Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 6  |  Issue : 2  |  Page : 104-107

Usefulness of desmoglein 1 and 3 in serodiagnosis of pemphigus vulgaris and its correlation with disease activity - ELISA study


1 Department of Oral Pathology and Microbiology, Pacific Dental College and Hospital, Udaipur, Rajasthan, India
2 Department of Preventive and Community Dentistry, Pacific Dental College and Hospital, Udaipur, Rajasthan, India
3 Department of Oral and Maxillofacial Pathology, 320 Field Hospital, Ambala, Punjab, India
4 Department of Oral Pathology and Microbiology, Maharishi Markandeshwar College of Dental Sciences and Research, Maulana, Ambala, Punjab, India

Date of Web Publication16-Oct-2014

Correspondence Address:
Neha Gandhi
Department of Oral Pathology and Microbiology, Pacific Dental College and Hospital, Udaipur
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0975-8844.143051

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  Abstract 

Background: Pemphigus is an autoimmune blistering disease with three subtypes, which includes pemphigus vulgaris (PV), pemphigus foliaceus (PF) and paraneoplastic pemphigus (PP), which are characterized by circulating autoantibodies to the desmosomal glycoproteins desmoglein 3 (Dsg 3) and Dsg 1. Detection and quantification of these antibodies can be useful in diagnosis of PV. Aims: The purpose of this study was to evaluate the practical application of ELISAs for diagnosis of PV based on immunological reactivity of Dsg 1 and Dsg3. Materials and Methods: Based on clinical presentation and histopathologic confirmation, 10 patients previously diagnosed of PV were included in the study, out of which, five were with active lesions and five were under medication and hence were at remission stages. Sera of the patients were tested for Dsg 1 and Dsg 3 titres by Desmoglien enzyme-linked immunosorbent assay (ELISA) test. Results: Desmoglien ELISA was positive in all the patients, with higher values of Dsg antibody titers in patients with active disease. Dsg 3 titers exceeded the cut off value in patients with oral lesions and titers of both Dsg 1 and Dsg 3 were greater than cut value in patients with mucocutaneous involvement. Conclusion: Thus, the Dsg 1 and Dsg 3 provided objective and quantitative data which allowed differentiation of PV, and in view of these advantages; they are likely to become a routine technique in diagnostic laboratories.

Keywords: Autoimmune disease, Desmoglien 1, Desmoglien 3, ELISA, pemphigus, pemphigus vulgaris


How to cite this article:
Gandhi N, Jain S, Choudhary K, Kumar M. Usefulness of desmoglein 1 and 3 in serodiagnosis of pemphigus vulgaris and its correlation with disease activity - ELISA study . J Orofac Sci 2014;6:104-7

How to cite this URL:
Gandhi N, Jain S, Choudhary K, Kumar M. Usefulness of desmoglein 1 and 3 in serodiagnosis of pemphigus vulgaris and its correlation with disease activity - ELISA study . J Orofac Sci [serial online] 2014 [cited 2020 Sep 20];6:104-7. Available from: http://www.jofs.in/text.asp?2014/6/2/104/143051


  Introduction Top


Pemphigus is an autoimmune blistering skin disease, which is characterized by intra-epithelial blistering (Lever 1953). The two major types of pemphigus are the more severe pemphigus vulgaris (PV), which accounts for 80-90% of cases, and pemphigus foliaceus (PF). PV is caused by autoantibodies against epithelial intercellular components, especially cadherins and particularly desmogleins, which are considered to play an important role in cell to cell adhesion in the stratified squamous epithelia. [1],[2] The major antigens in PV and PF are the desmosomal glyco-proteins desmoglein 3 (Dsg3) and desmoglein 1 (Dsg1), respectively.

