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, 231–236

Blackwell Science, Ltd

Comparison of three monoclonal and three polyclonal antibodies in the immunohistochemical diagnosis of canine autoimmune

skin diseases

JOSÉ PÉREZ,* CRISTINA ARCE,† ANGELA MORENO,† ELENA MOZOS,* FRANCISCO RODRÍGUEZ‡ and DIEGO LLANES†

*Departamento de Anatomía y Anatomía Patológica Comparadas, Edificio de Sanidad Animal, Córdoba, Spain †Unidad mixta CSIC-Universidad de Córdoba, Departamento de Genética, Córdoba, Spain ‡Unidad de

Anatomía y Anatomía Patológica Comparadas, ULGC, Spain

(

Received

26

February

2002;

accepted

13

June

2002)

Abstract

The aim of this study was to evaluate the utility of three monoclonal antibodies (mAbs), and two anti-canine IgG and one anticanine IgM polyclonal antibodies (pAbs) for the immunohistochemical diagnosis ofcanine autoimmune skin diseases. Skin biopsies from 11 cases of pemphigus (7 foliaceus, 3 vulgaris and 1 ery-thematosus), 12 cases of discoid lupus erythematosus (DLE) and 12 cases of chronic hyperplasic dermatitis wereused. The CA4E7 mAb (IgG

1

+ IgG

2

) showed similar sensitivity, but higher specificity and lower backgroundthan the two anti-IgG pAbs for the immunohistochemical diagnosis of pemphigus and DLE. The CA4F1 mAb(IgG

2

) and CA3H1 mAb (IgG

2

) showed moderate and low interepithelial reactivity, respectively, in autoimmuneskin diseases, but strong staining of the cytoplasm of plasma cells of the inflammatory infiltrates. These resultssuggest that the CA4E7 mAb may be valuable in the immunohistochemical diagnosis of such disorders.

Keywords

:

autoimmune skin diseases, discoid lupus erythematosus, dog, IgG

1

, IgG

2

, pemphigus

INTRODUCTION

Canine autoimmune skin diseases are relatively un-common, but clinically challenging because an accuratediagnosis is of crucial importance for correct therapyand management.

1

The diagnosis is often difficult,particularly when secondary lesions occur, and re-quires clinical evaluation and the demonstration of thetypical histopathological features in multiple skinbiopsies.

1–3

The demonstration of immunoglobulins (IgG orIgM) with an interepithelial pattern or in the basementmembrane of the epidermis has been used as a comple-mentary tool for the diagnosis of canine autoimmuneskin diseases. Both immunofluorescence and immuno-peroxidase methods have been used.

4–13

More recently,circulating autoantibodies have also been detected.

14,15

The immunoperoxidase method is more sensitiveand has higher morphologic quality than the immuno-fluorescence technique.

6,10

Despite the value of thesetechniques as complementary tools for the diagnosisof canine autoimmune skin diseases, they do have vari-able sensitivity, which ranges from 52 to 100%.

2,6–11

Inaddition, the specificity of these methods is also vari-able as numerous cases of chronic inflammatory skin

diseases, such as deep pyoderma,

8,11

hypersensivitydermatitis,

11

scabies, demodicosis and dermatomyco-sis,

8

may yield positive interepithelial staining similarto that found in autoimmune skin diseases. Moreover,normal nose and footpads of the dog reacted with anti-IgM in 73 and 45% of cases, respectively.

5,16

Only one study has evaluated the usefulness of threemonoclonal antibodies (mAbs), against IgG

2

, IgG

3

and IgG

4

, in the immunohistochemical diagnosis ofautoimmune skin diseases, and these had significantlylower sensitivity than polyclonal reagents.

12

The aim ofthis study was to analyse the distribution of three newmAbs which recognize IgG

1

and IgG

2

, two anticanineIgG polyclonal antibodies (pAbs), and one anticanineIgM pAb in skin biopsies of 23 cases of canine auto-immune skin diseases and 12 cases of nonautoimmunechronic dermatitis.

