Calcium enhances the sensitivity of immunofluorescence for pemphigus antibodies

Wendy L. Matis, Grant James Anhalt, Luis A. Diaz, Evandro A. Rivitti, Ciro R. Martins, Robert Berger

Research output: Contribution to journalArticle

Abstract

Indirect immunofluorescence (IF) to detect pemphigus and pemphigoid autoantibodies is commonly performed with monkey esophagus (ME) as substrate and phosphate-buffered saline (PBS) as a diluent. The purpose of this study was to evaluate comparative IF titers using human skin (HS) as substrate with variations in the buffers employed. Substrates (ME or HS) were incubated in PBS, Trisacetate-buffered saline (TAS), TAS with 5 mM CaC1+2 (TAS-Ca+2), and PBS or TAS with 1 mM EDTA, prior to incubation with pemphigus or pemphigoid sera for indirect IF. We examined sera from 11 patients with pemphigus vulgaris (PV), 10 patients with Brazilian pemphigus foliaceus (BPF), and 4 patients with bullous pemphigoid. In 20 of 21 pemphigus sera, endpoint indirect IF titers were highest on normal skin with TAS-Ca+2. Six sera (2 PV and 4 BPF) had endpoints that were 5 double dilutions higher than the endpoints obtained with ME and PBS. Six sera (3 PV and 3 BPF) were 4 double dilutions higher, 7 sera (3 PV and 4 BPF) were 2-3 double dilutions higher, and 2 PV sera were equivalent with both substrate/buffers. Preincubation of either tissue with EDTA prior to indirect IF abolished PV and BPF antibody binding completely. Exposure to EDTA after the tissue was incubated with PV or BPF sera did not affect indirect IF titers. In the presence of Ca+2, the antigen was resistant to trypsin in concentrations of 0.001%; however, in the absence of added Ca+2 it was destroyed by 0.0001% trypsin. These differences were not observed with bullous pemphigoid sera; all 4 sera had similar endpoint indirect IF titers. This study shows a significant increase in the sensitivity of indirect IF assays for pemphigus autoantibodies by the use of Ca+2-supplemented buffers on human skin. This finding may also have implications for procedures designed to purify and/or detect pemphigus antigens.

Original languageEnglish (US)
Pages (from-to)302-304
Number of pages3
JournalJournal of Investigative Dermatology
Volume89
Issue number3
StatePublished - Sep 1987

Fingerprint

Pemphigus
Fluorescent Antibody Technique
Skin
Phosphates
Edetic Acid
Dilution
Calcium
Buffers
Indirect Fluorescent Antibody Technique
Antibodies
Substrates
Autoantibodies
Trypsin
Bullous Pemphigoid
Serum
Tissue
Antigens
Esophagus
Haplorhini
Assays

ASJC Scopus subject areas

  • Dermatology

Cite this

Calcium enhances the sensitivity of immunofluorescence for pemphigus antibodies. / Matis, Wendy L.; Anhalt, Grant James; Diaz, Luis A.; Rivitti, Evandro A.; Martins, Ciro R.; Berger, Robert.

In: Journal of Investigative Dermatology, Vol. 89, No. 3, 09.1987, p. 302-304.

Research output: Contribution to journalArticle

Matis, Wendy L. ; Anhalt, Grant James ; Diaz, Luis A. ; Rivitti, Evandro A. ; Martins, Ciro R. ; Berger, Robert. / Calcium enhances the sensitivity of immunofluorescence for pemphigus antibodies. In: Journal of Investigative Dermatology. 1987 ; Vol. 89, No. 3. pp. 302-304.
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AU - Matis, Wendy L.

AU - Anhalt, Grant James

AU - Diaz, Luis A.

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AU - Berger, Robert

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AB - Indirect immunofluorescence (IF) to detect pemphigus and pemphigoid autoantibodies is commonly performed with monkey esophagus (ME) as substrate and phosphate-buffered saline (PBS) as a diluent. The purpose of this study was to evaluate comparative IF titers using human skin (HS) as substrate with variations in the buffers employed. Substrates (ME or HS) were incubated in PBS, Trisacetate-buffered saline (TAS), TAS with 5 mM CaC1+2 (TAS-Ca+2), and PBS or TAS with 1 mM EDTA, prior to incubation with pemphigus or pemphigoid sera for indirect IF. We examined sera from 11 patients with pemphigus vulgaris (PV), 10 patients with Brazilian pemphigus foliaceus (BPF), and 4 patients with bullous pemphigoid. In 20 of 21 pemphigus sera, endpoint indirect IF titers were highest on normal skin with TAS-Ca+2. Six sera (2 PV and 4 BPF) had endpoints that were 5 double dilutions higher than the endpoints obtained with ME and PBS. Six sera (3 PV and 3 BPF) were 4 double dilutions higher, 7 sera (3 PV and 4 BPF) were 2-3 double dilutions higher, and 2 PV sera were equivalent with both substrate/buffers. Preincubation of either tissue with EDTA prior to indirect IF abolished PV and BPF antibody binding completely. Exposure to EDTA after the tissue was incubated with PV or BPF sera did not affect indirect IF titers. In the presence of Ca+2, the antigen was resistant to trypsin in concentrations of 0.001%; however, in the absence of added Ca+2 it was destroyed by 0.0001% trypsin. These differences were not observed with bullous pemphigoid sera; all 4 sera had similar endpoint indirect IF titers. This study shows a significant increase in the sensitivity of indirect IF assays for pemphigus autoantibodies by the use of Ca+2-supplemented buffers on human skin. This finding may also have implications for procedures designed to purify and/or detect pemphigus antigens.

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