Anti-human Cd13 Monoclonal Antibodies

Objective To generate monoclonal antibodies against human CD13 molecule expressing on the surface of human myeloid cells. Methods 1) Using RT-PCR, cloned the gene of human CD13's enzymatic activity domain (amino acids from 69 to 480), and inserted the cloned cDNA into an eukaryotic expression vector pcDNA3.1(+) to construct the recombinant plasmid pCDNA3.1(+)/CD13.2) Using the intramuscular route of DNA (pcDNA3.1(+)/CD13) injection and intravenous route of HL-60 cells booster, immuned BALB/C mice. 3) To produce Mabs, spleen cells obtained from CD13 encoding DNA immuned mice were fused with myeloma cells NS1 with the standard hybridoma technique, then obtained hybridomas that can secrete anti- human CD13 Mabs constantly. 4) To assess Mabs' specificity and usefulness, used kinds of cell lines expressing or not expressing CD13, peripheral blood cells of normal people and 10 samples of fresh marrow cells of Leukemia patients through FCM, fluorescence microscope and Western Blotting, and then picked out 2E3, 5) Detected the type of anti human CD13 Mabs. 6) Macrocultured 2E3 in mice abdominal cavity and purified the ascitic fluid with affinity chromatograph of hydroxyapatite ceramic. After getting the purified 2E3, labeled 2E3 with FITC. 7) Used competitive inhibition experiment to find whether 2E3 can bind the same epitope as other commonly used anti human CD13 Mabs. Results 1) Successfully constructed recombinant eukaryotic expression plasmid pcDNA3.1(+)/CD13. 2) Aquired seven hybridomas secreting anti-CD 13 Mabs constantly. 3) The type of anti- human CD13 Mabs is IgM,...