A modified sandwich ELISA for accurate measurement of HbF in α-thalassemia carriers containing Hb Bart’s and Hb Portland 1

ABSTRACT

Hemoglobin F (HbF) in blood lysate can be accurately measured by various methods, including immunoassay. In this study, we have produced polyclonal antibody (pAb) against HbF and established a modified sandwich-type ELISA for HbF quantification in blood lysates. The modified sandwich ELISA utilized anti-γ-globin monoclonal antibody clones Thal N/B as the capture antibody (Ab) coated on solid-phase, fluorescein isothiocyanate (FITC)-labeled pAb as the detecting Ab, and HPR-labeled anti-FITC Ab as the signal-generating Ab. By using an optimized blood lysate dilution, the HbF could be measured with no interference from hemoglobin Bart’s (Hb Bart’s) and hemoglobin Portland (Hb Portland 1) presented in α-thalassemia carriers. HbF levels measured by the modified sandwich ELISA were comparable to those quantified by the standard cation-exchange high performance liquid chromatography. We suggested that this modified sandwich ELISA was able to accurately measure HbF levels even in α-thalassemia carriers containing Hb Bart’s and Hb Portland 1 and be an alternative method for HbF measurement.

KEYWORDS:

• Hemoglobin F
• hemoglobin Bart’s
• antibody
• α-thalassemia
• sandwich ELISA

Acknowledgments

This study was supported by the Thailand Research Fund (TRF) Senior Research Scholar (RTA5980007) and the TRF Thailand Office of Higher Education Commission (MRG6080269). We thank the laboratory staff at Biomedical Technology Research Center, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand, for the technical assistances. SK was a MS student of Medical Technology program, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand. We also thank Dr Denis Sweatman of Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Thailand for English proofreading this manuscript.

Conflict of interest

The authors declared no conflict of interest.

Additional information

Funding

This work was supported by the Thailand Office of Higher Education Commission [MRG6080269] and Thailand Research Fund (TFR) Senior Research Scholar [RTA5980007].