Peptide-MHC class I complex （pMHC） is a specific ligand for TCR recognition, and important for CD8^＋T cell activation. Here we described a genetically engineered divalent class I major histocompatibility complex （...Peptide-MHC class I complex （pMHC） is a specific ligand for TCR recognition, and important for CD8^＋T cell activation. Here we described a genetically engineered divalent class I major histocompatibility complex （MHC） molecule, H-2K^d/IgG2aFc, a fusion protein consisting of the extracellular domains of H-2K^d, a murine MHC class I molecule, and the Fc region of IgG2a. This fusion protein is expected to attach the H-2K^d molecule to the surface of murine macrophage （MФ） through its Fc portion binding to Fc receptor （FcR） of MФ. cDNAs coding for the extracellular domains of H-2K^d and the Fc region of IgG2a were cloned respectively, and then recombined into plasmid pcDNA3.1（＋）. The H-2K^d/IgG2aFc protein was expressed by the plasmid-transfected cell line J558L, and purified from its supernatant with a Staphylococcal Protein A （SPA） column. The fusion protein showed a 58.4 kDa band as revealed by SDS-PAGE and Western blotting with murine IgG-specific antibody, which consists with that expected for extracellular domains of H-2K^d heavy chain plus the Fc region of IgG2a. The sandwich ELISA assay with antibodies specific for Fc portion and for H-2K^d indicated the fusion protein consists of both Fc portion and H-2K^d. Peritoneal MФ of C57BL/6 （H-2K^b） can be stained with H-2K^d specific monoclonal antibody （mAb） after incubated with the H-2K^d/IgG2aFc fusion protein. These results demonstrate the fusion protein can be used to attach the H-2K^d molecule to the surface of murine MФ, and provides a novel means to manipulate the T cell recognized epitope on the surface of murine MФ, which can be applied to activate antigen-specific cytotoxic T lymphocyte （CTL）.展开更多
Objective: To analyze the dynamic evaluation of chemiluminescence, colloidal gold, and immunofluorescence chromatography in detecting antibodies in COVID-19 patients within four weeks of infection, and to provide evid...Objective: To analyze the dynamic evaluation of chemiluminescence, colloidal gold, and immunofluorescence chromatography in detecting antibodies in COVID-19 patients within four weeks of infection, and to provide evidence for clinical application. Method: 74 patients with confirmed SARS-COV-2 infection in the local area were selected as the experimental group, while 231 patients with negative SARS-COV-2 results but not vaccinated with Covid19 vaccine were selected as the control group;during the first, second, third, and fourth weeks after enrollment in the experimental group, three methods were used to detect SARS-COV-2 IgG and IgM in patients’ blood: chemiluminescence method, colloidal gold antibody method, and immuno-fluorescence chromatography. In the control group, three methods were used to detect SARS-COV-2 IgG and IgM during physical examination for SARS-COV-2 nucleic acids. The ROC curve was drawn to analyze the value of each indicator in predicting SARS-COV-2 infection, and the kappa method was used to analyze the consistency of the detection results of each indicator. Results: There was no significant difference in the positive rates of SARS-COV-2 IgM and IgG antibodies detected by chemiluminescence, colloidal gold, and immunofluorescence chromatography during the four-week period (P > 0.05). The positive rates of SARS-COV-2 IgM and IgG antibodies detected by the three methods during the first week of infection were not higher than 60%;when the three methods were used to detect SARS-COV-2 IgM and IgG in vivo, the AUC diagnosed by the test results was less than 0.80 at the first week, the diagnostic efficacy of the three methods was above 0.95 from the second week to the fourth week, and the diagnostic efficacy of the three methods was higher than 0.97 at the fourth week. The diagnostic efficacy of the three methods was comparable;the three methods for detecting SARS-COV-2 IgM and IgG antibodies showed high consistency in four cycles. Conclusion: Chemiluminescence, colloidal gold, and immunofluorescence chromatography are highly consistent in the detection of SARS-COV-2 IgM and IgG antibodies, and can be used as an auxiliary diagnosis and efficacy observation of novel coronavirus infections according to the needs, but the positive rate of infected people in the first week is low.展开更多
基金supported by a grant from the National Natural Science Foundation of China(NO.30271201).
