Abstract
Spleen cells from a BALB/cByJ mouse previously immunized with purified rat liver microsomal cytochrome P-450c were fused with myeloma cells (P3X63Ag8.653) and 10 hybridoma clones secreting antibody against cytochrome P-450c were selected for characterization. The monoclonal antibodies (C1-C10) were purified from mouse ascites fluid and nine were determined to be distinct immunoglobulins. C6 was an IgG2b, whereas the rest were of the IgG1 subclass. A competitive enzyme-linked immunoassay was used to show that the antibodies were directed against at least five spatially distinct epitopes on cytochrome P-450c. Additional evidence for the recognition of distinct epitopes was provided by Ouchterlony immunoprecipitation of cytochrome P-450c with mixtures of appropriate monoclonal antibodies. Differences in antibody reactivity provided evidence for a sixth overlapping epitope that was recognized by two antibodies (C4 and C6). Three monoclonal antibodies to the same epitope on cytochrome P-450c, (CD2, CD3, and CD5) cross-reacted strongly with cytochrome P-450d, another isozyme induced by 3-methylcholanthrene treatment of rats. The antibodies that did not cross-react with cytochrome P-450d contained kappa light chains, whereas the three cross-reacting antibodies contained lambda light chains. None of the monoclonal antibodies cross-reacted with purified cytochromes P-450a, P-450b, P-450e, P-450f, P-450g, or P-450h or any other cytochrome P-450 in "Western blots" of liver microsomes from untreated or 3-methylcholanthrene-treated rats. C8 was a potent inhibitor of metabolism catalyzed by cytochrome P-450c in a reconstituted system as well as microsomes from 3-methylcholanthrene-treated rats. This antibody effected maximal inhibition of catalytic activity at an approximately 0.5:1 molar ratio of IgG to cytochrome P-450c, i.e. one antibody-binding site per epitope on cytochrome P-450c.
Highlights
(P3X63Ag8.653) and 10 hybridoma clones secreting cinogenic substances
Ten of the 30 positive clones were selected for further study, three of which produced antibody that cross- 2000 reactedwith cytochromeP-450d.ELISAscreening of subclones derived fromclones which produceantibody that cross- 5 0 0 reacts with cytochrome P-450ddid not reveal any dissociation of reactivitytowardcytochromes P-45Oc and P-450d
Characterization of the 10 MAb directed against rat liver microsomal cytochrome P-45Oc has shown that nine are distinct antibodies
Summary
Materials-Pristane (2,6,10,14-tetramethylpentadecane)and 4chloro-1-naphthol were obtained from Aldrkh Chemical Co.; and polyethylene glycol 4000 was obtained from E. Tissue culture supernatant,ascites fluid or purified antibody was added to the wells, incubated for 1.5-3 h, and discarded For this and subsequent steps, antibodies were diluted in phosphate-buffered saline containing 0.1% bovine serum albumin and 1%normal rabbit serum. After 1-1.5 h of incubation, the antibody solution was discarded and the plate was washed three times with phosphate-buffered saline containing 0.05% Tween 20. The blots were rinsed three times with phosphatebuffered saline containing 0.05% Tween 20 and incubatedwith 1:2000 dilution of F(ab'h fragments of goat anti-mouse IgG (specific for both the heavy and light chains) which were conjugated with horseradish peroxidase. The peroxidase-conjugated antibody was allowed to react with the blots for 2 h at 37 "C in phosphate-buffered saline containing 2% horse serum, 5% goat serum, 2% bovine serum albumin, and 0.05% Tween 20. Substratebindingspectra of cytochrome P-45Oc were determinedas described for microsomal cytochrome P-450 [33]
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