ABSTRACT
AIM: To prepare monclonal antibody (mAb) against both hemoglobin A2 (HbA2) and hemglobin A (HbA), this antiboy can bind to delta globin chain and beta globin chain, but not to gamma globin chain. METHODS: Mice was immunized with recombinant Hb delta fusion protein, and hybridoma cells were generated by cell fusion techniques followed by screening with natural HbA2 and HbA separated by ion exchange chromatography. The purified monclonal antibody was identified by indirect ELISA, Western blot based on native PAGE and SDS-PAGE, surface plasmon resonance (SPR), flow cytometry and immunohistochemistry. RESULTS: The monoclonal antibody against both HbA2 and HbA was obtained and designated as 2C9 that shows no binding to fetal hemoglobin (HbF), alpha globin chain and recombinant zetaglobin chain. CONCLUSION: The mAb 2C9 was defined as specificity to hemoglobin delta globin chain and beta globin chain, which suggests that mAb 2C9 recognizes a common epitope on bothdeltaglobin chain and beta globin chain. This antibody would be expected to be an effective tool in research and clinical practice in hemoglobinopathies.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , delta-Globins/immunology , Animals , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Female , Hemoglobin A2/immunology , Hemoglobins/immunology , Hybridomas/immunology , Mice , Mice, Inbred BALB C , alpha-Globins/immunology , beta-Globins/immunologyABSTRACT
The human delta-globin chain, unique to the hemoglobin A2 (HbA2) heterotetramer, is important for the evaluation of hemoglobinopathy. However, there are no well-defined antibodies specific for the delta-globin chain, a fact that is attributed a striking similarity (93%) in amino acid sequence between delta-globin and beta-globin of the hemoglobin A (HbA). In this study, two monoclonal antibodies (mAbs) against the delta-globin chain were generated and designated as 2H4 and 1H11. These antibodies were specific to HbA2 and do not cross-react with HbA and HbF (fetal hemoglobin). Moreover, the expression of HbA2 in fetal liver and mature erythrocytes was determined using these two mAbs. In addition to being useful tools for research or diagnosis, these antibodies could be valuable for development of rapid and effective antibody-based immunoassays of HbA2 expression in erythroid cells and non-erythroid tissue.