MATERIALS AND METHODS

Tissue samples

Skin biopsies from 23 dogs with autoimmune skindiseases: pemphigus foliaceus (7 cases), pemphigus vul-garis (3 cases), pemphigus erythematosus (1 case) anddiscoid lupus erythematosus (DLE, 12 cases) wereused. Skin biopsies from 12 dogs with chronic nonau-toimmune skin diseases including allergic dermatitis(4 cases), chronic primary pyoderma (5 cases), chronicallergic dermatitis associated with pyoderma (3 cases)

Correspondence: J. Pérez, Department Anatomía y AnatomíaPatológica Comparadas, Edificio de Sanidad Animal, Campus deRabanales, Ctra. Madrid-Cádiz km 396, 14014 Córdoba, Spain.Tel.: +34-57-218178; Fax: +34-57-214488; E-mail: an1pearj@uco.es

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and 6 histologically normal canine skin biopsies werealso included. All skin biopsies were selected from thefiles of the Diagnostic Service of the Department ofComparative Pathology of the Veterinary Faculties ofCórdoba and Las Palmas de Gran Canaria (Spain).The initial diagnosis was based on clinical and his-topathological evaluations. After re-examination ofhaematoxylin and eosin (H&E)-stained tissue sections,cases with characteristic histopathological featuressuch as subcorneal pustules with acanthocytes, sub-corneal clefts, mononuclear or mixed interface derma-titis, hydropic changes or degenerate and eosinophilickeratinocytes, which may represent apoptotic cells,

2,3

were included in the study. Skin biopsies were fromdogs of different breed, age and sex. Anatomical loca-tions from which biopsies were taken were also vari-able, but none of them was from the nose or footpads.Skin biopsies were fixed in 10% neutral-buffered for-malin and embedded in paraffin wax. Four-micrometrethick tissue sections were stained with H&E and Tolu-idine Blue for routine histopathological examination.

Monoclonal antibodies

Details of the production and characterization ofthe three monoclonal antibodies (CA4E7, CA3H1 andCA3F1) have been described elsewhere.

17

The specifi-city of each mAb was determined by indirect enzyme-linked immunosorbent assay (ELISA) using variouscanine immunoglobulins and subfractions, includingIgG

1

, IgG

2

, IgM, IgA (Bethyl Laboratory, Inc, Mont-gomery, USA), heat-treated IgG (Sigma Chemical,St Louis, MO, USA) and acid-treated IgG (SigmaChemical).

17

CA3H1 and CA3F1 reacted only withIgG

2

, whereas CA4E7 reacted with IgG

1

, IgG

2

, IgMand IgA.

17

Immunohistochemistry

The avidin

−

biotin

−

peroxidase complex (ABC) methodwas used. Endogenous peroxidase activity was blockedby incubation with hydrogen peroxide 0.3% in meth-anol for 30 min. Tissue sections were incubated with0.1% pronase (Sigma Chemical) for 10 min at roomtemperature. Tissue sections were then incubated with5% normal goat serum for 30 min at room tempera-ture. Primary antibodies were incubated overnight at4

°

C (see details in Table 1). After three 10-min rinsesin 0.01

PBS (pH 7.2), a biotinylated goat antirabbit

immunoglobulin (Vector Laboratories, Burlingame,CA, USA) diluted 1 in 200 in PBS was applied for30 min at room temperature (20–25

°

C) for primarypAbs, and a biotinylated goat antimouse immuno-globulin (Dako, Glostrup, Denmark) diluted 1 in 100in PBS was used for 30 min at room temperature assecondary antibody for the primary mAbs. An ABCcomplex (Vector) diluted 1 in 50 in PBS was then appliedfor 1 h at room temperature. As chromogen, 3,3

′

-diaminobenzidine tetrahydrochloride (Sigma), dilutedto 0.035% in Tris-buffered saline containing hydrogenperoxide 0.01%, was applied for 1 min. Tissue sectionswere then rinsed in tap water, counterstained withMayer’s haematoxylin for 1 min, dehydrated andmounted under DPX. Tissue sections in which the spe-cific primary antibodies were replaced by PBS or rabbitnonimmune isotypic sera were used as negative con-trols. Tissue sections of canine lymph nodes were usedas positive controls to check the reactivity of theantibodies used. Prior to this study, different dilutions(1/50, 1/100, 1/200 and 1/500) of both primary and sec-ondary antibodies, as well as two different antigenretrieval methods (incubation with 0.1% pronase for10 min at room temperature, and incubation in 0.01

citric acid in microwave at 100

°

C for 5 min), weretested on canine lymph node tissue sections to achievethe optimal working conditions.

