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Development of the Duck CD25 Antigen Capture-ELISA and CD25 Kinetics of the Pathogen-infected Ducks

Author: FangJie
Tutor: ZhouJiYong
School: Zhejiang University
Course: Preventive Veterinary Medicine
Keywords: duck CD25 duck IL-2Rα AC ELISA soluble IL-2R
CLC: S858.32
Type: Master's thesis
Year: 2007
Downloads: 2
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Abstract


An antigen capture ELISA assay (AC-ELISA) for detection of duck soluble IL-2 receptor (duCD25) using monoclonal antibody (mAb) 6F2, which possesses high affinity to natural IL-2R on the cell surface, rabbit anti-duCD25 polyclonal antibody (pAb), and the recombinant duCD25 as a capture antibody, a detection antibody, and a reference protein, respectively. After optimization, the optimal working concentrations of mAb 6F2 and pAb were determined to be 5μg/ml and 1.25μg/ml, respectively. The detection limit of AC-ELISA for duCD25 was considered as 3.9-30 ng/ml by testing serially diluted the reference protein of duCD25, and coefficient of variation (CV) values of the intra-assay and inter-assay were calculated less than 10% and 11%, respectively.Soluble duCD25 was detected by AC-ELISA in cell supernatants of peripheral blood monouclear cells (PBMC) stimulated with 15μg/ml Con A for 8 days, whereas it not detectable in the 5μg/ml Con A activated cell supernatants. Analysis of the soluble and membrane duCD25 using anti-duCD25 pAb as a probe indicated that soluble CD25 in the supernatants is a cleavage form of the cell-associated CD25.In animal infection models, serum samples of ducks infected with anvian influenza virus (AIV) H5N1 and H9N2, and Riemerella anatipestifer sero type II respectively were collected and assayed for soluble duCD25 by the AC-ELISA at different time post infection (p.i.). In the AIV H5N1 infected ducks, soluble duCD25 was detected in serum at 3.987±0.942 ng/ml 72 h p.i. and 4.285±0.441 ng/ml 96h p.i. Serum soluble duCD25 in Riemerella anatipestifer infected ducks was shown to be 6.934±1.138 ng/ml on 2 d p.i., and increased greatly to 13.693±0.904 ng/ml on 3d p. i. However, the soluble duCD25 was not detected in duck serum after infected with AIV H9N2. Therefore, its is suggested that not all infection could be assessed by soluble CD25 levels, and analysis of CD25+ cells is necessary.Using AIV H9N2, H5N1, Riemerella anatipestifer sero typeⅡinfection as models, kinesis of duCD25+ cells in peripheral blood of the infected ducks was analyzed by FACS with mAb 6F2 specific for duCD25. The results showed that duCD25+ cells in peripheral blood of the normal ducks retains at a very low level (<5%). In the AIV H9N2 infected ducks, the frequency of duCD25+ cells in peripheral blood increased signicantly and reached the peak of 18.93%±0.66% within 24 h p.i., declined quickly at 48 h p.i. and back to the normal level at 72 h p.i., and then it began to rise again at 96h p.i. and maintained a stable level of 13.71%±3.73% at 144h p.i. In the AIV H5N1 infected ducks, CD25+ cells in peripheral blood reached the peak (11.13%±1.96%) within 12h p.i. and turned back to normal level at 24h p.i. without recovery from the infection. The frequency of duCD25+ cells of spleen in the dieing ducks was higher than 10% while the duCD25+ cells of peripheral blood in both dieing and recovered animals remained at a normal level.. The CD25+ cell frequency in Riemerella anatipestifer infected ducks continued to grow up and finally reached a peak of 24.8%±1.5% at 3 d p.i. With the analysis of soluble duCD25 in sera together, these results indicated that the duCD25+ cells and soluble CD25 played an important role in the immune response against pathogen infection. CD25 expressing on the cell surface represents the activation of immune response; on the other hand, the CD4+CD25+ Treg cells together with soluble CD25 in sera have an effect on suppressing proliferation of CD25+ cells in peripheral blood.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Poultry > Duck
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