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Interleukin_4 (IL_4), Bovine, recombinant ELISA

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[#YSRTPBP010] Interleukin_4 (IL_4), Bovine, recombinant ELISA


YSRTPBP010 | Interleukin_4 (IL_4), Bovine, recombinant ELISA, 1 ml.
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(1) Immunogenicity of a Staphylococcus aureus-cholera toxin A/B vaccine for bovine mastitis.[TOP]

Pubmed ID :29739718
Publication Date : //
Staphylococcus aureus causes a chronic, contagious disease of the udder, or mastitis, in dairy cows. This infection is often refractory to antibiotic treatment, and has a significant economic impact on milk production worldwide. An effective vaccine to prevent S. aureus mastitis would improve animal health, reduce antibiotic dependence and inform human vaccine approaches. The iron-regulated surface determinant A (IsdA) and clumping factor A (ClfA) are conserved S. aureus extracellular-matrix adhesins and target vaccine antigens. Here we report the results of two bovine immunogenicity trials using purified IsdA and ClfA-cholera toxin A/B chimeras (IsdA-CTA/B and ClfA-CTA/B). Cows were intranasally inoculated with IsdA-CTA/B + ClfA-CTA/B at dry off and followed for 70 days. Trial 1 utilized three groups with one or two booster doses at a total concentration of 600 or 900 μg. Trial 2 utilized two groups with one booster at a total concentration of 1200 μg. Humoral immune responses in serum and milk were examined by ELISA. Responses in serum were significant between groups and provide evidence of antigen-specific IgG induction after vaccination in both trials. Cellular proliferation was detected by flow cytometry using antigen-stimulated PBMCs from day 60 of Trial 2 and revealed an increase in CD4+ T cells from vaccinated cows. IsdA and ClfA stimulation induced IL-4 expression, but not IFN-γ or IL-17, in PBMCs from day 60 as determined by cytokine expression analysis. Opsonophagocytosis of S. aureus confirmed the functional in vitro activity of anti-IsdA antibodies from Trial 2 serum and milk. The vaccine was well tolerated and safe, and results support the potential of mucosally-delivered CTA/B chimeras to protect cows from mastitis caused by S. aureus.

Authors : Misra N, Wines T F, Knopp C L, Hermann R, Bond L, Mitchell B, McGuire M A, Tinker J K,

(2) [Preparation and identification of monoclonal antibodies against chicken interleukin 4].[TOP]

Pubmed ID :28712409
Publication Date : //
Objective To prepare and characterize the monoclonal antibodies (mAbs) against chicken interleuikin-4 (ChIL-4). Methods Using lymphocyte hybridoma technique, 8-week-old BALB/c mice were immunized with purified recombinant ChIL-4 (rChIL-4) with GST tag (GST-ChIL-4), and rChIL-4 with histidine tag (His-ChIL-4) was utilized to set up an indirect ELISA to screen positive hybridomas. Limited dilution methods were used to generate hybridoma cell lines stably secreting mAbs against ChIL-4. The specificity of the mAbs was identified by indirect ELISA, immunofluorescent assay (IFA) and Western blot. Results Four mAbs against rChIL-4 were obtained and named 6A8, 18F12, 19F1 and 20G3. ELISA titers of the 4 mAbs were (1.6~6.4)×10. ELISA, IFA and Western blot showed that the 4 mAbs could only react with rChIL-4, but not with the other proteins like bovine IL-4 (BovIL-4), chicken IFN-γ (ChIFN-γ). Conclusion The mAbs against rChIL-4 were successfully obtained.

