| Interleukin-4 (IL-4) is a multipotent T-cell-derived cytokine associated with the modulation of host defense and immunity by acting on a variety of cell type. IL-4 plays the pivotal role in regulating T and B lymphocyte differentiation which is required for the development of Th2 immune responses. In addition, IL-4 is well recognized as a modulator of monocyte or macrophage activation.The studies of IL-4 gene and its biology activity in human, mice, rats and pigs have been reported. In chicken, the detection of IL-4 and its functions during immune responses have been influenced by the lack of specific reagents to this important Th2 cytokine. In this study, the specific antibodies against ChIL-4 were developed, and an antibody sandwich ELISA was established. It may be a valuable tool for immunodetection, functional analysis of immune cell and immunoregulation in poultry.1. Production and characterization of monoclonal antibodies against ChIL-4To prepare monoclonal antibodies against chicken ChIL-4, the purified protein rGST-ChIL-4 was used as immunogen to immunize subcutaneously 8-week-old BALB/c mice. The immune dose was 100μg recombinant protein emulsified in complete Freund's adjuvant for the first subcutaneous immunization and in incomplete Freund's adjuvant for the second injection, the recombinant protein without adjuvant was used to boost immunized mice for the third time through intraperitoneal injection, the interval of two immunizations is 2 weeks. Then an intravenous dose of purified protein was administrated. After 3 days, splenocytes from immunized mice were fused with Sp2/0-Ag-14 myeloma cells. Purified rHis-ChIL-4 was used as detecting antigen, and the supernatant of hybridoma clones was screened by indirect ELISA. Nine hybridoma cell lines secreting McAbs against ChIL-4, named 16D8, 17D7, 18F7, 18F12, 19F1, 20C9, 20F2, 20G3, 6A8 were obtained. The immunoglobulin subclass of McAbs 16D8 and 20F2 were IgG2b, the others were IgG1. The ascitic titers of these McAbs were 1,280,000, 1,280,000, 640,000, 640,000, 640,000, 640,000, 640,000, 640,000, 160,000 and 160,000, respectively. In Dot-ELISA, all McAbs could only react with the immunogen and the detecting antigen. Western-blot analysis confirmed that all McAbs could only react with the corresponding recombinant proteins. All these results suggested that the specific McAbs against ChIL-4 were developed, which are very useful in both fundamental and practical studies.2. The establishment of an antibody sandwich ELISA for the detection of ChIL-4After screened from the six antibodies, the 20C9 (10μg/ml) was selected and used as coating antibody, and Bio-16D8 (2μg/ml) used as the second antibody in an antibody sandwich ELISA. Based on the optimization assay, the phosphate buffered saline(PBS) containing 10% FCS was determined as the blocking solution, and the PBS containing 4%PEG6000 and 0.05% Tween-20 was used as diluting solution. The optimal reaction time course determined as 2h as reaction time of antigen, 30min as reaction time of labeled antibody, 30min as reaction time of HRP-streptavidin, meanwhile 5min as substrate reaction time. The detecting limit of this antibody sandwich ELISA was 0.039μg/ml of ChIL-4.
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