[Preparation of polyclonal and monoclonal antibodies against CRF and change of CRF in the sleep-deprived rat brain].
OBJECTIVE
To produce polyclonal and monoclonal antibodies against corticotropin-releasing factor (CRF) and to study the changes of CRF in sleep-deprived rat brain with the antibodies acquired.


METHODS
Commercial CRF was linked to bovine throglobulin (BTG) and bovine serum albumin (BSA) respectively to produce immunogen and embedding antigen. New Zealand rabbits and BALB/c mice were immunized with the BTG-CRF immunogen to produce polyclonal and monoclonal antibodies, respectively. The acquired antibodies were appraised with ELISA and immnohistochemical staining. The characterized antibodies were used to observe the changes of CRF in the rat brain 48 h after sleep deprivation.


RESULTS
CRF polyclonal antibody and 9 clones of monoclonal antibodies were obtained with high titer, affinity and specificity. Among them, the polyclonal antibody and 2 monoclonal antibodies (1D10, 2F4) were excellent in immunocytochemical staining. The CRF-like immunoreactive substances were found more strongly expressed in the neurons of paraverticular hypothalamic nucleus (PVN), central subnucleus of amygdala (CeA), oval subnucleus of bed nucleus of stria terminalis (BNSTov), and nucleus of solitary tract (NTS) in sleep-deprived rat brain. While they were much weaker and even absent in the control.


CONCLUSIONS
The polyclonal and monoclonal antibodies against CRF were successfully produced for immunocytochemical studies. The results indicate that CRF may play an important role in stress-responsive modulation during sleep deprivation. PVN, CeA and BNSTov are integral part of brain circuit related to the stress modulation.
Duan
Xiao-li
XL
Institute of Neurosciences, Fourth Military Medical University, Xi'an 710032, China. xiaoliduan@fmmu.edu.cn
Wang
Bai-ren
BR
Yang
Tang-bin
TB
Yu
Hai-ming
HM
Zhu
Zheng-hua
ZH
Xu
Zhen
Z
Wang
Xi
X
Wang
Jia-tong
JT
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D001921:Brain / Q000166:cytology / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D003346:Corticotropin-Releasing Hormone / Q000276:immunology* / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000276:immunology; D007106:Immune Sera / Q000276:immunology*; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012892:Sleep Deprivation / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology
D000911:Antibodies, Monoclonal; D007106:Immune Sera; D003346:Corticotropin-Releasing Hormone