Development and application of novel immunoassays for eosinophil granule major basic proteins to evaluate eosinophilia and myeloproliferative disorders

Abstract

Background: During eosinophil differentiation, the granule eosinophil major basic protein 1 (eMBP1) is synthesized as a 32-kDa precursor form, referred to as proMBP1, which is processed into the 14-kDa mature form of eMBP1. The prevalence of these two forms of MBP1 in most pathological conditions remains unknown.

Objective: To develop the immunoassays that differentiate mature eMBP1 and proMBP1 and apply them to analyze their levels in biological fluids from patients with eosinophilia and hematologic disorders.

Methods: We produced a series of monoclonal antibodies and selected pairs capable of discriminating between the two molecular forms of eMBP1. Radioimmunoassay (RIA) was performed to simultaneously quantitate the levels of mature eMBP1 and proMBP1 in secretions from patients with chronic rhinosinusitis (CRS) and sera from patients with hypereosinophilic syndrome (HES) and other myeloproliferative disorders.

Results: The novel immunoassays possessed less than 1% crossreactivity between mature eMBP1 and proMBP1. Mature eMBP1, but not proMBP1, was found in nasal secretions of CRS patients. In contrast, elevated serum levels of mature eMBP1 and proMBP1 were observed in approximately 60% and 90% of HES patients, respectively, with proMBP1 present in greater quantities than mature eMBP1. Patients with several myeloproliferative disorders also showed high serum levels of proMBP1 while mature eMBP1 remained at basal levels.

Conclusion: The novel immunoassays successfully differentiated mature eMBP1 and proMBP1 in human biological fluids. Further studies addressing the clinical correlates of these assays will help to develop biomarkers to diagnose and monitor patients with eosinophilia and myeloproliferative disorders.

Keywords: Eosinophilia; Eosinophils; MBP; Myeloproliferative disorders; proMBP.

Figure 1.

Development of immunoassays specific for proMBP1 and mature eMBP1. (A) Plates were coated with the 163–15E10 antibody. Serial dilutions of recombinant proMBP-1 and native mature eMBP1 were added and detected using the radiolabeled 176–8H8 antibody. (B) Plates were coated with 173–14E10 antibody. Serial dilutions of recombinant proMBP-1 and native mature eMBP1 were added and detected by 171–8B2 antibody. The data are representative of at least 3 experiments showing similar results.

Figure 2.

Analyses of nasal secretions from chronic rhinosinusitis (CRS) patients and serum specimens from hypereosinophilic syndrome (HES) patients. (A) Radioimmunoassay analyses of proMBP11 and eMBP1 in nasal secretions from CRS patients. Subject numbers are indicated on the x-axis. (B) Radioimmunoassay analyses of proMBP1 and eMBP1 in serum specimens from HES patients and normal healthy subjects. Subject numbers and groups are indicated on the x-axis. (C) Western blot analyses for the presence of proMBP1 and eMBP1. Sera from two HES patients, nasal secretions from two CRS patients, and corresponding control healthy subjects were analyzed as described in the Methods section by using specific antibodies against mature eMBP1 (J6–8A4) and proMBP1 (J163–15E10). The control lanes for the mature eMBP1 blot and proMBP1 blot are mature eMBP1 isolated with eosinophils from HES patients and proMBP1 purified from supernatants of Chinese hamster ovarian cells transfected with the expression vector pSv/DHFR/ProMBP, respectively. The data presented are a representative of two experiments, which show similar findings.

Figure 3.

Analyses of serum specimens from patients with HES and other myeloproliferative disorders. The results of analyses for proMBP1 and eMBP1 in patients with myeloproliferative diseases are shown as box and whisker plots. The whiskers indicate the minimum and maximum values and the boxplots indicate medians and lower and upper quartiles. The median values for each group are described on the top of the whisker. Each dot represents one subject. HES; hypereosinophilic syndrome, CML; chronic myelogenous leukemia. ET; essential thrombocythemia, MDS; myelodysplastic syndrome, MMM; myelofibrosis with myeloid metaplasia, PV; polycythemia vera, and SMCD; systemic mast cell disease.