DEVELOPMENT OF MONOCLONAL ANTIBODIES AGAINST THE HUMAN CYP11B1 AND CYP11B2

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 723-757-Renin-Angiotensin-Aldosterone System/Endocrine Hypertension
Bench to Bedside
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-734
Celso E Gomez-Sanchez*1, Xin Qi2, Carolina Velarde-Miranda3, Elise P Gomez-Sanchez4, C Richard Parker Jr.5, Fumitoshi Satoh6, Takashi Maekawa7, Yasuhiro Nakamura8, Hironobu Sasano9 and William E Rainey II10
1G.V. (Sonny) Montgomery VA Medical Center, Jackson, MS, 2University of Mississippi Medical Center, Jackson, MS, 3University of Mississippi Medical Center, 4G,V (Sonny) Montgomery VA Medical Center, 5Univ of Alabama - Birmingham, Birmingham, AL, 6Tohoku University Hospital, Sendai, Japan, 7Tohoku University School of Medicine, 8Tohoku University Graduate School of Medicine, Sendai, Japan, 9Tohoku Univ Sch of Med, Miyagi, Japan, 10University of Michigan Med Center, Ann Arbor, MI
The final enzymatic steps in the synthesis of cortisol and aldosterone are exerted by the cytochrome P450 11b-hydroxylase (CYP11B1) and the cytochrome P450 aldosterone synthase (CYP11B2), respectively.  The CYP11B2 is located only in the zona glomerulosa; CYP11B1 is located in the zona fasciculata-reticularis.  The enzymes share approximately 95% homology at the aminoacid (AA) level, thus specific antibodies have been difficult to develop.  Multiple peptides were synthesized representing epitopes that were different between the two proteins, and conjugated to various immunogenic proteins for immunization.  Only those corresponding to AA 41-55 of the CYP11B2 and AA 80-90 of the CYP11B1 elicited specific monoclonal antibodies in mice and rats, respectively.  The antibodies detect a single band at the correct molecular mass for the enzymes and do not cross-react with the closely homologous enzyme.  Immunohistochemistry using sections of paraffin embedded samples required antigen retrieval by heating in pH8 EDTA buffer for 45 min and permeabilization with 0.5% SDS in the blocking buffer.   Immunohistochemistry showed CYP11B1 immunoreactivity in the zona fasciculata that in some areas of the gland extended to the capsule.  The CYP11B2 enzyme detected only a few scattered single cells and clusters of cells next to the capsule which were often completely surrounded by cells expressing CYP11B1 as demonstrated using double labeling. There was wide variation in the number of cells and cell clusters expressing the CYP11B2 enzyme in different adrenals. ZF cells expressing CYP11B1 enzyme also co-express the 17ahydroxylase, but cells expressing the CYP11B2 do not. 

Some aldosterone-producing adenomas show a very uniform staining with the CYP11B2 enzyme and often few nests of cells that only express the CYP11B1 within the adenoma.

The availability of a constant supply of specific mouse and rat monoclonal antibodies is a significant resource for the study of adrenal disorders.

Disclosure: WER II: Speaker, Pfizer, , Lilly. Nothing to Disclose: CEG, XQ, CV, EPG, CRP Jr., FS, TM, YN, HS

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: National Institutes of Health grant HL27255, HL105383, Dept Veterans Affairs Award Number 1018X007080.