Criteria for a positive reaction were a consistentintercellular labelling and/or staining of the basalmembrane of the epidermis. Diffuse cytoplasmic stain-ing of keratinocytes and/or focal intercellular stainingwas not considered a positive reaction.

RESULTS

None of the five antibodies used reacted with the epi-dermis of the control skin samples with an interepithe-lial pattern. The three mAbs and the two anticanineIgG pAbs reacted strongly with numerous plasmacells of the medullary cords of control lymph nodes,whereas the anticanine IgM pAb labelled smaller num-bers of plasma cells of the control lymph nodes.

Autoimmune skin diseases

The results of the immunohistochemical study aresummarized in Table 2. The CA4E7 mAb yielded a

AntibodyProtein conc* (mg mL−1) Dilution Specificity Source

CA4E7 2.13 1 : 100 IgG1, IgG2† Biovet-UCO‡CA3H1 1.6 1 : 100 IgG2 Biovet-UCO‡CA4F1 1.06 1 : 100 IgG2 Biovet-UCO‡anti-IgG (pAb1) NA 1 : 200 IgG (H + L) Nordic§anti-IgG (pAb2) NA 1 : 200 IgG (H + L) ICN Biomedical¶Anti-IgM NA 1 : 100 IgM (Fc) Nordic§

*Determined by Bio-Rad Protein Assay (Bio-Rad Laboratories GmbH, München, Germany). †This antibody also yielded cross-reactivity with IgM and IgA.17 ‡Córdoba, España. §Tilburg, the Netherlands. ¶Aurora, OH, USA. NA: not available in the manufacturer’s specification sheet.

Table 1. Details of primary monoclonal andpolyclonal antibodies for canine IgG used inthe immunohistochemical study

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Diagnosis of canine autoimmune skin diseases 233

positive interepithelial reaction with the 11 cases ofpemphigus (Fig. 1a) analysed and with 11 of the 12cases of DLE. The reaction was intercellular and/orin the basement membrane of the epidermis, either inzones with typical histopathological lesions of auto-immune disease, such as subcorneal pustules with acan-tholysis, or in zones in which the epidermis was devoidof these changes. Diffuse cytoplasmic labelling ofkeratinocytes was absent or weak in all skin biopsies.

The CA4F1 mAb labelled 6 cases of pemphigusfoliaceus (Fig. 1b), 2 of pemphigus vulgaris, and 6 ofthe 12 cases of DLE. This mAb presented a very cleanbackground and interpretation of the results was there-fore easy. However, the reactivity in the epidermis wasweaker than that obtained with the CA4E7 mAb.

The CA3H1 mAb labelled only two cases of pemphigusfoliaceus, one of pemphigus vulgaris and four of DLE,whereas the remaining cases were negative. This mAbshowed very low background in the epidermis anddermis. However, it strongly stained the cytoplasm ofnumerous plasma cells in the inflammatory infiltrates ofboth autoimmune and nonautoimmune skin diseases.

The anticanine IgG pAb1 labelled six cases ofpemphigus foliaceus, two of pemphigus vulgaris, oneof pemphigus erythematosus and eight of DLE. Themajority of skin biopsies analysed showed a diffusestaining of the epidermis that made interpretationdifficult (Fig. 1c). This antibody also labelled plasmacells in the inflammatory infiltrate of both autoimmuneand nonautoimmune skin diseases, but the backgroundstaining of collagen fibres was higher than thoseobtained with the three mAbs.

The anticanine IgG pAb2 labelled 11 cases of pem-phigus, 10 of DLE and 6 of nonautoimmune chronic

dermatitis with an interepithelial pattern. This pAbyielded less background in the epidermis and dermisthan the pAb1, but background was still higher thanthe three mAbs. The immunostaining of plasma cellswas less strong and the background staining of dermalcollagen was higher than that obtained with the threemAbs.

