Abstract
A stabilized hybridoma cell line secreting anti-retinoic acid monoclonal antibodies of subclass IgG1 with κ chains was produced by fusing NS-1 myeloma cells with the spleen cells from BALB/c female mice immunized with all- trans-4-oxoretinoic acid-oxime-chicken IgG conjugate. The antibody titer of mice ascitic fluid ranged from 1 12,800 to 1 25,600 , as determined by competitive indirect enzyme-linked immunosorbent assay (ELISA). 50% inhibition dosage of all- trans-retinoic acid at a 1 20,000 dilution of mice ascitic fluid was 6.6 ng/ml, as determined by ELISA. The anti-retinoic acid monoclonal antibody was generated in mice ascitic fluid and purified by protein G affinity chromatography. Cross-reactivity of the monoclonal antibody was determined at 0.1 μg/ml concentration of retinoids and indicated high specificity to both all- trans-retinoic acid (86% inhibition) and 13- cis-retinoic acid (87% inhibition), and strong cross-reactivity with 4-oxoretinoic acid (77%) and 4-oxoretinoic acid oxime (109%). Specificity was confirmed by the horseradish peroxidase-linked immunostaining method and immunoradioassay. The affinity constant of the monoclonal antibody, K, was determined to be 3.6 × 10 9 1/mol. A calibration curve for retinoic acid using the monoclonal antibody to retinoic acid was developed; the detection limit for all- trans-retinoic acid is 1 ng/ml in the competitive indirect ELISA. The antibody counteracts the effect of retinoic acid on growth inhibition and differentaition in HL-60 cells.
Published Version
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