Development And Characterization Of Monoclonal Antibodies Specific For Bovine Interferon-Gamma

Interferon-gamma (IFN-y, also termed immune interferon or type II interferon) is an important mediator of cellular immune reactions, primarily secreted by natural killer (NK) cells and natural killer T (NKT) cells of the innate immune response, and CD8+and CD4+Thl effector T cells of the adaptive immune system upon stimulation with antigens or nonspecific mitogens. Interferon-y has broad and complex biological functions, such as involving in tumor control, defensing viral and intracellular bacterial infections, and exerting a range of immunoregulatory activities.IFN-y has been shown to play a key role in the regulation of immune responses, specifically in the induction and maintenance of Thl responses which are necessary for the defense against intracellular pathogens. The appearance of IFN-y-secreting T lymphocytes is distinctive of Thl responses, and the level of IFN-y in biological samples or the enumeration of IFN-y-producing cells is frequently used as indicator of an ongoing Thl immune response. Therefore the need to be able to detect and quantify IFN-y in biological samples is particularly evident in animal models of intracellular infections such as tuberculosis, listeriosis, chlamydial infections, and many others.The monoclonal antibodies (McAbs) which are able to specifically recognize natural bovine interferon-gamma (BoIFN-y) can be used to purify the natural BolFN-y and various recombination BoIFN-γ as ligands, or to research the BoIFN-y biological activity and immunoregulatory activity as research tools in vivo; in addition, a variety of immunological detection methods (such as antigen capture ELISA, ELISPOT, Immuno-PCR, ICC etc.) established based-on these McAbs can be capable of detecting BoIFN-gamma levels in biological samples, to evaluate the status of cellular immunity, as well as diagnose or auxiliarily diagnose diseases (such as tuberculosis, paratuberculosis, etc.). To this end, high specific BoIFN-y McAbs were developed which are able to recognize natural BoIFN-y in an inhibition ELISA, and provided the above-mentioned objectives with a principal, crucial and the most basic material foundation.Six-week-old BALB/c mice were immunized with the purified rHis-BoIFN-γ; subsequently, cell fusions were performed through the lymphocyte hybridoma technique. Then30hybridoma cell lines which can steadily secrete BoIFN-y monoclonal antibody screened by indirect ELISA with rGST-BoIFN-γ were acquired after three subclones by limiting dilution, and they were named as1B4,1D2,1D5,1E7,1G5,1H2,1H5,2B4,2B12,2D7,2D8,2E2,2E3,2G5,3A8,3A10,3C12,3E3,3E9,3F1,3F2,4C8,4D3,5B8,5F5,6C7,6E8,7A6,7D10and7E5, respectively.Isotype analysis revealed1B4,1D2,1D5,1E7,1H2,2B12,2D7,2D8,2G5,3C12,3E3,3F1,3F2,4C8,4D3,5F5and6E8are IgGl,1G5,1H5,2B4,2E2,2E3,3A8,3A10,3E9,5B8,7A6and7E5as IgG2a,6C7and7D10two McAbs as IgG2b. The indirect ELISA results showed that the titers of all McAbs were very high, with l*104~8xl04in culture supernatant and2×l06~1.6×l07in ascitic fluids, besides1H2and3F1as1:1280,5B8as1:2560in culture supernatant.Western blotting analysis revealed that1B4,1D2,1E7,1G5,1H2,1H5,2B12,2D8,2E2,2E3,2G5,3E3,3E9,4D3,5B8and6C7sixteen McAbs presented two specific bands of molecular weights23KDa and43KDa, rHis-BoIFN-y and rGST-BoIFN-y, respectively, and this suggested that the sixteen McAbs had good reactivity with BoIFN-y.In the reference BoIFN-y indirect ELISA,1B4,1D2,1E7,1G5,1H2,2B12and2D8seven McAbs culture supernatant optical density were very low, bordering on negative;1D5,1H5,2B4,2D7,2E2,2E3,2G5,3A8,3A10,3C12,3E3,3E9,3F1,3F2,4C8,4D3,5B8,5F5,6C7,6E8,7A6,7D10and7E5twenty-three McAbs revealed positive reaction.In specificity of McAbs assay, all30McAbs only react with BoIFN-γ except for cross-reactivity of most McAbs with rHis-CerIFN-γ (cervus interferon-gamma), not other recombinant cytokines expressed in prokaryotic expression system.In indirect immunofluorescence assay the reactivity of all30McAbs with rBac-BoIFN-γ expressed in the baculovirus expression vector system through Sf9cells transfected by recombinant baculovirus Bacmid-preBoIFN-γ were identified, the results showed that1B4,1D2,1E7,2B12,2D8,4D3,5B8,5F5and7D10nine McAbs had negative fluorescence;1D5,1G5,1H2,1H5,2B4,2D7,2E2,2E3,2G5,3A8,3A10,3C12,3E3,3E9,3F1,3F2,4C8,6C7,6E8,7A6and7E521McAbs possessed significant specific fluorescence on the cell membrane and appeared positive reaction with rBac-BoIFN-γ.An inhibition ELISA using natural bovine IFN-γ collected from bovine whole blood plasma stimulated with PWM as the competitor was developed to identify30McAbs, and inhibition rate of eleven McAbs (1G5,1H2,1H5,2E2,2E3,2G5,3E3,3E9,4D3,5B8and6C7) were over50%; their maximal inhibition rates were75.09%,79.10%,79.55%,71.35%,83.40%,84.76%,83.33%,76.10%,93.26%,74.90% and85.43%, respectively. In other words, the eleven McAbs had the capability to react with natural bovine IFN-y, and possessed a good practicality prospect.In addition, in an inhibition ELISA in which rFLAG-BoIFN-γ expressed by secreting type Flp-In-293-BoIFN-γ-FLAG cells as the competitor, and the results displayed that2G5,3E3and3E9showed positive reaction for rFLAG-BoIFN-y, the maximal inhibition rates95.10%,91.64%and94.14%, respectively.In summary, thirty hybridoma cell lines secreted BoIFN-γ McAbs were acquired, and among them,1G5,1H2,1H5,2E2,2E3,2G5,3E3,3E9,4D3,5B8and6C7eleven McAbs have ability to react to natural BoIFN-γ, which suggested these McAbs application value and provided subsequent research for perfect biological materials; besides,2G5,3E3and3E9McAbs had good reactivity with rFLAG-BoIFN-γ expressed by Flp-In-293-BoIFN-γ-FLAG cell line, and can provide favorable ligand McAbs for the cell line application.