The anticanine IgM showed interepithelial and/or basal membrane staining in two cases of pemphigusfoliaceus, one of pemphigus vulgaris and two of DLE.

Nonautoimmune skin diseases

Results of the immunohistochemical study are summa-rized in Table 3. The CA4E7 mAb stained only with aninterepithelial pattern one case of nonautoimmune skindisease of the 12 cases analysed, and it labelled plasmacells of the inflammatory infiltrate both in autoimmuneand nonautoimmune skin diseases. The CA4F1 andthe CA3H1 antibodies did not show interepithelial orbasement membrane staining in the 12 cases of non-autoimmune chronic dermatitis analysed, but bothlabelled plasma cells of the inflammatory infiltrates inautoimmune and nonautoimmune skin diseases.

The anti-IgG (pAb1 and pAb2) showed interepithe-lial and/or basal membrane staining in 6 of the 12 casesof nonautoimmune chronic dermatitis, and the anti-IgM pAb in 2 cases.

DISCUSSION

The results of this study showed that the CA4E7 mAbreacted with 100% of cases of pemphigus and with 91.7%of cases of DLE. Moreover, it had good specificity as

Table 2. Number of positive cases and (sensitivity) obtained with the different antibodies for the immunohistochemical diagnosis of canineautoimmune skin diseases

Skin diseaseAntibodies CA4E7 CA3H1 CA4F1 IgG pAb1 IgG pAb2 IgM

Pemphigus foliaceus (n = 7) 7 (100)* 2 (28.6) 6 (85.7) 6 (85.7) 7 (100) 1 (14.3)Pemphigus vulgaris (n = 3) 3 (100) 1 (33.3) 2 (66.7) 2 (66.7) 3 (100) 1 (33.3)Pemphigus erythematosus (n = 1) 1 (100) 0 (0) 0 (0) 1 (100) 1 (100) 0 (0)Total pemphigus (n = 11) 11 (100) 3 (27.3) 8 (72.3) 9 (81.8) 11 (100) 2 (5.5)Discoid lupus erythematosus (n = 12) 11 (91.7) 4 (33.3) 6 (50) 8 (66.7) 10 (83.3) 1 (9.1)

N, number of cases analysed. *Number of positive cases (percentage of positive cases or sensitivity for the immunohistochemical diagnosis).

Table 3. Number of positive cases and (percentage of positive cases) obtained with the different antibodies in normal skin and nonautoimmunecanine skin diseases

Skin Disease/Specificity†

Antibodies CA4E7 CA3H1 CA4F1 IgG pAb1 IgG pAb2 IgM

Normal skin (n = 6) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)Allergic dermatitis (n = 4) 0 (0)* 0 (0) 0 (0) 1 (25) 1 (25) 0 (0)Pyoderma (n = 5) 0 (0) 0 (0) 0 (0) 3 (60) 3 (60) 1 (20)Allergic dermatitis and pyoderma (n = 3) 1 (33.3) 0 (0) 0 (0) 2 (66.7) 2 (66.7) 1 (33.3)Total nonautoimmune skin diseases (n = 12) 1 (8.3) 0 (0) 0 (0) 6 (50) 6 (50) 2 (16.7)Specificity† 91.7% 100% 100% 50% 50% 83.3%

N, number of cases analysed. *Number of positive cases (percentage of positive cases). †The specificity of the antibodies for the immunmohistochemical diagnosis of autoimmune skin diseases was calculated as follows: 100 – percentage of positive cases of nonautoimmune skin diseases.