文摘Peptide-MHC class I complex （pMHC） is a specific ligand for TCR recognition, and important for CD8^＋T cell activation. Here we described a genetically engineered divalent class I major histocompatibility complex （MHC） molecule, H-2K^d/IgG2aFc, a fusion protein consisting of the extracellular domains of H-2K^d, a murine MHC class I molecule, and the Fc region of IgG2a. This fusion protein is expected to attach the H-2K^d molecule to the surface of murine macrophage （MФ） through its Fc portion binding to Fc receptor （FcR） of MФ. cDNAs coding for the extracellular domains of H-2K^d and the Fc region of IgG2a were cloned respectively, and then recombined into plasmid pcDNA3.1（＋）. The H-2K^d/IgG2aFc protein was expressed by the plasmid-transfected cell line J558L, and purified from its supernatant with a Staphylococcal Protein A （SPA） column. The fusion protein showed a 58.4 kDa band as revealed by SDS-PAGE and Western blotting with murine IgG-specific antibody, which consists with that expected for extracellular domains of H-2K^d heavy chain plus the Fc region of IgG2a. The sandwich ELISA assay with antibodies specific for Fc portion and for H-2K^d indicated the fusion protein consists of both Fc portion and H-2K^d. Peritoneal MФ of C57BL/6 （H-2K^b） can be stained with H-2K^d specific monoclonal antibody （mAb） after incubated with the H-2K^d/IgG2aFc fusion protein. These results demonstrate the fusion protein can be used to attach the H-2K^d molecule to the surface of murine MФ, and provides a novel means to manipulate the T cell recognized epitope on the surface of murine MФ, which can be applied to activate antigen-specific cytotoxic T lymphocyte （CTL）.
文摘Objective: To analyze the dynamic evaluation of chemiluminescence, colloidal gold, and immunofluorescence chromatography in detecting antibodies in COVID-19 patients within four weeks of infection, and to provide evidence for clinical application. Method: 74 patients with confirmed SARS-COV-2 infection in the local area were selected as the experimental group, while 231 patients with negative SARS-COV-2 results but not vaccinated with Covid19 vaccine were selected as the control group;during the first, second, third, and fourth weeks after enrollment in the experimental group, three methods were used to detect SARS-COV-2 IgG and IgM in patients’ blood: chemiluminescence method, colloidal gold antibody method, and immuno-fluorescence chromatography. In the control group, three methods were used to detect SARS-COV-2 IgG and IgM during physical examination for SARS-COV-2 nucleic acids. The ROC curve was drawn to analyze the value of each indicator in predicting SARS-COV-2 infection, and the kappa method was used to analyze the consistency of the detection results of each indicator. Results: There was no significant difference in the positive rates of SARS-COV-2 IgM and IgG antibodies detected by chemiluminescence, colloidal gold, and immunofluorescence chromatography during the four-week period (P > 0.05). The positive rates of SARS-COV-2 IgM and IgG antibodies detected by the three methods during the first week of infection were not higher than 60%;when the three methods were used to detect SARS-COV-2 IgM and IgG in vivo, the AUC diagnosed by the test results was less than 0.80 at the first week, the diagnostic efficacy of the three methods was above 0.95 from the second week to the fourth week, and the diagnostic efficacy of the three methods was higher than 0.97 at the fourth week. The diagnostic efficacy of the three methods was comparable;the three methods for detecting SARS-COV-2 IgM and IgG antibodies showed high consistency in four cycles. Conclusion: Chemiluminescence, colloidal gold, and immunofluorescence chromatography are highly consistent in the detection of SARS-COV-2 IgM and IgG antibodies, and can be used as an auxiliary diagnosis and efficacy observation of novel coronavirus infections according to the needs, but the positive rate of infected people in the first week is low.
