[Production and property identification of monoclonal antibodies against DAF molecules by DNA immunization followed by a single boost with cell antigen]
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Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood
Published on: February 6, 2018
Na Pan1, Lin Shi, Li-hua Huang
1Department of Immunology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences Peking Union Medical College, Tianjin 300020, China.
Aim:
To produce mAb against DAF by DNA immunization followed by a single boost with cell antigen and to characterize its property.
Methods:
Recombinant plasmid pcDNA3.1/DAF was constructed by molecular cloning technique and injected into mice quadriceps muscle of thigh. To boost the DAF immunized mice, HPB-All cell antigen were injected on day 3 before cell fusion. The affinity and property of mAb to natural membrane protein and denatured protein were identified by FCM, fluorescence microscope and Western blot.
Results:
Two mAb against DAF were obtained, namely 2B6B and 2B6E. The affinity constant of 2B6E was 1.81x10(-7) mol/L. The isotype of the two mAbs were IgG2a, and the epitope of them was different. FCM, fluorescence microscope and Western blot indicated that the obtained mAb had affinity to natural membrane protein and denatured protein with high specificity.
Conclusion:
This study indicates that the DNA immunization and cell antigen boost method enables mice to produce mAb against DAF. The low frequency of nonspecific mAb is one of the advantages of this method compared to the conventional cell immunization method. Moreover the mAb generated by this method has satisfactory binding activity and specificity.
