Pub Date : 2020-02-02DOI: 10.1016/B978-0-12-374984-0.01314-0
B. Mittal, P. Chaturvedi, Sonam Tulsyan
{"title":"Restriction fragment length polymorphism.","authors":"B. Mittal, P. Chaturvedi, Sonam Tulsyan","doi":"10.1016/B978-0-12-374984-0.01314-0","DOIUrl":"https://doi.org/10.1016/B978-0-12-374984-0.01314-0","url":null,"abstract":"","PeriodicalId":77124,"journal":{"name":"Experimental and clinical immunogenetics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-02-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/B978-0-12-374984-0.01314-0","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46910405","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A. Császár, J. Duba, B. Melegh, J. Kramer, C. Szalai, Z. Prohászka, I. Karádi, M. Kovács, K. Méhes, L. Romics, G. Füst
The aim of the present study was to compare the frequencies of the F allele of C3 complement component and the Leiden mutation of coagulation factor V in patients with severe coronary heart disease (CHD) who survived myocardial infarction (MI; group A), and those who had no MI in their case history (group B). We have determined the C3 allele frequencies by electrophoresis, and Leiden mutation by PCR in 338 patients with severe CHD and in 490 and 523 healthy controls, respectively. The C3*F allele frequency was significantly (p = 0.006) higher in group A (0.213) that in group B (0.132). A significant (p = 0.045) difference was found between ≤60-year group A (0.077) and group B (0.029) patients in the frequency of Leiden mutation. These findings indicate that the C3*F allele and the Leiden mutation may be associated with an increased risk of developing myocardial infarction in CHD patients.
{"title":"Increased Frequency of the C3*F Allele and the Leiden Mutation of Coagulation Factor V in Patients with Severe Coronary Heart Disease Who Survived Myocardial Infarction","authors":"A. Császár, J. Duba, B. Melegh, J. Kramer, C. Szalai, Z. Prohászka, I. Karádi, M. Kovács, K. Méhes, L. Romics, G. Füst","doi":"10.1159/000049199","DOIUrl":"https://doi.org/10.1159/000049199","url":null,"abstract":"The aim of the present study was to compare the frequencies of the F allele of C3 complement component and the Leiden mutation of coagulation factor V in patients with severe coronary heart disease (CHD) who survived myocardial infarction (MI; group A), and those who had no MI in their case history (group B). We have determined the C3 allele frequencies by electrophoresis, and Leiden mutation by PCR in 338 patients with severe CHD and in 490 and 523 healthy controls, respectively. The C3*F allele frequency was significantly (p = 0.006) higher in group A (0.213) that in group B (0.132). A significant (p = 0.045) difference was found between ≤60-year group A (0.077) and group B (0.029) patients in the frequency of Leiden mutation. These findings indicate that the C3*F allele and the Leiden mutation may be associated with an increased risk of developing myocardial infarction in CHD patients.","PeriodicalId":77124,"journal":{"name":"Experimental and clinical immunogenetics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000049199","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64622153","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
K. W. Eriksen, M. Nielsen, K. Kaltoft, A. Svejgaard, M. H. Nissen, C. Röpke, N. Ødum
Signal transducer and activator of transcription 6 (STAT6) is essential for the biological activities of interleukin-4 (IL-4) and the development of allergic responses in mice. Here we report on a sensitive and specific assay for STAT6 activation in response to IL-4. We took advantage of double-stranded oligonucleotide probes containing a STAT6-binding gene-sequence from the promotor of the immunoglobulin heavy chain germline Ε transcript to study the IL-4-induced DNA binding of STAT6. Using these probes, we show that repeated adjacent STAT6-binding sites result in enhanced STAT6-DNA binding. Moreover, the distance between the binding sites is critical for STAT-DNA binding, i.e. STAT6 binding is decreased at distances above 20 nucleotides between neighbouring binding sites. Using this assay to study cross-talk between IL-4 and chemokines, we provide evidence that MIP-1β and MIG inhibit IL-4-induced STAT6 activation, whereas other chemokines and cytokines do not. In conclusion, our data show that oligonucleotide fishing is a supplementary tool for studying cytokine cross-talk at a genomic level.
