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[Production and property identification of monoclonal antibodies against DAF molecules by DNA immunization followed by a single boost with cell antigen]. 2007

Na Pan, and Lin Shi, and Li-hua Huang, and Li Zhou, and Da-shui He, and Yu-guang Zhang
Department of Immunology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences Peking Union Medical College, Tianjin 300020, China.

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.

UI MeSH Term Description Entries
D007114 Immunization Deliberate stimulation of the host's immune response. ACTIVE IMMUNIZATION involves administration of ANTIGENS or IMMUNOLOGIC ADJUVANTS. PASSIVE IMMUNIZATION involves administration of IMMUNE SERA or LYMPHOCYTES or their extracts (e.g., transfer factor, immune RNA) or transplantation of immunocompetent cell producing tissue (thymus or bone marrow). Immunologic Stimulation,Immunostimulation,Sensitization, Immunologic,Variolation,Immunologic Sensitization,Immunological Stimulation,Sensitization, Immunological,Stimulation, Immunologic,Immunizations,Immunological Sensitization,Immunological Sensitizations,Immunological Stimulations,Sensitizations, Immunological,Stimulation, Immunological,Stimulations, Immunological,Variolations
D008807 Mice, Inbred BALB C An inbred strain of mouse that is widely used in IMMUNOLOGY studies and cancer research. BALB C Mice, Inbred,BALB C Mouse, Inbred,Inbred BALB C Mice,Inbred BALB C Mouse,Mice, BALB C,Mouse, BALB C,Mouse, Inbred BALB C,BALB C Mice,BALB C Mouse
D008856 Microscopy, Fluorescence Microscopy of specimens stained with fluorescent dye (usually fluorescein isothiocyanate) or of naturally fluorescent materials, which emit light when exposed to ultraviolet or blue light. Immunofluorescence microscopy utilizes antibodies that are labeled with fluorescent dye. Fluorescence Microscopy,Immunofluorescence Microscopy,Microscopy, Immunofluorescence,Fluorescence Microscopies,Immunofluorescence Microscopies,Microscopies, Fluorescence,Microscopies, Immunofluorescence
D010957 Plasmids Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS. Episomes,Episome,Plasmid
D005260 Female Females
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D000911 Antibodies, Monoclonal Antibodies produced by a single clone of cells. Monoclonal Antibodies,Monoclonal Antibody,Antibody, Monoclonal
D000915 Antibody Affinity A measure of the binding strength between antibody and a simple hapten or antigen determinant. It depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. It includes the concept of "avidity," which refers to the strength of the antigen-antibody bond after formation of reversible complexes. Affinity, Antibody,Antibody Avidity,Avidity, Antibody,Affinities, Antibody,Antibody Affinities,Antibody Avidities,Avidities, Antibody
D000918 Antibody Specificity The property of antibodies which enables them to react with some ANTIGENIC DETERMINANTS and not with others. Specificity is dependent on chemical composition, physical forces, and molecular structure at the binding site. Antibody Specificities,Specificities, Antibody,Specificity, Antibody

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