Authors : Dai Hua, Chen Junhua, Zhang Haixia, Shen Ying, Chen Xiang, Jiao Xin'an,

(3) Development of a bead-based multiplexed assay for simultaneous quantification of five bovine cytokines by flow cytometry.[TOP]

Pubmed ID :28700121
Publication Date : //
Quantifying cytokines is extremely important in studies of host-pathogen interactions. Multiplex assays are commercially available but only for human and mouse cytokines. Here a method for the simultaneous quantification of five important bovine cytokines IFNγ, IL-4, IL-10, IL-12, and TNFα in cell culture supernatants, using flow cytometry was reported. Functional beads from BD Biosciences expressing specific APC intensity were used. Commercially available antibodies against bovine cytokines were covalently coupled to beads as capture antibodies. Fixed recombinant cytokines were revealed with a second monoclonal antibody coupled with biotin, then revealed with streptavidin-PE. This complex was analyzed using a standard flow cytometer. Experiments were performed to check no cross reactions had occurred. The limits of detection ranged between 0.08 and 0.4 ng/ml depending on the cytokine, and the linearity between the lower and higher limits was remarkable (R  > 99.8%). Finally, native cytokines from cell culture supernatants were tested. Results were compared using the standard ELISA test and showed that concentrations of native cytokine in cell culture supernatants were comparable with the two methods, with a wider dynamic range using beads and flow cytometry than with ELISA assays. Bovine IFNγ, IL-4, IL-10, IL-12, and TNFα in culture supernatants can be now simultaneously detected in a single assay, using a standard flow cytometer for both basic and high-throughput analyses. © 2017 International Society for Advancement of Cytometry.

Authors : Rodrigues Valérie, Baudier Jean Baptiste, Chantal Isabelle,

(4) Immunogenicity of amino acids 1-150 of Streptococcus GapC displayed on the surface of Escherichia coli.[TOP]

Pubmed ID :28179116
Publication Date : //
Streptococcus is one of the main pathogens that cause bovine mastitis. They includes into S.agalactiae, S.dysgalactiae, and S.uberis. The GapC protein is a virulence factor that is expressed on the surface of Streptococcus species. GapC is highly antigenic and immunization with GapC confers cross-protection against all three species. Our previous data showed that amino acids 1-150 of GapC (GapC) of S. dysgalactiae conferred similar immunoprotection compared to full-length GapC. Thus, the present study aimed to construct a recombinant Escherichia coli XL1-Blue strain that displayed GapC on its surface, and to investigate the immunogenicity of the surface-localized GapC. To do so, the ompA gene of the E. coli XL1-Blue strain was replaced with the lpp'-ompA-gapC1 or lpp'-ompA genes by λ Red recombination, the former of which fused GapC to an Lpp lipoprotein signal peptide and amino acids 1-159 of OmpA; the recombinant strains were named XL1-Blue/LOG76 and XL1-Blue/LO11, respectively. GapC was confirmed to localize to the surface of the XL1-Blue/LOG76 strain by an indirect enzyme-linked immunosorbent assay (ELISA), a fluorescence-activated cell sorter analysis, and laser-scanning confocal microscopy. Then, ICR mice were immunized intramuscularly with the XL1-Blue/LOG76 or XL1-Blue/LO11 strains, or recombinant GapC. The sera of the immunized mice were collected and the anti-GapC antibody levels were detected by ELISA. Lymphocytes secreting interleukin (IL)-4 and interferon-γ were detected by an enzyme-linked ImmunoSpot assay, as was the level of IL-17A level in the supernatant of cultured splenic lymphocytes. The mice immunized with the XL1-Blue/LOG76 strain or GapC exhibited better cellular and humoral immunity. Lastly, the immunized mice were challenged with S. uberis, S. dysgalactiae, and S. agalactiae strains, and mice that were immunized with the XL1-Blue/LOG76 strain were better protected than those that were immunized with the XL1-Blue/LO11 strain. These results indicate that it is feasible to display GapC on the E. coli surface as a vaccine against Streptococcus species.

