Abstract
Antigen Used for Immunization
Prokaryotically expressed FurA protein.
Method of Immunization
BALB/c mice (6 weeks old) were immunized subcutaneously with 50 μg of the purified protein His6-FurA emulsified in Freund's complete adjuvant. Three booster injections were given at 2-week intervals.
Parental Cell Line Used for Fusion
Sp2/0-Ag-14 mouse myeloma cells.
Selection and Cloning Procedure
Three days after the last injection, the spleen was removed and spleen cells were fused with Sp2/0-Ag-14 mouse myeloma cells. The culture supernatant from the wells with growing cells was examined for the presence of antibodies to FurA. The colonies producing anti-FurA antibodies were cloned twice at a density of 0.4 per well by limiting dilution. The cloned cells were injected into the mouse belly cavity and ascitic fluid with anti-FurA MAbs were generated.
Heavy and Light Chains of Immunoglobulin
Full protein.
Specificity
Four monoclonal antibodies (H9H6, H9E12, H10H6, and H10H8) were produced. Specificities were tested by Western blot analysis and cell fluorescence microscopy assay; the four antibodies could recognize the FurA protein, respectively.
Specific Antigen Identified
Mycobacterium tuberculosis ferric uptake regulator A.