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. 2012 Apr;31(2):111-7.
doi: 10.1089/hyb.2011.0102.

Functional Characterization of an IgE-class Monoclonal Antibody Specific for the Bullous Pemphigoid Autoantigen, BP180

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Free PMC article

Functional Characterization of an IgE-class Monoclonal Antibody Specific for the Bullous Pemphigoid Autoantigen, BP180

Kelly A N Messingham et al. Hybridoma (Larchmt). .
Free PMC article

Abstract

BP180 (collagen XVII) is the target antigen in several autoimmune diseases including bullous pemphigoid (BP). Both IgE and IgG class autoantibodies have been shown to be pathogenic in BP; however, studies designed to elucidate the patho-mechanisms mediated specifically by the IgE-class autoantibodies are limited by the low levels (ng/mL) of IgE in human sera. In this report, we developed mouse IgE class monoclonal antibodies (MAbs) against the immunodominant NC16A domain of the human BP180 protein and characterized two of the resultant MAbs, designated 395A5 and 395D2. Epitope mapping studies revealed that both MAbs target segment 2 of NC16A, as was described for IgE and IgG class BP autoantibodies. Also similar to BP IgE, MAb 395A5 showed indirect immunofluorescence labeling of the basement membrane zone (BMZ) of human skin, stimulated histamine release from mast cells when triggered with NC16A, and induced keratinocyte production of IL-8. The 395D2 MAb was also able to trigger antigen-specific histamine release from mast cells; however, in contrast to BP IgE and 395A5, 395D2 did not label the cutaneous BMZ, nor did it induce IL-8 production in keratinocytes. In summary, these studies underscore the importance of functionally characterizing MAbs generated for use in human disease models. The 395A5 IgE class murine MAb was shown to share several key functional properties with the pathogenically active IgE produced by BP patients. We therefore expect that this MAb will prove to be a useful tool for dissecting the mechanisms used by BP180-NC16A-specific IgE antibodies in the induction of BP skin lesions.

Figures

FIG. 1.
FIG. 1.
Mapping of the bullous pemphigoid (BP) IgE MAb reactive sites by immunoblot. (A) Supernatants of our 395 hybridoma, Clones A5 and D2, were screened for IgE reactivity to either affinity-purified NC16A protein or lysates of bacteria expressing one of five subregions: NC16A1, NC16A2, NC16A2.5, NC16A3, or NC16A1–3. Proteins or lysates were electrophoresed and transferred to a nitrocellulose membrane. Membranes were probed with hybridoma supernatants (diluted 1:2) as the primary antibody. Anti-human IgE was used to detect specific reactivity to purified NC16A protein (left panel) and the NC16A segment 2 subdomain (right panel). 395 clone A5 is shown. Similar results were obtained with 395 clone D2. (B) To ensure specificity, 395 clone A5 and 395 clone D2 were used as primary antibodies on protein blots containing equimolar amounts of NC16A-GST, GST, and NC16A segment 2, followed by secondary detection with either anti-mouse IgE or IgG. HD18 (control) is mouse IgG specific for NC16A.
FIG. 2.
FIG. 2.
395A5, but not 395D2, MAbs bind to the basement membrane zone of human skin. Cryosections of normal human skin were incubated with MAbs 395A5, 395D2, IgELB4 (IgE isotype control), a BP serum (diluted 1:10), a BP180 specific IgG MAb (HD18), or a murine IgG isotype control. Antibody binding to the BMZ was visualized with anti-mouse IgE, anti-mouse IgG, anti-human IgE, or anti-human IgG, as indicated. Antibody binding to BP180 in human skin was evidenced by linear staining at the basement membrane zone (arrows) using 395A5 MAbs, BP sera, and HD18, but was absent with the 395D2 MAb or the isotype controls.
FIG. 3.
FIG. 3.
395A5, but not 395D2, MAbs trigger IL-8 production by human keratinocytes. Primary human keratinocytes were treated with 100 ng/mL 395A5, 395D2, IgELB4, or IgE purified from a BP patient (BP IgE) or normal control serum (NH IgE) for 20 h. Cell-free supernatants were collected and IL-8 was measured by ELISA. (A) Treatment keratinocytes with 395A5 MAbs, but not 395D2 or IgELB4, triggered release of IL-8. (B) Similarly, BP IgE, but not NH IgE, triggered IL-8 secretion.
FIG. 4.
FIG. 4.
395A5 and 395D2 MAbs mediate NC16A-specific histamine release. RBL-SX-38 basophils were coated with 395A5, 395D2, or IgELB4 MAbs or IgE purified from two BP patients (BP IgE1 or BP IgE2) or normal control serum (NH IgE) for 30 min and washed, and degranulation was triggered by exposure to 100 nM NC16A or GST control protein for 30 min. (A) MAbs 395A5 and 395D2 triggered greater histamine release in response to NC16A protein, but not GST control protein, similar to BP IgE1 and IgE2 (B). Control antibodies did not trigger NC16A-specific histamine release.

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