The diagnosis of pemphigus rests upon the demonstration of these autoantibodies by immunofluorescence techniques whilst differentiation of PV and PF usually relies upon the combination of clinical and histological features. [3] The method of detecting the autoantibodies largely relies on immunofluorescence testing utilizing both direct immunofluorescence (DIF) and indirect immunofluorescence (IIF). The cell staining pattern using DIF or IIF is virtually identical, making it difficult to distinguish between PV and PF. [2] Enzyme-linked immunosorbent assay (ELISA) had several other advantages including analysis of large numbers of samples in a relatively short time, the data are objective as the optical density of each well can be read automatically and expressed as a numerical value from a continuous scale. This contrasts with IIF which is subjective and in which results are expressed from a series of discontinuous serum dilutions. Other advantages of ELISA are it provides information on antigen identity and can allow PV and PF to be distinguished, provides reproducible, quantitative data which can be beneficial in guiding patient management and it is simple, easily applied. [3] Availability of cDNA clones for the pemphigus antigens has helped to produce recombinant proteins which represent epitopes of the native antigens, including conformational ones. Using these recombinant antigens (rDsg1 and rDsg3), a sensitive and highly specific ELISA system for detection of autoantibodies against Dsg1 and Dsg3 has been developed. The purpose of this study was to evaluate the practical application of these ELISA tests for the serological diagnosis for PV and PF. The demonstration of reactivity against either Dsg3 or Dsg1 indicates a diagnosis of pemphigus. Moreover, combined ELISA results against Dsg3 and Dsg1 may help to differentiate PV and PF. [2]


  Materials and methods Top


Clinically, patients with PV show majors signs of vesicles formation along with positive Nikolsky sign. Histopathologically, there is formation of intraepithelial cleft and cytological findings shows the presence of Tzanck cells in the smear. Ten patients, clinically and cytopathologically diagnosed as PV earlier, were included in the study. Five patients were having active lesions, of which three were with mucosal (oral) lesions and two with both oral and cutaneous lesions. Another five patients included were at remission stages of which, again, three with mucosal (oral) lesions and two with oral and cutaneous lesions. 3-4 ml of blood samples were collected to carry out Desmoglein ELISA test for detection and quantification of Dsg autoantibodies in sera. (MESACUP Desmoglein Test 'Dsg3' and Dsg1').

Serum samples were sent to laboratory and ELISA test was performed according to the manufacturer's instructions. [4] Calibrators and patient sera are added to microwells coated with rDsg1 and rDsg3. Antibodies react with immobilized antigen (Sample Incubation). After washing, horseradish peroxidase conjugated anti-human IgG monoclonal antibody is added (Conjugate incubation). Then peroxidase substrate was added for color development (Substrate incubation). Acid solution was then used to terminate enzyme reaction and to stabilize the color development. The color developed was read as the absorbance of each well at 450 nm with microtitre plate reader in optical density (OD) units. Intensity of color quantifies the antigen-antibody reaction. [2],[4]

Calculations

Unit value of antibody titers (U/ml) = {OD of serum sample - OD of negative Calibrator/OD of Dsg calibrator - OD of negative Calibrator} × 100

OD (optical density) = absorbance value at 450 nm

The cut-off (index) value as established by the manufacturer for anti-Dsg3 was 20 U/ml and that for anti-Dsg1 was also 20 U/ml. This was established with assaying 179 normal serums, 60 PV patient serums and 246 patient serums other than PV.

The test interpretation as set by the manufacturer was as follows: [4]

Anti-Dsg 3 Value (U/ ml)

≥20 positive

20 > − ≥7 intermediate

7 > negative

Anti-Dsg 1 Value (U/ ml)

≥20 positive

20 > − ≥14 intermediate

14 > negative


  Results Top


Dsg3 was positive (greater than cut off value) in all the patients with clinically diagnosed PV.

In patients with active oral lesions: Unit values of Dsg3 ELISA were greater than cut off values and the values of Dsg1 were less than the cut off values [Table 1].
Table 1: Patients with active lesions (Cut off value: >20 U/ml)

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In patients with active mucocutaneous lesions unit values of both Dsg3 and Dsg1 were greater than cut off values [Table 1].

In patients at remission stages, lesions involving both mucosal and mucocutaneous showed that the unit values of Dsg3 were greater than cut off values. And the values for Dsg1 were less than the cut-off values [Table 2].
Table 2: Patients with remission (Cut off value: >20 U/ml)

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Comparing unit values for Dsg3 and Dsg1 in patients with active lesions and those at remission stages, the Dsg titers were relatively higher in former [Table 2].


