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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.

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