Preparation And Characterization Of Monoclonal Antibody Against Chicken CD8α And Its Application

Monoclonal antibodies (mAbs) against the surface molecules including CDs of T lymphocytes are valuable tools to study the development and function of distinct subpopulations of chicken T lymphocytes, to purify lymphocytes in order to analyze immune responses, to define the role of CD molecules after blocking these molecules based on mAbs in vivo, to analyze the structure and function of CD molecules. In the present study, the specific monoclonal mAb against chicken CD8α(ChCD8α) was developed.Three six-week old female BALB/c mice were immunized intraperitoneally with 1×106 lymphocytes of White Leghorn chicken every two weeks interval. Three days prior to fusion, mice were boosted with five times of the immunization dosage .Then the mice were sacrificed and splenocytes were harvested. Two weeks after fusion, culture supernatants of hybrid cells were screened by indirect immunofluorescence FACS using the cell line Flp-in-293-CD8α-flag as detecting immunogen.Positive hybrids were subcloned twice by limiting dilution to ensure that antibody producing cells were truly monoclonal and that the antibody secretion could be stable. One positive clone named 6E5. The hybridoma 6E5 was injected into pristine-primed mice (approximately 1×106 hybridoma cells/mouse) for ascite production. And then the subclass of mAb were determined as IgG1 by antigen-mediated ELISA employing. The titers of mAb was up to 1:1204000 in indirect immunofluorescent FACS assay.Using CD3/CD8 double staining FACS analysis, the results of 6E5 reacted with lymphocytes of White Leghorn chicken was coincided well with those of commercial mAb CT8. It suggests that 6E5 is a specific mAb against chicken CD8α.The epitopes recognized by 6E5 and commercial mAb CT8 were evaluated by FACS with the competitive immunological staining. It was shown that there was no inhibition between 6E5 and CT8 to binding lymphocytes White Leghorn chicken. The results demonstrated that the recognized epitopes were different for both of two mAbs.The specificity of mAb 6E5 was determined through detecting the reactivity with lymphocytes from different lymph tissues, including thymus, spleen and bursa of White Leghorn chicken. The results showed that mAb 6E5 could react with CD8+ T cells from thymus and spleen, but not bind to lymphocytes of bursa, which belong to B cells.The reactivity spectrum of mAb 6E5 was further evaluated to detecting the binding with peripheral blood mononuclear cells(PBMC) from different species of poultry, including Recessive White chicken, Shack-kee chicken, Langshan chicken, Anka chicken, Longxi partridge chicken, Campbell duck, Crow duck, Yangzhou goose. The results showed that mAb 6E5 could react with PBMC from all chicken species tested, but not with those from duck and goose. These results further demonstrated that mAb 6E5 was a useful antidody with good reactivity and specificity in FACS.Moreover, a FACS method based on mAb 6E5 was develop to measure the ratio of CD4+ lymphocytes with CD8+ ones. Hy-line White layer were immunized with Salmonella choleraesuis C500△asd(pYA3334) and Salmonella choleraesuis C500△asd(pYA3334-F), respectively. The ratio of CD4+ lymphocytes with CD8+ ones was monitored at different time points in different immunized groups. The results were coincided well between both 6E5 and CT8.All these results suggested that mAb 6E5 has been successfully developed, and is a very useful reagent in further studies.