  Discussion Top


Pemphigus is a group of potentially life-threatening autoimmune mucocutaneous diseases characterized by epithelial blistering affecting cutaneous and/or mucosal surfaces, the term being derived from the Greek 'Pemphix' (bubble ot blister). [1] PV and PF are characterized by antibodies to the desmosomal proteins Dsg3 and Dsg1, respectively. [5]

Histopathology is still used as the routine diagnostic test for pemphigus. Though DIF is essential in the diagnostic work-up of pemphigus diseases, its sensitivity is not 100%. In patients with the disease duration less than 3 months, it is shown than DIF provided sensitivity as low as 46.7%. [6],[7]

The Dsg1 and Dsg3 ELISAs have provided a sensitive and specific diagnostic tool for both PV and PF and these immunoassays are widely used in dermatology. These ELISAs have also been shown to be useful for monitoring disease status. [8],[9] The ELISA is based on an enzyme reaction. In our assay, tetramethyl-benzidine (TMB) is oxidized in the presence of horseradish peroxidase (HRP) conjugated with antihuman IgG antibodies. This reaction results ina detectable colour change, measured by absorbance or optical density (OD), which in turn is related to the amount of HRP and thus indirectly to the quantity of anti-Dsg antibodies. The maximum OD depends on the epitope density of the antigen coated on the plate. Therefore, there is a particular antibody concentration range that results in a linear, dose-dependent correlation between the amount of anti-Dsg antibodies and final OD. When sera contain an excess amount of anti-Dsg antibodies, however, the antigen-antibody reaction may be saturated so that the final OD obtained does not necessarily represent an accurate estimate of the concentration of anti-Dsg autoantibodies.

ELISA was run with the appropriately diluted sera and true index values were calculated by the manufacturer. [10] In the present study, ELISA kit with the index value more than 20 U/ml was considered as positive result. [4]

In this study we studied 10 patients, histologically diagnosed as pemphigus, out of which five were with active disease and five were under medication at remission stages. We demonstrated that the unit values of Dgs antibodies were higher than the cut off values in both group of patients. We also demonstrated that index values obtained with appropriately diluted sera do reflect disease activity. Anti Dsg3 antibodies values were positive in all patients of both groups of mucosal as well as mucocutaneous involvement (100%). Anti Dsg1 were positive in three out four patients (75%) with mucocutaneous lesions while in patients with oral mucosal involvement, it was negative. Our results were in accordance with similar studies carried out by various authors. [2],[3],[5] The study also suggested that sera of patients with mucocutaneous lesion reacted either with Dsg3 alone or with both Dsg3 and Dsg1, while sera of patient with mucosal involvement react exclusively with Dsg3. These findings were similar to the study carried out by Komai A. et al., [11] and Herman K. E et al. [12]

When the unit values of Dsg1 and 3 were compared in patients with active disease and in the patients under medication, it was found that antibody titers were higher in patients with active disease. These findings were supported by a recent study using an ELISA that allows quantitative measurementof Dsg1 and Dsg3 antibody levels, which demonstrated an association between Dsg3 antibody levels and oral disease severity which suggested that there was a general trend of increasing oral disease severity and increasing Dsg3 ELISA value. [5],[13] The provision of such quantitative data may ultimately prove to be beneficial in guiding patient management.

Thus, in mucocutaneous lesions antibodies are directed against both Dsg1 and 3 and mucosal lesions involve antibodies against Dsg3. Thus a positive Dsg3 result was indicative of PV, regardless of associated Dsg1 result. No case was found solely positive for Dsg1 in this study.

The Dsg1 and Dsg3 ELISAs, are useful in cases of pemphigus with autoantibodies to either Dsg1 or Dsg3 but a negative result does not dismiss the possibility of a rarer subtype of pemphigus with autoantibodies to other antigens. [12] The cases of PV and PF chosen for this evaluation study had not posed diagnostic difficulty and the ELISAs simply confirmed the diagnosis.

Therefore, these ELISAs are useful diagnostic and disease monitoring tools but more traditional diagnostic techniques may be needed in some cases. The cases of PV chosen for this evaluation study had not posed diagnostic difficulty and the ELISAs simply confirmed the diagnosis.


  Conclusion Top


In summary, the Dsg1 and Dsg3 ELISAs using recombinant pemphigus antigens, when used together, provide a sensitive, specific and quantitative diagnostic tool for the detection of anti-Dsg1 and anti-Dsg3 autoantibodies. This new diagnostic tool may be useful to make a proper diagnosis and evaluate patients with pemphigus to understand the fundamental pathophysiological mechanisms of pemphigus. Although the diagnosis of pemphigus is mainly based on histopathologic examinations and immunofluorescence tests, our study demonstrated that the addition of Dsg ELISA to the above-mentioned tests can increase the diagnostic yield.