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it reacted with only 1 of the 12 cases of chronic non-autoimmune dermatitis. These results suggest that thesensitivity and specificity of this antibody for the diag-nosis of canine autoimmune skin diseases was higherthan that obtained with the anticanine IgG (pAb1 andpAb2) and anticanine IgM polyclonal antibodies. Thehigh variability of the sensitivity of immunohistochem-ical techniques in the diagnosis of canine autoimmuneskin diseases reported from 52

2

to 100%

6

may havebeen due, at least in part, to the different reagents (parti-cularly primary antibodies) used. In our study, differ-ences among the various antibodies were consistent.Thus, the CA4E7 mAb showed similar sensitivity tothe pAb2 and moderately higher sensitivity to thepAb1 for the diagnosis of different types of pemphigusand DLE. However, the number of false-positive casesin the group of chronic hyperplastic nonautoimmunedermatitis was markedly higher with both pAbs (50%)than with the CA4E7 mAb (8.3%).

The CA4F1 and CA3H1 mAbs showed moderateand low sensitivity for the diagnosis of canine autoim-mune skin disease, respectively. These results are similarto that reported previously with anticanine IgG

2

,

12

although the mAbs used in this study and used by Day& Mazza

12

were characterized using IgG subfractionsfrom different companies, and therefore may not beequivalent. The CA4E7 mAb reacted with both IgG

1

and IgG

2

and also showed weak cross-reaction withcanine IgM and IgA.

17

However, the low numbers ofcases of pemphigus and DLE positive with the anti-canine IgM, and the very low prevalence of both IgMand IgA in the epidermis of canine autoimmune skin dis-eases,

6,10

suggest that the higher interepithelial reactiv-ity obtained with the CA4E7 mAb was not due to IgMand IgA deposition.

The high number of plasma cells stained with thethree mAbs, particularly in cases of DLE, coincideswith the abundant infiltration of IgG

2

- and IgG

4

-positive plasma cells in the inflammatory infiltrates ofcanine autoimmune skin diseases.

12

However, all thesemAbs showed low reactivity with interepithelial immu-noglobulins in autoimmune skin diseases. It is possiblethat the epitope detected by such anti-IgG

2

mAbs isaltered in IgG autoantibodies that bind to keratino-cytes, but not in immunoglobulins contained in thecytoplasm of plasma cells.

12

Figure 1.

Pemphigus foliaceus. (a)

CA4E7. Interepithelial immunoreactivity (arrow) in the epidermis adjacent to a pustule

×

100. (b)

CA3F1.Similar area showing weak interepithelial staining (arrow)

×

200. (c) CA3H1. Labelling is only observed in some plasma cells of the infiltrate(arrowhead)

×

200. (d)

pAb1. The immunostaining of the epidermis is very weak (arrow)

×

100. ABC method.

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Diagnosis of canine autoimmune skin diseases 235

The interepithelial reactivity of the two anticanineIgG (pAb1 and pAb2) was high in pemphigusfoliaceus, vulgaris and DLE, coinciding with theresults of previous studies using immunofluorescenceand immunoperoxidase techniques.

2,6–9,11

Comparedwith the anti-IgG mAbs tested here, the pAb1 andpAb2 showed a higher background in keratinized lay-ers of the epidermis, which made the observation of theinterepithelial pattern difficult. Moreover, the specifi-city obtained with both antibodies was low (50% offalse positive in cases of chronic nonautoimmune skindermatitis). In previous studies using anticanine IgGfrom the same commercial source as the pAb1 usedhere, interepithelial reactivity was obtained in 11 of 14samples of dogs with nonautoimmune chronic inflam-matory skin lesions,

11

in 12 of 13 dogs with deeppyoderma and in 3 of 8 cases of hypersensitivitydermatitis.

12

In another study a sensitivity of 38 and21% of false-positive cases was obtained with poly-clonal antibodies and the direct immunofluorescencemethod.

2

These results confirm that the use of suchantibodies is valuable only together with clinical andhistopathological evaluation.

The low interepithelial and/or basal membranestaining of the anti-IgM pAb in both pemphigusdiseases and DLE agrees with the results of previousstudies,

6,8,10,11

and confirms that IgM antibodies areof low value in the immunohistochemical diagnosis ofcanine autoimmune skin diseases.

In conclusion, the higher specificity, sensitivity andless background obtained with the CA4E7 mAb com-pared with that of the two anti-IgG pAbs, suggests thatthis mAb may be of value in the immunohistochemicaldiagnosis of canine autoimmune skin diseases, togetherwith the clinical and histopathological features. Theresults of this study suggest that IgG

1

rather than IgG

2

is predominantly involved in the pathogenesis of caninepemphigus and DLE.

