A predominant IgG4 subclass may be responsible for false-negative direct immunofluorescence in bullous pemphigoid.
BACKGROUND
Bullous pemphigoid (BP) is an immune-mediated blistering disease, usually characterized immunopathologically by the linear deposition of IgG and C3 along the basement membrane zone (BMZ) of skin. However, positive deposition of C3 but negative staining for IgG on direct immunofluorescence (DIF) studies has been noted in some patients.


METHODS
Twelve patients known to have BP but with absence of staining for IgG were included in this study. Frozen sections of skin specimens from the 12 patients were subjected to IgG DIF, as well as a sandwich double antibody method of staining for IgG, IgG subclasses, and light chains. Enzyme-linked immunosorbent assay (ELISA) using commercially available human IgG subclasses was used to analyze the subclass restriction of FITC-labeled antihuman IgG conjugates.


RESULTS
Of the 12 skin specimens with positive C3 and negative IgG on DIF, nine were positive for IgG with the double antibody sandwich method. In addition, all 12 specimens had positive linear staining for the subclass IgG4 along the BMZ with this method. There was no IgG light chain restriction. Two commercially obtained antihuman IgG conjugates, both commonly used in our laboratory for DIF testing, were analyzed for separate IgG subclass specificity by ELISA. Both conjugates showed high reactivity to IgG1 and IgG3 with less reactivity to IgG2 and IgG4.


CONCLUSIONS
These results suggest that the following factors contribute to false-negative staining for IgG on DIF in some BP patients: (i): subthreshold IgG in skin specimens; (ii) limited reactivity of commercial antihuman IgG conjugates to the IgG4 subclass; and (iii) decreased sensitivity of DIF compared with double antibody methods for the detection of IgG. The use of sandwich double antibody immunofluorescence methods to test for IgG and/or IgG subclasses may be helpful in definitively diagnosing BP in patients with negative IgG and positive C3 staining on DIF.
Buschman
Kerry E
KE
Department of Dermatology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
Seraly
Mark
M
Thong
H Y
HY
Deng
Jau-Shyong
JS
Draviam
Rose P
RP
Abernethy
John L
JL
D001323:Autoantibodies / Q000032:analysis; D003176:Complement C3 / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005188:False Negative Reactions; D019085:Fluorescent Antibody Technique, Direct; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis*; D007147:Immunoglobulin Light Chains / Q000032:analysis; D010391:Pemphigoid, Bullous / Q000175:diagnosis / Q000276:immunology*; D012867:Skin / Q000276:immunology; D013194:Staining and Labeling / Q000379:methods*
D001323:Autoantibodies; D003176:Complement C3; D007074:Immunoglobulin G; D007147:Immunoglobulin Light Chains