Clinical and Experimental Dermatology
Increased sensitivity and high speciﬁcity of indirect
immunoﬂuorescence in detecting IgG subclasses for diagnosis of
O. N. Horv
Department of Dermatology and Allergology, Ludwig Maximilian University, Munich, Germany;
Department of Dermato-Venerology, Teaching Hospital
e Vinohrady, Third Faculty of Medicine, Charles University in Prague, Prague, Czech Republic;
Department of Dermatology, Allergology and
ubeck Institute of Experimental Dermatology (LIED), University of L
Background. Indirect immunoﬂuorescence (IIF) microscopy on monkey oesopha-
gus is an important assay for the diagnosis of bullous pemphigoid (BP). Its relatively
low sensitivity (60–80%) may be partly due to insufﬁcient detection of minor IgG
Aim. To determine the operating characteristics of an IgG subclass in IIF.
Methods. We designed a retrospective, dual-centre, controlled cohort study on sera
from 64 BP sera that had been rated as false negatives by traditional IIF microscopy,
and assessed circulating IgG
Results. The sensitivities of IIF in detecting IgG
and all three in combi-
nation were 45.3%, 18.8%, 32.8% and 48.4%, respectively. Speciﬁcities were
Conclusion. Detection of IgG subclass (especially IgG
) autoantibodies by
IIF on monkey oesophagus can signiﬁcantly improve diagnostic performance of IIF
microscopy for diagnosis of BP.
Diagnosis of bullous pemphigoid (BP) is generally
based on clinical criteria, histology [including direct
immunoﬂuorescence (DIF) microscopy], detection of
circulating IgG autoantibodies by indirect immunoﬂuo-
rescence (IIF) microscopy, and ELISA for BP180 and
All IgG subclasses have been
shown to play a role, but IgG
may be more
important pathogenetically than other subtypes.
IIF has a sensitivity of 60–80%.
that this relatively low sensitivity may be partly due to
undetectably low serum concentrations of circulating
IgG autoantibodies or to insufﬁcient reactivity of com-
mercial anti-human IgG conjugates to minor IgG sub-
classes such as IgG
. Indeed, we previously
that ampliﬁcation of the signal on salt-split
skin by detection of complement-ﬁxing antibodies
resulted in positivity in almost 50% of BP serum sam-
ples that had been rated as false negatives by IIF
microscopy on oesophagus. Furthermore, Kumar et al.
showed increased sensitivity of IIF microscopy using
secondary antibodies directed against IgG subclasses.
However, in that study, a control group was not
therefore the speciﬁcity of IgG subtype IIF
microscopy could not be determined and false-positive
results were possible. Lamb et al. postulated that a
sandwich double antibody IIF microscopy using anti-
human IgG subclass antibodies would amplify the sig-
and indeed found that this technique increased
the sensitivity of traditional IIF from 36.6% to 56.6%
in patients with BP.
However, that study also did not
include a control group.
Correspondence: Dr Mikl
ardy, Department of Dermatology and
Allergology, Ludwig Maximilian University, Frauenlobstr. 9-11, D-80337
Conﬂict of interest: the authors declare that they have no conﬂicts of
Accepted for publication 15 January 2017
ª 2018 British Association of Dermatologists
Clinical and Experimental Dermatology (2018) 43, pp248–253