ACKNOWLEDGEMENTS

This work was supported by grants: AGR137 (Juntade Andalucía) and C., Arce was supported by grants ofthe Spanish Ministry to Science and, Technology andBIOVET-UCO.

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Résumé Le but de ce travail était d’évaluer l’intérêt de trois anticorps monoclonaux (mAbs), de deux anticorpspolyclonaux anti-IgG canines et d’un anticorps polyclonal antiIgM canin (pAbs) pour le diagnostic immuno-histochimique des maladies auto-immunes chez le chien. Des biopsies cutanées de 11 cas de pemphigus (7foliaceus, 3 vulgaris et 1 erythematosus), 12 cas de lupus érythémateux discoïde (DLE) et 12 cas de dermatitehyperplasique chronique ont été utilisés. L’anticorps monoclonal CA4E7 mAb (IgG1 + IgG2) a présenté une sen-sibilité semblable, mais une spécifité plus élévée que les deux anticorps polyclonaux anti-IgG pour le diagnosticimmunohistochimique de pemphigus et de DLE. Les anticorps monoclonaux CA4F1 (IgG2) et CA3H1 mAb(IgG2) ont présenté respectivement une réactivité modérée et faible au niveau de l’épithélium pour les maladiesauto-immunes, mais un marquage important du cytoplasme des plasmocytes des infiltrats inflammatoires. Cesrésultats suggèrent que l’anticorps monoclonal CA4E7 est intéressant dans le diagnostic immunohistochimiquede ces maladies.

Resumen El objetivo de este trabajo fue evaluar la utilidad de tres anticuerpos monoclonales (mAbs), y dosanticuerpos policlonales (pAbs) anti-IgG y anti-IgM caninas para el diagnóstico inmunohistoquímico lasenfermedades cutáneas autoinmunes caninas. Se utilizaron biopsias cutáneas de 11 casos de pénfigo (7 foliáceo,3 vulgar y 1 eritematoso), 12 casos de lupus eritematoso discoide (DLE) y 12 casos de dermatitis hiperplásicacrónica. El mAb CA4E7 (IgG1 + IgG2) mostró sensibilidad similar, pero mayor especificidad y menos fondo quelos dos pAb anti-IgG para el diagnóstico inmunohistoquímico de pénfigo foliáceo y DLE. El mAb CA4F1 (IgG2)y mAb CA3H1 (IgG2) mostraron reactividad moderada e interepitelial, respectivamente, en enfermedadescutáneas autoinmunes, pero intensa tinción del citoplasma de células plasmáticas de los infiltrados inflamatorios.Estos resultados sugieren que el mAb CA4E7 puede ser válido para el diagnóstico inmunohistoquímico de estasenfermedades.

Zusammenfassung Das Ziel dieser Arbeit war die Bewertung von 3 monoklonalen Antikörpern (mAk), undpolyklonalen Antikörpern (pAk), zwei anti-caninen IgD und einem anti-caninen IgM Antikörper, für die immuno-histochemische Diagnose von autoimmunen Hauterkrankungen des Hundes. Hautbiopsien von 11 Fällen mitPemphigus (7 foliaceus, 3 vulgaris und 1 erythematodes), 12 Fällen mit diskoidem Lupus erythematodes (DLE)und 12 Fällen mit chronischer hyperplastischer Dermatitis wurden bewertet. Die CA4E7 mAk (IgG1 + IgG2)zeigten gleiche Sensitivität, aber höhere Spezifizität und schwächeren Hintergrund als die zwei anti-IgG pAk inder immunhistochemischen Diagnose von Pemphigus und DLE. Die CA4F1 (IgG2) und CA3H1 mAk (IgG2)zeigten moderate und schwache interepitheliale Reaktivität, färbten aber das Zytoplasma von Plasmazellen imEntzündungsinfiltrat stark an. Diese Ergebnisse deuten darauf hin, dass CA4E7 mAb in der immunhistoche-mischen Diagnose solcher Erkrankungen wertvoll sein könnte.

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