Authors : Song Baifen, Yang Xijing, Sun Hunan, Yu Liquan, Ma Jinzhu, Wu Zhijun, Cui Yudong,

(5) Brucella abortus 2308ΔNodVΔNodW double-mutant is highly attenuated and confers protection against wild-type challenge in BALB/c mice.[TOP]

Pubmed ID :28131952
Publication Date : //
Brucellosis is an important zoonotic disease of worldwide distribution, which causes animal and human disease. However, the current Brucella abortus (B. abortus) vaccines (S19 and RB51) have several drawbacks, including residual virulence for animals and humans. Moreover, S19 cannot allow serological differentiation between infected and vaccinated animals. We constructed double deletion (ΔNodVΔNodW) mutant from virulent B. abortus 2308 (S2308) by deleting the genes encoding two-component regulatory system (TCS) in chromosome II in S2308.2308ΔNodVΔNodW was significantly reduced survival in murine macrophages (RAW 264.7) and BALB/c mice. Moreover, the inoculated mice showed no splenomegaly. The mutant induced high protective immunity in BALB/c mice against challenge with S2308, and elicited an anti-Brucella-specific immunoglobulin G (IgG) response and induced the secretion of gamma interferon (IFN-γ) and interleukin-4 (IL-4). Moreover, NODV and NODW antigens would allow the serological differentiation between infected and vaccinated animals. These results suggest that 2308ΔNodVΔNodW mutant is a potential live attenuated vaccine candidate and can be used effectively against bovine brucellosis.

Authors : Li Zhiqiang, Wang Shuli, Zhang Jinliang, Yang Guangli, Yuan Baodong, Huang Jie, Han Jincheng, Xi Li, Xiao Yanren, Chen Chuangfu, Zhang Hui,

(6) Schistosoma japonicum cystatin attenuates murine collagen-induced arthritis.[TOP]

Pubmed ID :27393379
Publication Date : //
Recombinant SjCystatin (rSjCystatin), a recombinant protein of Schistosoma japonicum cystatin, has been reported to have an effect on immunoregulation mediated by IL-10 induction. Rheumatoid arthritis (RA) is a common autoimmune inflammatory arthropathy, and recombinant immune-modulating drugs for RA treatment are under development. We aimed to study the putative immune regulation of rSjCystatin and its prophylactic/therapeutic effects on murine collagen-induced arthritis (CIA). CIA was induced in DBA/1 mice by inoculation with bovine collagen II (CII). rSjCystatin was administered prior or post development of CIA. The severity of CIA was assessed using established clinical and histopathological scoring systems. The incidence was also determined. The CII-specific antibodies in sera and cytokines in splenocyte culture supernatants were measured by ELISA. Th1/Th2/Th17 cells and Tregs development in splenocytes were monitored by flow cytometry. The inflammatory mediators in the diseased joint were semiquantitated by qPCR. Prophylactic injection of rSjCystatin attenuated paw clinical scores, incidence, and histopathology scores of joints in CIA mice. The arthritis-alleviative effects were closely associated with the augmentation of IL-4, IL-10, and collagen-specific IgG1, and with the distinct reduction of IFN-γ, collagen-specific IgG2a, and the marked decrease of proinflammatory cytokines IL-6, IL-17, and TNF-α and RANKL. The data indicate that rSjCystatin may prevent cartilage destruction and inflammation of joints in CIA mice. The effects are related to the inhibitory modulation of Th1 and Th17 and upregulation of Tregs and Th2 via a shift of cytokines profiling from Th1 to Th2 response.

Authors : Liu Fang, Cheng Weisheng, Pappoe Faustina, Hu Xiaodong, Wen Huiqin, Luo Qingli, Wang Shushu, Deng Fang, Xie Yuanyuan, Xu Yuanhong, Shen Jilong,

(7) [Immunogenicity evaluation of Mycobacterium tuberculosis MPT83 protein and establishment of serological diagnostic method for bovine tuberculosis detection].[TOP]

Pubmed ID :25958703
Publication Date : //
The aim of this study was to express Mycobacterium tuberculosis MPT83 protein and to evaluate its immunogenicity in murine model as well as the serological diagnosis potential value for bovine tuberculosis.

