Western blotting as a tool for the serodiagnosis of farmer’s lung disease: validation with Lichtheimia corymbifera protein extracts

Abstract

Electrosyneresis and double diffusion are immunoprecipitation techniques commonly used in the serological diagnosis of Farmer's lung disease (FLD). These techniques are reliable but lack standardization. The aim of this study was to evaluate Western blotting for the serodiagnosis of FLD. We carried out Western blotting with an antigenic extract of Lichtheimia corymbifera, an important aetiological agent of the disease. The membranes were probed with sera from 21 patients with FLD and 21 healthy exposed controls to examine the IgG antibody responses against purified somatic antigens. Given the low prevalence of the disease, 21 patients could be considered as a relevant series. Four bands were significantly more frequently represented in membranes probed with FLD sera (bands at 27.7, 40.5, 44.0 and 50.5 kDa) than those probed with control sera. We assessed the diagnostic value of different criteria alone or in combination. The diagnostic accuracy of the test was highest with the inclusion of at least two of the following criteria: at least five bands on the strip and the presence of one band at 40.5 or 44.0 kDa. Sensitivity, specificity and positive and negative predictive values were all 81 %, and the odds ratio was 18.06. Inclusion of bands of high intensity diminished rather than improved the diagnostic value of the test. We concluded that Western blotting is a valuable technique for the serodiagnosis of FLD. The industrial production of ready-to-use membranes would enable the routine use of this technique in laboratories, and provide reliable and standardized diagnostic results within a few hours.

The value of serum precipitins against specific antigens in patients diagnosed with hypersensitivity pneumonitis – retrospective study

“…Molds can induce farmer's lung in large agriculture regions, especially during wet and warm seasons [24]. Most frequently involved are Alternaria, Aspergillus, and Botrytis species [24], and in some geographic regions, Absidia (Lichtheimia corymbifera) and Wallemia were suggested as the cause of farmer's lung disease [26,27]. As molds' antigens were not included in our laboratory set, this type of allergy could be missed out in our material.…”

“…We did not confirm the allergy to thermophilic actinomycetes in most of our patients who reported the exposition to hay or hay products. The data from literature indicate that farmer's lung may be caused either by thermophilic bacteria or by molds [10,24,26,27]. Molds can induce farmer's lung in large agriculture regions, especially during wet and warm seasons [24].…”

Identification of Antigenic Proteins from Lichtheimia corymbifera for Farmer’s Lung Disease Diagnosis

“…Protein extracts from L . corymbifera (reference strain BBCM/IHEM 3809 isolated from FLD-linked hay) were prepared as previously described [ 15 ]. Briefly, the process consisted of the enzymatic lysis of the fungal cell wall with a recombinant β-1,3-glucanase (Lyticase from Arthrobacter luteus , Sigma-Aldrich ® , St Louis, MO, USA), followed by protein precipitation with trichloroacetic acid in presence of deoxycholate and protein purification with the SDS-Page Clean-Up Kit (GE Healthcare ® , Piscataway, USA).…”

“…Briefly, the process consisted of the enzymatic lysis of the fungal cell wall with a recombinant β-1,3-glucanase (Lyticase from Arthrobacter luteus , Sigma-Aldrich ® , St Louis, MO, USA), followed by protein precipitation with trichloroacetic acid in presence of deoxycholate and protein purification with the SDS-Page Clean-Up Kit (GE Healthcare ® , Piscataway, USA). A set of sera from FLD patients (n = 7) and from HEC (n = 9) which detect bands representative of each group with the weakest background signals was selected from a previous 1D-WB experiment [ 15 ]. Protein extracts (25 μg) were separated by two dimensional SDS-PAGE on small size gels, and immunoreactive proteins were detected by WB using the selected sera.…”

“…Comparative western blotting (WB) analysis, performed with L . corymbifera protein extract and sera from patients with FLD and healthy exposed controls (HEC), identified bands specific to FLD at 27.7, 40.3, 44.0, and 50.5 kDa [ 15 ]. Proteins included in these bands might be suitable candidates.…”