The Journal of Practical Medicine >

2025 , Vol. 41 >Issue 2: 271 - 277

DOI: https://doi.org/10.3969/j.issn.1006-5725.2025.02.018

Medical Examination and Clinical Diagnosis

Screening of anti⁃fetal hemoglobin monoclonal antibodies based on trailing count method and its application in preliminary diagnostic method for β⁃thalassemia

  • Moli YIN ,
  • Jingzhe XU ,
  • Yu YAN ,
  • Zhenxiao TONG ,
  • Lei LIU ,
  • Huiyan. WANG
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  • Jilin Medical University，Jilin Collaborative Innovation Center for Antibody Engineering，Jilin 132013，Jilin，China

Received date: 2024-09-06

  Online published: 2025-01-26

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Abstract

Objective To establish an initial diagnostic method for β-thalassemia （BT） using a double antibody sandwich ELISA approach. Methods The hybridoma producing monoclonal antibodies against anti-HbF were screened using a trailing count method. The mAbs were evaluated through ELISA， modified immunocytochemistry， and Western blot analysis. A double antibody-sandwich ELISA assay was established by labeling the pairs of mAbs with ALP using the glutaral method， and this detection system was used to analyze 40 serum samples. Results The results demonstrate the successful generation of nine hybridoma cell lines capable of secreting highly active anti-HbF monoclonal antibodies （mAbs）. Specifically， four mAbs （3F7， 4G1， 6C1， and 9H7） exhibited exclusive reactivity towards HbF without any cross-reactivity with hemoglobin variants （HbA and HbA2）. These four HbF-specific mAbs displayed exceptional specificity and sensitivity， with a maximum titer of 1∶256 000 and the highest affinity constant （Ka） recorded at 2.36×108 L/mol. Among these mAbs， optimal antibody pairing was achieved using capture antibody 3F7 in conjunction with ALP-4G1 for the development of a sandwich ELISA detection method. By employing this approach， fetal and healthy human blood samples were successfully quantified for HbF levels with an impressive detection sensitivity reaching up to 80%. Conclusion This sandwich ELISA demonstrated precise quantification of HbF levels， making it suiTab.for both research and diagnostic purposes in the field of β-thalassemia.

Key words： fetal hemoglobin; monoclonal antibody; quantitative detection; trailing count method

Cite this article

Moli YIN , Jingzhe XU , Yu YAN , Zhenxiao TONG , Lei LIU , Huiyan. WANG . Screening of anti⁃fetal hemoglobin monoclonal antibodies based on trailing count method and its application in preliminary diagnostic method for β⁃thalassemia[J]. The Journal of Practical Medicine, 2025 , 41(2) : 271 -277 . DOI: 10.3969/j.issn.1006-5725.2025.02.018

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