Aim 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. Conclusion 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.
Monoclonal antibody techniques are very important tools in modern life science research. Despite extensive research efforts paid in recent years, and promising results yielded in the study on the structure and function of genes and proteins, there is still a great need for further researches on the definition, principle and applicability of some immunological methods. This review gives an overview of the advances in immunological researches, including DNA immunization, cellular immunization and preparation of monoclonal antibodies. Using methods of modem molecular immunology, such as genetic immunization, cellular immunization, subtractive immunization and repetitive immunization multiple sites (RIMMS), to construct eukaryotic expression vector and to prepare high-affinity monoclonal antibodies in short time, the conventional method which is time-consuming and laborious could be improved. It is meaningful to the field of basic research and application, such as proteomics, biochip, clinical medicine and diagnosis and therapy of diseases.
The immunologic phenotypes of 68 cases of acute nonlymphoblastic leukemia (ANLL) were analysed with a panel of monoclonal antibodies. The results showed that the frequencies of myeloid antigens expressed on ANLL cells are CD15 > CD33 > CD13 > CD14 and there were few cases expressing lymphoid antigens. The expression of myeloid antigen on leukemia cells is not only the evidence for diagnosis ANLL, but also is valuable predicting prognosis. In cases with the ratio of CD33/CD13 < 1, the complete remission (CR) rate was higher than those with CD33/CD13 > 1 (P < 0.05). Patients with expression of CD15 antigen on their leukemia cells appeared to be associated with long survival.
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