 
  References Top

1.
Scully C, Challacombe SJ. Pemphigus vulgaris: Update on etiopathogenesis, oral manifestations, and management. Crit Rev Oral Biol Med 2002;13:397-408.  Back to cited text no. 1
    
2.
Amagai M, Komai A, Hashimoto T, Shirakata Y, Hashimoto K, Yamada T, et al. Usefulness of enzyme-linked immunosorbent assay using recombinant desmogleins 1 and 3 for serodiagnosis of pemphigus. Br J Dermatol 1999;140:351-7.  Back to cited text no. 2
    
3.
Harman KE, Gratian MJ, Seed PT, Bhogal BS, Challacombe SJ, Black MM. Diagnosis of pemphigus by ELISA: A critical evaluation of two ELISAs for the detection of antibodies to the major pemphigus antigens, desmoglein 1 and 3. Clin Exp Dermatol 2000;25:236-40.  Back to cited text no. 3
    
4.
MESACUP desmolglein 1 and 3 ELISA Test (CE marked). MBL medical and biological laboratories pvt. Ltd. Registered office: White leaf Business Centre, 11 Little Balmer, Buckingham, MK18 1TF.  Back to cited text no. 4
    
5.
Harman KE, Seed PT, Gratian MJ, Bhogal BS, Challacombe SJ, Black MM. The severity of cutaneous and oral pemphigus is related to desmoglein 1 and 3 antibody levels. Br J Dermatol 2001;144:775-80.  Back to cited text no. 5
    
6.
Aithal V, Kini U, Jayaseelan E. Role of immunofluorescence on Tzank smears in pemphigus vulgaris. Diagn Cytopathol 2007;35:403-7.  Back to cited text no. 6
    
7.
Helander SD, Rogers RS 3rd. The sensitivity and specificity of direct immunofluorescence testing in disorders of mucous membranes. J Am Acad Dermatol 1994;30:65-75.  Back to cited text no. 7
    
8.
Mortazavi H, Shahdi M, Amirzargar AA, Naraghi ZS, Valikhani M, Daneshpazhooh M, et al. Desmoglein ELISA in the diagnosis of pemphigus and its correlation with the severity of pemphigus vulgaris. Iran J Allergy Asthma Immunol 2009;8:53-6.  Back to cited text no. 8
    
9.
Ishii K, Amagai M, Hall RP, Hashimoto T, Takayanagi A, Gamou S, et al. Characterization of autoantibodies in pemphigus using antigen-specific ELISAs with baculovirus expressed recombinant desmogleins. J Immunol 1997;159:2010-7.  Back to cited text no. 9
    
10.
Cheng SW, Kobayashi M, Tanikawa A, Kinoshita-Kuroda K, Amagai M, Nishikawa T. Monitoring disease activity in pemphigus with enzyme-linked immunosorbent assay using recombinant desmogleins 1 and 3. Br J Dermatol 2002;147:261-5.  Back to cited text no. 10
    
11.
Komai A, Amagai M, Ishii K, Nishikawa T, Chorzelski T, Matsuo I, et al. The clinical transition between pemphigus foliaceus and pemphigus vulgaris correlates well with the changes in autoantibody profile assessed by an enzyme-linked immunosorbent assay. Br J Dermatol 2001;144:1177-82.  Back to cited text no. 11
    
12.
Vu TN, Lee TX, Ndoye A, Shultz LD, Pittelkow MR, Dahl MV, et al. The pathophysiological significance of nondesmoglein targets of pemphigus autoimmunity. Development of antibodies against keratinocyte cholinergic receptors in patients with pemphigus vulgaris and pemphigus folliaceus. Arch Dermatol 1998;134:971-80.  Back to cited text no. 12
    
13.
Abasq C, Mouquet H, Gilbert D, Tron F, Grassi V, Musette P, et al. ELISA testing of anti-desmoglein 1 and 3 antibodies in the management of pemphigus. Arch Dermatol 2009;145:529-35.  Back to cited text no. 13
    



 
 
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