Abstract
An antipeptide antibody was raised against a 14-mer synthetic peptide (CDFRANPNEPA KMN) corresponding to the amino acid sequence from 491 to 504 of human cytochrome P-450 (CYP)1B1. Rabbit-derived antisera demonstrated the ability to induce moderately high antibody titers (>1:105) as judged by enzyme-linked immunosorbent assay. In Western blot analysis, the purified antibody recognized a single protein band (estimated as 56 kDa) in microsomes prepared from human and rodent tissues. No significant cross-reactivity to either human CYP1A1 or human CYP1A2 protein was detected. Titration studies using recombinant human CYP1B1 and an enhanced chemiluminescence-based detection method demonstrated a minimal detection sensitivity for this antiserum at about 0.34 ng/band in 8 × 7-cm minigels. The immunoprecipitation and immunoinhibition results indicate that this antisera recognizes the nondenatured human CYP1B1 protein but does not inhibit its enzyme activity. Using this antibody, CYP1B1 protein was detected in nine different human tissues and in cultured cells induced by various chemicals. This highly specific, highly sensitive antibody provides an important tool to study tissue distribution and cellular expression levels of CYP1B1, with negligible cross-reactivity from the other members of the CYP1 family.
Footnotes
-
Send reprint requests to: George Y. Tang, Ph.D., National Center For Toxicological Research, HFT-100, 3900 NCTR Road, Jefferson, AR 72079. E-mail: ytang{at}nctr.fda.gov
-
Preliminary data were presented previously at the 88th Annual Meeting of American Association for Cancer Research (1997) in San Diego, CA, and published in Proceedings Vol. 38, no. 856.
- Abbreviations used are::
- CYP
- cytochrome P-450
- TCDD
- 2,3,7,8-tetrachlorodibenzo-p-dioxin
- E2
- 17β-estradiol
- HPLC
- high-performance liquid chromatography
- KLH
- keyhole limpet hemocyanin
- PAGE
- polyacrylamide gel electrophoresis
- Received June 22, 1998.
- Accepted October 12, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|