[Production and property identification of monoclonal antibodies against DAF molecules by DNA immunization followed by a single boost with cell antigen].
OBJECTIVE
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.


CONCLUSIONS
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.
Pan
Na
N
Department of Immunology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences Peking Union Medical College, Tianjin 300020, China.
Shi
Lin
L
Huang
Li-hua
LH
Zhou
Li
L
He
Da-shui
DS
Zhang
Yu-guang
YG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000915:Antibody Affinity / Q000235:genetics; D000918:Antibody Specificity / Q000235:genetics; D000941:Antigens / Q000276:immunology*; D015153:Blotting, Western; D018958:CD55 Antigens / Q000235:genetics* / Q000276:immunology*; D005260:Female; D006801:Humans; D007114:Immunization / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000941:Antigens; D018958:CD55 Antigens; D019444:Vaccines, DNA