Authors : Meng Chuang, Wan Ting, Xu Zhengzhong, Shan Fa, Fan Feng, Chen Xiang, Jiao Xin'an,

(8) The effect of bovine viral diarrhea virus (BVDV) strains on bovine monocyte-derived dendritic cells (Mo-DC) phenotype and capacity to produce BVDV.[TOP]

Pubmed ID :24607146
Publication Date : //
Dendritic cells (DC) are important antigen presentation cells that monitor, process, and present antigen to T cells. Viruses that infect DC can have a devastating impact on the immune system. In this study, the ability of bovine viral diarrhea virus (BVDV) to replicate and produce infectious virus in monocyte-derived dendritic cells (Mo-DC) and monocytes was studied. The study also examined the effect of BVDV infection on Mo-DC expression of cell surface markers, including MHCI, MHCII, and CD86, which are critical for DC function in immune response.

Authors : Rajput Mrigendra K S, Darweesh Mahmoud F, Park Kaci, Braun Lyle J, Mwangi Waithaka, Young Alan J, Chase Christopher C L,

(9) Fasciola hepatica - the pilot study of in vitro assessing immune response against native and recombinant antigens of the fluke.[TOP]

Pubmed ID :24338305
Publication Date : //
Fasciola hepatica is a liver fluke that infects 2.4 million of people and causes great economical loss in animal production. To date a 100% effective vaccine has not been developed and the disease is controlled by drug therapy. Great efforts are put into development of effective vaccine against parasite what is difficult since Fasciola spp. (like other helmints) during evolutionary process has developed sophisticated and efficient methods to evade immune response. During preliminary experiments it is convenient to use cell lines which are relatively cheap and allow for reproducible comparison of results between laboratories. We stimulated BOMA (bovine monocyte/macrophage cell line) and BOMAC (bovine macrophage cell line) with native or recombinant antigens of Fasciola hepatica and assessed IFN-γ, IL-4 and TNF-α level upon stimulation. We observed diminished secretion of proinflammatory TNF-α in LPS activated BOMA cells stimulated with Excretory/Secretory products of adult fluke (Fh-ES). We also observed greater changes in gene expression in LPS activated BOMA cells than in non activated BOMA cells upon stimulation using Fh-ES. The results show possibility of using cell lines for in vitro research of bovine immune response against liver fluke, although this model still requires validation and further characterization.

Authors : Bąska Piotr, Zawistowska-Deniziak Anna, Zdziarska Anna M, Wasyl Katarzyna, Wiśniewski Marcin, Cywińska Anna, Klockiewicz Maciej, Januszkiewicz Kamil, Wędrychowicz Halina,

(10) Cloning and expression of Bos indicus interleukin-4 in mammalian cells.[TOP]

Pubmed ID :23821822
Publication Date : //
Dendritic cells (DC) which are located at the interface of innate and adaptive immunity are targets of infection by many RNA and DNA viruses. Advances in the ex vivo generation of monocyte derived non proliferating dendritic cells have been used for clinical application like immunotherapy. IL-4 cytokine plays essential role in the maturation and generation of DCs. Bos indicus interleukin 4 (boIL-4) 408 bp was amplified from PBMC's and cloned in pBSIIKS+ vector. The sequence analysis showed N terminal 69 bp signal sequence and one N-glycosylation site. The phylogenetic tree analysis showed that Bos indicus IL-4 is closely related to the ruminant IL-4 and least sharing of genetic line of human and mouse IL-4. The recombinant bolL-4 protein was expressed in CHO cells which secreted a 16 kDa protein which was confirmed by SDS PAGE and western blotting. The rec-boIL-4 protein proliferated the bovine PBMC's, decreased production of nitric oxide in antigen stimulated macrophages, and phagocytosed the micro particles confirming its activity on dendritic cells.

Authors : Prashanth T, Reddy G R, Suryanaryana V V S, Dechamma H J,