Evaluation of two homemade ELISA assays for monitoring exacerbations in patients with allergic broncho-pulmonary aspergillosis

Patrice Le Pap, Isabelle Ourliac Garnier, Olivia meynial, Alix de Mont Martin


Background: ABPA is an immunologic bronchopulmonary inflammation due to immune response of
the lower respiratory tract against Aspergillus fumigatus. The evolution of ABPA comprises five stages
going through exacerbation episodes. Early detection of exacerbations is of major importance due to
negative effects on patients, resulting in degradation of health status.
Material/methods: A prospective follow-up over a 24 months period of a cohort of 18 ABPA patients
with initial ABPA diagnosis allowed us to screen serums for IgE levels, circulating precipitins by
immunoelectrophoresis (gold standard method) and A. fumigatus-specific antibodies by IgG ELISA
Serion. Clinical outcomes from each patient were also collected and analyzed. In parallel, homemade
Elisa assays using semi-purified antigens to detect IgG isotypes (ELISA1) and IgG4 (ELISA2) were
evaluated. Western blot using the same antigen was used to explore the antibody response. A control
group of serums from healthy subjects (n= 30) and patients diagnosed with atopic dermatitis (n= 5),
cystic fibrosis (n= 3), COPD (n=9), and candidiasis (n=4) was screened to conclude about the
specificity of our homemade-ELISA assays.
Results: During the course of the study, 84 serums from 18 ABPA patients were included for
detection of antibodies. Ten exacerbation episodes were notified for 10 patients. Through healthy
subjects, a cut-off was established for ELISA1. All the exacerbations well correlated with high ELISA1
antibody levels above the cut-off, whereas often associated with negative precipitins. Exacerbation
episodes were also characterized by a high level of anti-IgG4 and a special signature (a band of
10KDa) in Western blot. Some patients who were in remission still had a high level of ELISA1
antibodies that could be discriminated from a persistent exacerbation by ELISA2 and Western blot.
Monitoring IgG isotype and IgG4 levels for patients from the control group showed that the cut-off is
highly selective since all healthy subjects, atopic dermatitis and COPD patients are under the cut-off .
However, one of the 3 patients with invasive candidiasis was above the cut-off for ELISA1 but easily
excluded as a false positive by ELISA2. Cystic fibrosis patients without ABPA but positive with the
gold standard method and ELISA Serion also have a very high level of IgG by ELISA1. Performing
ELISA2 and Western blot allowed us to confirm negativity for two of these patients.
Conclusions: The ELISA1 assay evaluated in this study appeared to be efficient to point out
exacerbation episodes in patients with ABPA. False positive results obtained for cystic fibrosis and
candidiasis patients could be ruled out performing anti-IgG4 ELISA2 assay or Western blot.


abstract No: 


Full conference title: 

26th European Congress of Clinical Microbiology and Infectious Diseases