Development of a double-monoclonal antibody sandwich ELISA: Tool for chicken interferon-γ detection ex vivo.

The aim of the present work was to develop reagents to set up a chicken interferon-γ (ChIFN-γ) assay. Four monoclonal antibodies (mAbs) specific for ChIFN-γ were generated to establish sandwich ELISA based on 2 different mAbs. To improve the detection sensitivity of ChIFN-γ, a double-monoclonal antibody sandwich ELISA was developed using mAb 3E5 as capture antibody and biotinylated mAb 3E3 as a detection reagent. The results revealed that this ELISA has high sensitivity, allowing for the detection of 125 to 500 pg/mL of recombinant ChIFN-γ, and also has an excellent capacity for detecting native ChIFN-γ. This ELISA was then used to detect ChIFN-γ level in chickens immunized with a Newcastle disease virus (NDV) vaccine, the immunized chicken splenocytes were stimulated by NDV F protein as recall antigen. From our results, it appears that the sensitivity range of this sandwich ELISA test is adequate to measure the ex vivo release of ChIFN-γ.

L'objectif de la présente étude était de développer des réactifs afin de mettre au point une épreuve de détection de l'interféron-γ de poulet (ChIFN-γ). Quatre anticorps monoclonaux (AcMo) spécifiques pour ChIFN-γ ont été produits afin d'avoir un ELISA sandwich reposant sur deux AcMo différents. Afin d'améliorer la sensibilité de détection de ChIFN-γ, un test ELISA sandwich a deux anticorps monoclonaux a été développé utilisant l'AcMo 3E5 comme anticorps de capture et l'AcMo 3E3 biotinylé comme réactif de détection. Les résultats ont démontré que ce test ELISA possède une sensibilité élevée, permettant la détection de 125 à 500 pg/mL de ChIFN-γ recombinant, et ayant également une excellente capacité à détecter le ChIFN-γ original. Ce test ELISA a par la suite été utilisé pour détecter les quantités de ChIFN-γ chez des poulets immunisés avec un vaccin contre la maladie de Newcastle (NDV), les cellules de la rate des poulets immunisés ont été stimulées par la protéine F du NDV comme antigène de rappel. À partir de nos résultats, il semble que la plage de sensibilité de ce test ELISA sandwich est adéquate pour mesurer la libération ex vivo de ChIFN-γ.(Traduit par Docteur Serge Messier).

Evaluation of Immunogenicity and Protective Efficacy Elicited by Mycobacterium bovis BCG Overexpressing Ag85A Protein against Mycobacterium tuberculosis Aerosol Infection.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is currently the only vaccine available for preventing tuberculosis (TB), however, BCG has varying success in preventing pulmonary TB. In this study, a recombinant BCG (rBCG::Ag85A) strain overexpressing the immunodominant Ag85A antigen was constructed, and its immunogenicity and protective efficacy were evaluated. Our results indicated that the Ag85A protein was successfully overexpressed in rBCG::Ag85A, and the Ag85A peptide-MHC complexes on draining lymph node dendritic cells of C57BL/6 mice infected with rBCG::Ag85A were detectable 4 h post-infection. The C57BL/6 mice infected with this strain had stronger antigen-specific interferon-gamma (IFN-γ) responses and higher antibody titers than those immunized with BCG, and the protective experiments showed that rBCG::Ag85A can enhance protection against Mycobacterium tuberculosis (M. tuberculosis) H37Rv infection compared to the BCG vaccine alone. Our results demonstrate the potential of rBCG::Ag85A as a candidate vaccine against TB.

Preparation of monoclonal antibodies against Mycobacterium tuberculosis TB10.4 antigen.

TB10.4 protein is a member of the ESX family that is necessary for Mycobacterium tuberculosis survival and plays a vital role in mycobacterial pathogenesis. In this study, the gene encoding TB10.4 was cloned into prokaryotic expression vecters pET-30(a) and pGEX-6p-1. The two recombinant proteins His-TB10.4 and GST-TB10.4 were then expressed in vitro in prokaryotic expression systems to develop monoclonal antibodies (MAbs) against TB10.4 protein. The purified rHis-TB10.4 protein was used to immunize BALB/c mice, and eight MAbs were produced. An immunoblotting analysis indicated that all these MAbs specifically recognize the TB10.4 protein. These new MAbs provide powerful reagents for further functional research into TB10.4 protein.

