Both HIV-1 integrase (IN) and the central catalytic domain of IN (IN-CCD) catalyze the disintegration reaction in vitro.In this study,IN and IN-CCD proteins were expressed and purified,and a high-throughput format enz...Both HIV-1 integrase (IN) and the central catalytic domain of IN (IN-CCD) catalyze the disintegration reaction in vitro.In this study,IN and IN-CCD proteins were expressed and purified,and a high-throughput format enzyme-linked immunosorbent assay (ELISA) was developed for the disintegration reaction.IN exhibited a marked preference for Mn2+ over Mg2+ as the divalent cation cofactor in disintegration.Baicalein,a known IN inhibitor,was found to be an IN-CCD inhibitor.The assay is sensitive and specific for the study of disintegration reaction as well as for the in vitro identification of antiviral drugs targeting IN,especially targeting IN-CCD.展开更多
Systemic lupus erythematosus （SLE） is a complex autoimmune disease characterized by the production of autoantibodies to a broad range of self-antigens. Profiling the autoantibody repertoire using array-based technol...Systemic lupus erythematosus （SLE） is a complex autoimmune disease characterized by the production of autoantibodies to a broad range of self-antigens. Profiling the autoantibody repertoire using array-based technology has emerged as a powerful tool for the identification of biomarkers in SLE and other autoimmune diseases. Proteomic microarray has the capacity to hold large number of self-antigens on a solid surface and serve as a high-throughput screening method for the determination of autoantibody specificities. The autoantigen arrays carrying a wide variety of self-antigens, such as cell nuclear components （nucleic acids and associated proteins）, cytoplas- mic proteins, phospholipid proteins, cell matrix proteins, mucosal/secreted proteins, glomeruli, and other tissue-specific proteins, have been used for screening of autoantibody specificities associated with different manifestations of SLE. Arrays containing synthetic peptides and molecular modified proteins are also being utilized for identification of autoantibodies targeting to special antigenic epi- topes. Different isotypes of autoantibodies, including IgG, IgM, IgA, and IgE, as well as other Ig subtypes, can be detected simultaneously with multi-color labeled secondary antibodies. Serum and plasma are the most common biologic materials for autoantibody detection, but other body fluids such as cerebrospinal fluid, synovial fluid, and saliva can also be a source of autoantibody detection.展开更多
Influenza remains a world wide health threat, thus the need for a high-throughput and robust assay to quantify both seasonal and avian in-fluenza A strains. Therefore, a 384-well plate format was developed for the med...Influenza remains a world wide health threat, thus the need for a high-throughput and robust assay to quantify both seasonal and avian in-fluenza A strains. Therefore, a 384-well plate format was developed for the median tissue culture infectious dose assay (TCID50) utilizing the detection of nucleoprotein by an in situ en-zyme linked immunosorbent assay (ELISA) which was optimized for sensitivity in this assay. Highly pathogenic avian influenza, A/Vietnam/ 1203/04 (H5N1), and interpandemic strains, A/ New Caledonia/20/99 (H1N1) and A/Brisbane/ 10/07 (H3N2), were quantified using this high- throughput assay. Each 384-well plate can be used to analyze ten viral samples in quadrupli-cate, eight dilutions per sample, including all necessary assay controls. The results obtained from 384-well plates were comparable to tradi-tional 96-well plates and also demonstrate re-peatability, intermediate precision, and assay linearity. Further, the use of 384-well plates in-creased the throughput of sample analysis and the precision and accuracy of the resulting titer.展开更多
基金supported by the National Natural Science Foundation of China (Grant No. 30670497)the Beijing Natural Science Foundation (Grant No. 5072002)
文摘Both HIV-1 integrase (IN) and the central catalytic domain of IN (IN-CCD) catalyze the disintegration reaction in vitro.In this study,IN and IN-CCD proteins were expressed and purified,and a high-throughput format enzyme-linked immunosorbent assay (ELISA) was developed for the disintegration reaction.IN exhibited a marked preference for Mn2+ over Mg2+ as the divalent cation cofactor in disintegration.Baicalein,a known IN inhibitor,was found to be an IN-CCD inhibitor.The assay is sensitive and specific for the study of disintegration reaction as well as for the in vitro identification of antiviral drugs targeting IN,especially targeting IN-CCD.
基金supported by the National Natural Science Foundation of China(Grant No.81270852)
文摘Systemic lupus erythematosus （SLE） is a complex autoimmune disease characterized by the production of autoantibodies to a broad range of self-antigens. Profiling the autoantibody repertoire using array-based technology has emerged as a powerful tool for the identification of biomarkers in SLE and other autoimmune diseases. Proteomic microarray has the capacity to hold large number of self-antigens on a solid surface and serve as a high-throughput screening method for the determination of autoantibody specificities. The autoantigen arrays carrying a wide variety of self-antigens, such as cell nuclear components （nucleic acids and associated proteins）, cytoplas- mic proteins, phospholipid proteins, cell matrix proteins, mucosal/secreted proteins, glomeruli, and other tissue-specific proteins, have been used for screening of autoantibody specificities associated with different manifestations of SLE. Arrays containing synthetic peptides and molecular modified proteins are also being utilized for identification of autoantibodies targeting to special antigenic epi- topes. Different isotypes of autoantibodies, including IgG, IgM, IgA, and IgE, as well as other Ig subtypes, can be detected simultaneously with multi-color labeled secondary antibodies. Serum and plasma are the most common biologic materials for autoantibody detection, but other body fluids such as cerebrospinal fluid, synovial fluid, and saliva can also be a source of autoantibody detection.
文摘Influenza remains a world wide health threat, thus the need for a high-throughput and robust assay to quantify both seasonal and avian in-fluenza A strains. Therefore, a 384-well plate format was developed for the median tissue culture infectious dose assay (TCID50) utilizing the detection of nucleoprotein by an in situ en-zyme linked immunosorbent assay (ELISA) which was optimized for sensitivity in this assay. Highly pathogenic avian influenza, A/Vietnam/ 1203/04 (H5N1), and interpandemic strains, A/ New Caledonia/20/99 (H1N1) and A/Brisbane/ 10/07 (H3N2), were quantified using this high- throughput assay. Each 384-well plate can be used to analyze ten viral samples in quadrupli-cate, eight dilutions per sample, including all necessary assay controls. The results obtained from 384-well plates were comparable to tradi-tional 96-well plates and also demonstrate re-peatability, intermediate precision, and assay linearity. Further, the use of 384-well plates in-creased the throughput of sample analysis and the precision and accuracy of the resulting titer.
