenous domain (NC2) (Fig. 1A). Within the extracellular space type VII collagen forms an antiparallel tail-to-tail dimers stabilized by disulphide bonding at NC2 region. The antiparallel dimers aggregate laterally to form globular NC1 domains at both ends of the structure [6], [7], [8].

The major antigenic epitopes of type VII collagen are located within the NC1 domain of type VII collagen [9], [10]. NC2 domain of type VII collagen contains minor antigenic epitopes [11], [12]. Moreover, some EBA cases bind to the central collagenous domain of type VII collagen [13], [14], [15].

Conventional assays used for the diagnosis of EBA are direct immunofluorescence (IF) using the patient's own biopsy, indirect IF using the patient's own serum, salt-split skin IF and immunoblot analysis using dermal extract. Direct and indirect IF cannot differentiate EBA from bullous pemphigoid (BP) because IF shows linear deposits of IgG in the BMZ in both diseases. Salt-split skin IF is highly specific for the diagnosis of EBA [5], [16], however it is not routinely performed in many centers. Immunoblot cannot detect autoantibodies against conformation-dependent epitopes because it uses denatured and reduced antigens as substrates.

In this study, we sought to develop a practical ELISA to detect autoantibodies against type VII collagen for the diagnosis of EBA. Several different ELISAs have been reported previously for characterizing autoantibodies in EBA [9], [10], [12], [17]. However the previous ELISAs utilized only the NC1 domain or the subdomain of the NC1 of type VII collagen. Here we evaluated autoantibodies in EBA using NC1 domain ELISA and NC2 domain ELISA and found that small percentage of EBA sera bound exclusively to the NC2 domain. Therefore, we developed an ELISA using a combination of the NC1 and NC2 domains of type VII collagen and evaluated the usefulness of the ELISA for the diagnosis and monitoring of the disease activity in EBA.

Section snippets

Patients and sera

Serum samples were obtained from 49 EBA patients. All patients examined in this study had typical clinical and histological features of EBA. The diagnosis of all patients was confirmed by detection of IgG deposits at the dermal side of dermo-epidermal junction by indirect IF of salt-split skin. Healthy control sera were obtained from 55 normal subjects. Disease control sera were obtained from 20 patients with BP and 20 patients with pemphigus vulgaris. The diagnosis was confirmed by the

ELISA coated recombinant NC1 domain

To develop a sensitive and specific ELISA for detecting the autoantibodies against Type VII collagen in EBA patients, we first produced recombinant protein of NC1 which contains the entire 1253 residues of the NC1 domain of type VII collagen by mammalian expression system. The purified recombinant NC1 domain produced by stably transfected CHO cells were immobilized on microtiter plates (Fig. 1B). We tested NC1 mammalian expression type VII collagen domain ELISA using EBA sera, normal control

Discussion

EBA is considered a challenging disease both in the diagnosis and in the treatment. In this study, we tried to develop a sensitive and specific ELISA to detect anti-type VII collagen autoantibodies in EBA patients for practical use. We developed 3 ELISAs: NC1 domain (mammalian), NC2 domain (mammalian or bacterial), combination of NC1(mammalian) + NC2 (bacterial). We evaluated them with 49 EBA sera, disease control sera and normal control sera. We found that combination of NC1 (mammalian) + NC2

Conflict of interest

Ms Kim and Dr. Murakami belong to Medical and Biological Laboratories Co. Ltd. who supply anti-type VII collagen ELISA kit described in this study.

Acknowledgements

We would like to thank Mrs. Minae Suzuki for technical assistance. We also would like to thank Dr. Tetsuya Yoshida and Dr. Jun Yamagami for valuable discussion. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Health and Labour Sciences Research Grants for Research on Measures for Intractable Diseases from Ministry of Health, Labour and Welfare of Japan.

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Present Address: Department of Dermatology, Teikyo University Chiba Medical Center, 3426-3 Anesaki , Ichihara, Chiba 299-0111 Japan.

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