Loss of NR1 subunit of NMDARs in primary sensory neurons leads to hyperexcitability and pain hypersensitivity: involvement of Ca(2+)-activated small conductance potassium channels.

It is well established that activation of NMDARs plays an essential role in spinal cord synaptic plasticity (i.e., central sensitization) and pain hypersensitivity after tissue injury. Despite prominent expression of NMDARs in DRG primary sensory neurons, the unique role of peripheral NMDARs in regulating intrinsic neuronal excitability and pain sensitivity is not well understood, in part due to the lack of selective molecular tools. To address this problem, we used Advillin-Cre driver to delete the NR1 subunit of NMDARs selectively in DRG neurons. In NR1 conditional knock-out (NR1-cKO) mice, NR1 expression is absent in DRG neurons but remains normal in spinal cord neurons; NMDA-induced currents are also eliminated in DRG neurons of these mice. Surprisingly, NR1-cKO mice displayed mechanical and thermal hypersensitivity compared with wild-type littermates. NR1-deficient DRG neurons show increased excitability, as indicated by increased frequency of action potentials, and enhanced excitatory synaptic transmission in spinal cord slices, as indicated by increased frequency of miniature EPSCs. This hyperexcitability can be reproduced by the NMDAR antagonist APV and by Ca(2+)-activated slow conductance K(+) (SK) channel blocker apamin. Furthermore, NR1-positive DRG neurons coexpress SK1/SK2 and apamin-sensitive afterhyperpolarization currents are elevated by NMDA and suppressed by APV in these neurons. Our findings reveal the hitherto unsuspected role of NMDARs in controlling the intrinsic excitability of primary sensory neurons possibly via Ca(2+)-activated SK channels. Our results also call attention to potential opposing effects of NMDAR antagonists as a treatment for pain and other neurological disorders.

[Prokaryotic expression of recombinant bovine IL-4 and development of monoclonal antibodies against bovine IL-4].

AIM: To express recombinant bovine IL-4 (rBoIL-4) in Escherichia coli and prepare monoclonal antibody (mAb) against rBoIL-4. METHODS: The IL-4 gene without coding signal peptides was amplified from pSP73-BoIL-4 by PCR, then inserted into prokaryotic expression vector pGEX-6p-1 and pET-30a(+). The recombinant plasmids pGEX-6p-1-BoIL-4 and pET-BoIL-4 were transformed into DH5α for sequencing. After sequencing confirmation, the two recombinant plasmids were transformed into expression bacteria BL21 and BL21(DE3) respectively. BALB/c mice were immunized with the purified protein rHis-BoIL-4. With the purified rGST-BoIL-4 as detecting antigen, mAb-produced hybridoma cells against BoIL-4 were screened by indirect ELISA. The specificity of the mAbs was characterized by indirect ELlSA, Dot-ELlSA and Western blot. RESULTS: The recombinant bacteria BL21(pGEX-6p-1-BoIL-4) and BL21(DE3)(pET-BoIL-4) were developed. After induced by IPTG, SDS-PAGE analysis showed that the expression products of rGST-BoIL-4 and rHis-BoIL-4 had a molecular weight of 39 kD and 19 kD respectively, and expressed in inclusion body form. Seven hybridoma cell lines secreting mAbs against BoIL-4, named 2B8, 4A10, 5D6, 5D8, 7G10, 8B7 and 10F8 were obtained. The immunoglobulin subclasses were IgG1. The ascitic titers of these mAbs were 5 000, 16 0000, 10 000, 640 000, 5 000, 40 000 and 40 000, respectively. In Dot-ELISA, all mAbs could only react to the immunogen and the detecting antigen. Western-blot analysis confirmed that mAbs could only react to the corresponding recombinant proteins. The mAbs also reacted to the standard recombinant boIL-4 with biological activity. CONCLUSION: Seven mAbs specific to rBoIL-4 protein are obtained, which may have important application value in further study on diagnosis and pathogenesis of cattle diseases.