Development of monoclonal antibodies against human CYP11B1 and CYP11B2

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.

Figure 1

Comparative alignment of the protein sequence between human CYP11B1 and CYP11B2. The underlined letters indicate the amino acid differences between the sequences. The red letters are the sequences used for synthesis of peptides that were conjugated for immunization. The green –C represents a cysteine that was added to the synthetic peptide for conjugation.

Figure 2

Representative immunoblots used for antibody screening of wells that had tested positive using ELISA. EGFP-CYP11B1 or EGFP-CYP11B2 (0.8-1 mg) was separated in a single lane 12% PAGE Criterion gel and transferred to a PVDF membrane and used in a 21 lane SurfBlot. Lane 20 of the CYP11B1 blot was probed with an anti-GFP antibody. The CYP11B1-80-thyroglobulin conjugate rat monoclonal antibodies had two positive clones by immunoblot (#2 and #7). The CYP11B2-41-thyroglobulin conjugate mouse monoclonal antibodies had two positive cones by immunoblot (#13 and # 17). Supernatant from the cultured hybridomas were used at 1/50 dilution.

Figure 3

Immunoblot analysis using the CYP11B2-41-13 antibody to detect GFP fusion proteins for CYP11B2 and CYP11B1, lysated from 293TN cells transfected with cDNA for either CYP11B1 or CYP11B2 enzymes and HAC15 cells stimulated for 24 h with Angiotensin II, Forskolin or potassium.

Figure 4

Immunoblot analysis using the CYP11B1-80-7 antibody to detect EGFP fusion proteins for CYP11B2 and CYP11B1 from homogenate of H293 transfected cells, as well as from isolated mitochondria from HAC15 cells from untreated cells and those incubated with angiotensin II, forskolin and potassium. Lanes were loaded with 30 μg. The EGFP-CYP11B1 gave a strong band, while the EGFP-B2 did not give a band even after long overexposure.

Figure 5

CYP11B1 and CYP11B2 use for adrenal immunohistochemistry: Panel A and C are two different areas of the same adrenal, one showing scattered cells and the other clusters stained with the CYP11B2-41-17B antibody. Panel B and D are similar but at higher magnification. Panel E shows scattered cells with variable degree of staining. Panel F is an adrenal from a 5 day infant. Panel G and H are staining at two magnifications with the CYP11B1-80-2-2 antibody. Panels I-K are double staining for CYP11B1 and CYP11B2.

Figure 6

Triple immunofluorescence for CYP11B1, CYP11B2 and 17α-hydroxylase. The top panels are individual fluorescence for each protein and the bottom panels are the merged images as indicated.