טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentSezin Tatiana
SubjectHuman Amnion Membrane as a Substrate and an Antigenic
Source for the Detection of Auto Antibodies in
Autoimmune Bullous Skin Diseases
DepartmentDepartment of Medicine
Supervisor Professor Reuven Bergman
Full Thesis textFull thesis text - English Version


Abstract

Human amnion membrane (HAM) was suggested to be a superior antigenic substrate for immunoblotting (IB) in detecting auto antibodies (Abs) of autoimmune bullous skin diseases (AIBSDs). Therefore, the goal of this study was to determine the properties of HAM as a substrate and an antigenic source for the detection of auto Abs in AIBSDs diseases. Immunomapping & tandem liquid chromatography mass spectrometry (LC-MS/MS) were used to delineate the antigenic structure of HAM. Immunoblotting & indirect immunofluorescence (IIF) were used to study the diagnostic utility of HAM, and the results were compared to those of IIF on monkey esophagus; IB using normal human skin (NHS), and enzyme linked immunosorbent assay (ELISA) in 76 patients & 36 controls. Immunomapping demonstrated the following antigens (Ags) in both NHS and HAM: Desmogleins (Dsgs) 1&3, desmocollin 3 (Dsc3), periplakin, envoplakin, desmoplakin, plakoglobin, bullous pemphigoid antigens 1&2 (BP230 & BP180, respectively), laminin 332, integrin α6β4, collagen 7, laminin γ1 subunit and p170. LC-MS/MS demonstrated all of these Ags in both NHS and HAM, except for the absence of BP230, and low threshold levels of Dsg1, Dsg3 & Dsc3 in HAM. IIF using HAM as a substrate demonstrated anti basement membrane zone (BMZ) Abs in 20 (48.7%) of the 41 BP patients, and anti intercellular space (ICS) Abs in 18 (72%) of the 25 PV patients. IB using HAM as the antigenic source did not demonstrate anti BP230 Abs in any of the BP cases, but detected anti BP180 Abs in 22 (53.6%) of the 41 BP patients. It did not demonstrate anti Dsg1 and/or anti Dsg3 Abs in any of the 25 studied PV cases. Overall, the diagnostic accuracies of IIF & IB using HAM were inferior to IIF using monkey esophagus, in detecting anti BMZ & ICS Abs, and to ELISA in detecting anti BP180, BP230, Dsgs 1&3 Abs, respectively. In conclusion, HAM does not offer advantages in detecting auto Abs in AIBSDs over the other standard methods. This is most likely due to the low levels or the absence of several relevant Ags in HAM. The most accurate methods in detecting auto Abs are ELISA in BP, and both ELISA & IIF using monkey esophagus in PV.