Characterization of the Anti-BP180 Autoantibody Reactivity Profile and Epitope Mapping in Bullous Pemphigoid Patients11Tables 1, 2, 3 and 5 can be found at http://www.blackwellpublishing.com/products/journals/suppmat/jid/jid22126/jid22126sm.htm

Abstract

Bullous pemphigoid is a subepidermal bullous disease of skin and mucosae associated with autoantibodies to BP180. To characterize the humoral response to BP180, we generated a random BP180 epitope library displayed on lambda bacteriophage. After validation of the library by epitope mapping of three BP180-specific monoclonal antibodies, 15 novel or known BP180 epitopes were identified using 10 bullous pemphigoid serum samples. Fifty-seven bullous pemphigoid and 81 control sera were then assayed against the selected epitopes. Thirty-one out of 57 (54%) bullous pemphigoid sera reacted with at least an additional antigenic site other than the NC16A, within the extracellular (37%) and intracellular (28%) domains of BP180. In addition, the reactivity with extracellular epitopes of BP180 contained within the residue stretches 508-541 and 1331-1404 appeared to be related to the presence of both skin and mucosal involvement. Finally, a preliminary analysis of the epitope pattern in the disease course indicated that bullous pemphigoid patients exhibit a specific reactivity pattern, and that binding to intracellular epitopes of BP180, in addition to NC16A, may be detectable at an early clinical stage. Our findings provide novel insights into the pathophysiology of bullous pemphigoid and show the potential of the utilized approach as a tool for a rapid diagnosis of bullous pemphigoid patients and their management.

Missing the target: Characterization of bullous pemphigoid patients who are negative using the BP180 enzyme-linked immunosorbant assay

“…6,7,20,29 It is also well known that, in addition to NC16A, the majority of BP sera recognize other regions of the BP180 protein. 25,30,31 It has been proposed that reactivity to these other non-NC16A epitopes is a secondary event resulting from epitope spreading, a process in which inflammation triggered by autoimmunity damages the target tissue, which induces antibodies on the same or different antigens. 25,32 By definition, the initial reactivity with the NC16A region of BP180 would accompany, or even precede, reactivity to other sites in the molecule or other antigens, such as BP230.…”

“…25,30,31 It has been proposed that reactivity to these other non-NC16A epitopes is a secondary event resulting from epitope spreading, a process in which inflammation triggered by autoimmunity damages the target tissue, which induces antibodies on the same or different antigens. 25,32 By definition, the initial reactivity with the NC16A region of BP180 would accompany, or even precede, reactivity to other sites in the molecule or other antigens, such as BP230. 25 Our study indicates that, at the time of diagnosis, there is a cohort of BP with autoantibody reactivity exclusively outside of NC16A, indicating that antibodies to these regions can also be pathogenic.…”

“…25,32 By definition, the initial reactivity with the NC16A region of BP180 would accompany, or even precede, reactivity to other sites in the molecule or other antigens, such as BP230. 25 Our study indicates that, at the time of diagnosis, there is a cohort of BP with autoantibody reactivity exclusively outside of NC16A, indicating that antibodies to these regions can also be pathogenic.…”

“…15,19 The constructs of AA 1080 to 1107 and AA 1331 to 1404 were made using previously described methods. 15,19,25 The corresponding GST fusion proteins and GST alone were expressed in Escherichia coli, strain DH5a (Invitrogen, Grand Island, NY) or strain Rosetta 2 (Merck KGaA, Darmstadt, Germany) and purified from bacterial lysates using glutathione-agarose affinity chromatography. 15 Epitope mapping studies indicate this series of proteins will detect more than 90% of antibody reactivity with the extracellular domain.…”

Anti-Skin Specific Autoantibodies Detected by a New Immunofluorescence Multiplex Biochip Method in Patients with Autoimmune Bullous Diseases

“…BP180, also known as type XVII collagen or BP antigen 2, is a transmembrane glycoprotein of about 1,500 amino acids that ultrastructurally spans the lamina lucida before kinking back from the lamina densa into the lamina lucida [14]. The extracellular portion of the 16th noncollageneous domain (NC16A) has been identified as the immunodominant region of BP180 [14] and the recombinant NC16A domain has been employed in highly sensitive and specific ELISA systems to detect anti-BP180 autoantibodies [15,16,17,18]. …”