ABSTRACT
RBL-2H3 mediator release assay, developed for specific IgE screening studies, was not as sensitive as passive cutaneous anaphylaxis (PCA) assay in the polyclonal antibody detection. In the present investigation, the detection sensitivity of RBL-2H3 assay was elevated by modifying the experiment protocols from choosing the proper releasing medium and optimizing the sensitization manner. The polyclonal antibody was generated from Brown Norway (BN) rats exposed to Ovalbumin (OVA). In contrast to Tyrode buffer A, RBL-2H3 cells cultured in DMEM had a lower spontaneous secretion and a higher response to antigen stimulation, both of which could help to increase the detection sensitivity. The rat sera used in the sensitization process should be diluted appropriately to avoid the proliferation-promoting effect on RBL-2H3 cells. The results of the kinetics of sensitization showed that prolonging the sensitization time and then reculturing the cells in IgE free medium for a further 24 h after the removal of rat sera could reach a marked increase in the degree of sensitization. The highest anti-OVA antibody titer detected by the modified RBL-2H3 assay was 4096, while PCA assay was 1024. These data provide evidence that the modified RBL-2H3 mediator release assay has a promising prospect in the determination of the biologic activity of polyclonal antibody.
Subject(s)
Immunoglobulin E/analysis , Leukemia, Basophilic, Acute/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Ovalbumin/immunology , Passive Cutaneous Anaphylaxis , Rats , Rats, Inbred BNABSTRACT
Chlorogenic acid (CGA), a naturally occurring polyphenol compound, has a number of biological activities. However, roles of CGA in the mast cell-dependent anaphylactic reaction have not been fully examined. In the present study, the effect and mechanism of CGA on mast cell-dependent anaphylactic reaction were investigated using in vivo and in vitro models. CGA inhibited compound 48/80-induced systemic anaphylactic shock in mice and skin vascular permeability in rats. CGA also inhibited anti-dinitrophenyl (DNP) IgE-mediated passive cutaneous anaphylaxis (PCA). Moreover, CGA dose-dependently reduced histamine and TNF-alpha release from RBL-2H3 cells activated by anti-DNP IgE. Pretreatment with CGA suppressed IgE-antigen complex induced calcium uptake into RBL-2H3 cells. When CGA was added, the level of intracellular cyclic adenosine monophosphate (cAMP) in RBL-2H3 cells was significantly elevated compared with the untreated cells. Decreased calcium uptake and increased cAMP level might be involved in the inhibitory effect of CGA on mast cell activation. These results suggest a possible therapeutic application of CGA in allergic diseases.