{"title":"Oligonucleotide Fishing for STAT6: Cross-Talk between IL-4 and Chemokines","authors":"K. W. Eriksen, M. Nielsen, K. Kaltoft, A. Svejgaard, M. H. Nissen, C. Röpke, N. Ødum","doi":"10.1159/000049202","DOIUrl":"https://doi.org/10.1159/000049202","url":null,"abstract":"Signal transducer and activator of transcription 6 (STAT6) is essential for the biological activities of interleukin-4 (IL-4) and the development of allergic responses in mice. Here we report on a sensitive and specific assay for STAT6 activation in response to IL-4. We took advantage of double-stranded oligonucleotide probes containing a STAT6-binding gene-sequence from the promotor of the immunoglobulin heavy chain germline Ε transcript to study the IL-4-induced DNA binding of STAT6. Using these probes, we show that repeated adjacent STAT6-binding sites result in enhanced STAT6-DNA binding. Moreover, the distance between the binding sites is critical for STAT-DNA binding, i.e. STAT6 binding is decreased at distances above 20 nucleotides between neighbouring binding sites. Using this assay to study cross-talk between IL-4 and chemokines, we provide evidence that MIP-1β and MIG inhibit IL-4-induced STAT6 activation, whereas other chemokines and cytokines do not. In conclusion, our data show that oligonucleotide fishing is a supplementary tool for studying cytokine cross-talk at a genomic level.","PeriodicalId":77124,"journal":{"name":"Experimental and clinical immunogenetics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000049202","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64622574","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Objective: The balance between mucosal CD4+ T cells producing IFN-γ or IL-10 is essential in the maintenance of intestinal homeostasis. We aimed to investigate how in situ activated T cells were expanded in vitro according to a new cell culture protocol and if it selected for continuous clonal CD4+ T cells capable of producing mainly IFN-γ or IL-10. Methods: T cell cultures were established from colonic biopsy specimens of 27 patients with Crohn’s disease and from 10 healthy controls in a medium supplemented with IL-2 and IL-4 but without addition of exogenous antigen or mitogen. Cytokine production was measured after stimulation (IL-12, super antigen) and inhibition (ciclosporin and methylprednisolone). Results: Cytokine production was increased in cultures from patients with Crohn’s disease compared to controls (IFN-γ, p = 0.005; IL-10, p = 0.003; TNF-α, p = 0.03). Early cultures were highly responsive to IL-12 stimulation. We established CD4+ T-cell clones escaping cellular senescence with an inflammatory or regulatory cytokine profile. Discussion: The data indicate that cultures of in situ activated inflammatory and regulatory subpopulations of intestinal T lymphocytes with pathogenic importance in Crohn’s disease can be established preserving their functional properties during growth.
目的:粘膜CD4+ T细胞产生IFN-γ或IL-10之间的平衡在维持肠道稳态中至关重要。我们的目的是研究原位活化T细胞是如何根据新的细胞培养方案在体外扩增的，以及它是否被选择为能够主要产生IFN-γ或IL-10的连续克隆CD4+ T细胞。方法:选取27例克罗恩病患者和10例健康对照者的结肠活检标本，在添加IL-2和IL-4但不添加外源抗原或丝裂原的培养基中培养T细胞。在刺激(IL-12，超抗原)和抑制(环孢素和甲基强的松龙)后测量细胞因子的产生。结果:与对照组相比，克罗恩病患者培养物中细胞因子的产生增加(IFN-γ， p = 0.005;IL-10, p = 0.003;TNF-α， p = 0.03)。早期培养物对IL-12刺激反应强烈。我们建立了CD4+ t细胞克隆逃避细胞衰老与炎症或调节细胞因子谱。讨论:这些数据表明，在克罗恩病中具有致病性重要性的肠道T淋巴细胞原位激活炎症和调节亚群的培养可以在生长过程中保持其功能特性。
{"title":"In situ Activated Intestinal T Cells Expanded in vitro – without Addition of Antigen – Produce IFN-γ and IL-10 and Preserve Their Function during Growth","authors":"J. Agnholt, K. Kaltoft","doi":"10.1159/000049200","DOIUrl":"https://doi.org/10.1159/000049200","url":null,"abstract":"Objective: The balance between mucosal CD4+ T cells producing IFN-γ or IL-10 is essential in the maintenance of intestinal homeostasis. We aimed to investigate how in situ activated T cells were expanded in vitro according to a new cell culture protocol and if it selected for continuous clonal CD4+ T cells capable of producing mainly IFN-γ or IL-10. Methods: T cell cultures were established from colonic biopsy specimens of 27 patients with Crohn’s disease and from 10 healthy controls in a medium supplemented with IL-2 and IL-4 but without addition of exogenous antigen or mitogen. Cytokine production was measured after stimulation (IL-12, super antigen) and inhibition (ciclosporin and methylprednisolone). Results: Cytokine production was increased in cultures from patients with Crohn’s disease compared to controls (IFN-γ, p = 0.005; IL-10, p = 0.003; TNF-α, p = 0.03). Early cultures were highly responsive to IL-12 stimulation. We established CD4+ T-cell clones escaping cellular senescence with an inflammatory or regulatory cytokine profile. Discussion: The data indicate that cultures of in situ activated inflammatory and regulatory subpopulations of intestinal T lymphocytes with pathogenic importance in Crohn’s disease can be established preserving their functional properties during growth.","PeriodicalId":77124,"journal":{"name":"Experimental and clinical immunogenetics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000049200","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64622201","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Author Index Vol. 18, 2001","authors":"","doi":"10.1159/000049205","DOIUrl":"https://doi.org/10.1159/000049205","url":null,"abstract":"","PeriodicalId":77124,"journal":{"name":"Experimental and clinical immunogenetics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000049205","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64622280","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
P. Ramsland, A. Kaushik, J. Marchalonis, A. Edmundson
Available data suggest that ‘primitive’ antibody-combining sites often include longer than average HCDR3s. Long HCDR3 sequences have been reported in diverse vertebrates, including humans, cattle, camels and sharks. These long HCDR3 segments contain unusual sequence features such as stretches of Gly or Pro residues and multiple Cys residues. We examined how longer than average HCDR3s were accommodated in the V domains of human, murine and camel antibodies with known three-dimensional structures. The main conclusions were that (1) HCDR3s longer than 12 residues should protrude outward from the V domains; (2) descending HCDR3 polypeptides may utilize VL (including LCDR3) constituents as a platform, supporting the protruding segments; (3) intra- and inter-HCDR disulfides are frequently formed to rigidify the structure of HCDR3 or the combining site, and (4) V and C domains were possibly more similar in primordial antibodies than they are in their present day counterparts.
