Development of monoclonal antibodies against human CYP11B1 and CYP11B2

Mol Cell Endocrinol. 2014 Mar 5;383(1-2):111-7. doi: 10.1016/j.mce.2013.11.022. Epub 2013 Dec 8.

Abstract

1. The final enzymes in the biosynthesis of aldosterone and cortisol are by the cytochrome P450 CYP11B2 and CYP11B1, respectively. The enzymes are 93% homologous at the amino acid level and specific antibodies have been difficult to generate. 2. Mice and rats were immunized with multiple peptides conjugated to various immunogenic proteins and monoclonal antibodies were generated. The only peptide sequences that generated specific antibodies were amino acids 41-52 for the CYP11B2 and amino acids 80-90 for the CYP11B1 enzyme. 3. The mouse monoclonal CYP11B2-41 was specific and sensitive for use in western blots and produced specific staining of the zona glomerulosa of normal adrenal glands. The rat monoclonal CYP11B1-80 also detected a single band by western blot and detected only the zona fasciculata. Triple immunofluorescence of the adrenal demonstrated that the CYP11B1 and the CYP11B2 did not co-localize, while as expected the CYP11B1 co-localized with the 17α-hydroxylase.

Keywords: 17α-Hydroxylase; Adrenal cortex; CYP11B1; CYP11B2; Immunofluorescence; Monoclonal antibodies.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adult
  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / biosynthesis*
  • Antibodies, Monoclonal / isolation & purification
  • Cytochrome P-450 CYP11B2 / immunology*
  • Cytochrome P-450 CYP11B2 / metabolism
  • Humans
  • Immunohistochemistry / methods
  • Infant, Newborn
  • Mice
  • Molecular Sequence Data
  • Peptides / administration & dosage
  • Peptides / chemical synthesis
  • Peptides / immunology*
  • Rats
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Steroid 11-beta-Hydroxylase / immunology*
  • Steroid 11-beta-Hydroxylase / metabolism
  • Steroid 17-alpha-Hydroxylase / immunology
  • Steroid 17-alpha-Hydroxylase / metabolism
  • Zona Fasciculata / immunology
  • Zona Fasciculata / metabolism
  • Zona Fasciculata / ultrastructure*
  • Zona Glomerulosa / immunology
  • Zona Glomerulosa / metabolism
  • Zona Glomerulosa / ultrastructure*

Substances

  • Antibodies, Monoclonal
  • Peptides
  • Steroid 17-alpha-Hydroxylase
  • Cytochrome P-450 CYP11B2
  • Steroid 11-beta-Hydroxylase