Production of a hybridoma cell line secreting retinoic acid-specific monoclonal antibody.
A stabilized hybridoma cell line secreting anti-retinoic acid monoclonal antibodies of subclass IgG1 with kappa 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 microgram/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 X 10(9) l/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 differentiation in HL-60 cells.
Zhou
H R
HR
Department of Food Science and Human Nutrition, Michigan State University, East Lansing 48824-1224.
Abouzied
M M
MM
Zile
M H
MH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D000918:Antibody Specificity / Q000276:immunology; D001667:Binding, Competitive; D002460:Cell Line; D003429:Cross Reactions / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006825:Hybridomas / Q000276:immunology*; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D014212:Tretinoin / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D014212:Tretinoin