IgE binding epitopes of blag 6 from german cockroach

Bla g 6, a German cockroach allergen, shows homology to muscle protein troponin C. It contains four calciumbinding domains at 20-30, 56-67, 96-107, and 132-143 amino acid (aa) residues, and its immunoglobulin E (IgE) reactivity is dependent upon calcium ion level. However, the IgE binding epitopes of Bla g 6 have not been investigated. This study aimed to analyze the IgE binding epitopes from the five peptide fragments of Bla g 6. The full-length of three Bla g 6 isoallergens (Bla g 6.0101, Bla g 6.0201, and Bla g 6.0301) and five peptide fragments (P1: aa 1-111, P2: aa 1-95, P3: aa 33-111, P4: aa 80-151, and P5: aa 33-151) of Bla g 6.0101 were generated by polymerase chain reaction (PCR) and expressed in Escherichia coli. Enzyme-linked immunosorbent assay (ELISA) was performed on 24 patients' sera that adjusted the final concentration 10 mM of CaCl2 to determine the IgE activities of Bla g 6. Eight sera (33.3%), 9 sera (37.5%), and 11 sera (45.8%) showed IgE reactivity to Bla g 6.0101, Bla g 6.0201, and Bla g 6.0301, respectively. Among the sera from the positive IgE reactivity, three patients' sera were selected and the IgE reactivity was measured by ELISA with the five peptide fragments of Bla g 6. Based on IgE responses, one patient's serum exhibited the strongest IgE reactivity. We assumed that the aa between 96-151 residues, including the calcium binding domains III and IV, would be important for IgE binding. These results may provide information that will yield safe diagnostic methods and immunotherapeutics.