{"title":"Incorporation of Long CDR3s into V Domains: Implications for the Structural Evolution of the Antibody-Combining Site","authors":"P. Ramsland, A. Kaushik, J. Marchalonis, A. Edmundson","doi":"10.1159/000049197","DOIUrl":"https://doi.org/10.1159/000049197","url":null,"abstract":"Available data suggest that ‘primitive’ antibody-combining sites often include longer than average HCDR3s. Long HCDR3 sequences have been reported in diverse vertebrates, including humans, cattle, camels and sharks. These long HCDR3 segments contain unusual sequence features such as stretches of Gly or Pro residues and multiple Cys residues. We examined how longer than average HCDR3s were accommodated in the V domains of human, murine and camel antibodies with known three-dimensional structures. The main conclusions were that (1) HCDR3s longer than 12 residues should protrude outward from the V domains; (2) descending HCDR3 polypeptides may utilize VL (including LCDR3) constituents as a platform, supporting the protruding segments; (3) intra- and inter-HCDR disulfides are frequently formed to rigidify the structure of HCDR3 or the combining site, and (4) V and C domains were possibly more similar in primordial antibodies than they are in their present day counterparts.","PeriodicalId":77124,"journal":{"name":"Experimental and clinical immunogenetics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000049197","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64621973","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
‘Nomenclature of the Human Immunoglobulin Lambda (IGL) Genes’, the 18th report of the ‘IMGT Locus in Focus’ section, provides the first complete list of all the human IGL genes. The total number of human IGL genes per haploid genome is 87–96 (93–102 if the orphons are included), of which 37–43 genes are functional. IMGT/Human Genome Organization (HUGO) gene names and definitions of the human IGL genes on chromosome 22q11.2 and IGL orphons on chromosomes 8 and 22 are provided with the gene functionality and the number of alleles, according to the rules of the IMGT Scientific chart, with the accession numbers of the IMGT reference sequences and with the accession ID of the Genome Database GDB and NCBI LocusLink databases, in which all the IMGT human IGL genes have been entered. The tables are available at the IMGT Marie-Paule page of IMGT, the international ImMunoGeneTics database (http://imgt.cines.fr) created by Marie-Paule Lefranc, Université Montpellier II, CNRS, France.
{"title":"Nomenclature of the Human Immunoglobulin Lambda (IGL) Genes","authors":"M. Lefranc","doi":"10.1159/000049203","DOIUrl":"https://doi.org/10.1159/000049203","url":null,"abstract":"‘Nomenclature of the Human Immunoglobulin Lambda (IGL) Genes’, the 18th report of the ‘IMGT Locus in Focus’ section, provides the first complete list of all the human IGL genes. The total number of human IGL genes per haploid genome is 87–96 (93–102 if the orphons are included), of which 37–43 genes are functional. IMGT/Human Genome Organization (HUGO) gene names and definitions of the human IGL genes on chromosome 22q11.2 and IGL orphons on chromosomes 8 and 22 are provided with the gene functionality and the number of alleles, according to the rules of the IMGT Scientific chart, with the accession numbers of the IMGT reference sequences and with the accession ID of the Genome Database GDB and NCBI LocusLink databases, in which all the IMGT human IGL genes have been entered. The tables are available at the IMGT Marie-Paule page of IMGT, the international ImMunoGeneTics database (http://imgt.cines.fr) created by Marie-Paule Lefranc, Université Montpellier II, CNRS, France.","PeriodicalId":77124,"journal":{"name":"Experimental and clinical immunogenetics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000049203","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64622621","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Janitz, L. Reiners-Schramm, A. Muhlethaler‐Mottet, Mark Rosowski, R. Lauster
The class II transactivator is a major transcriptional factor acting on the promoters of MHC class II genes. Transcription of the CIITA gene is driven by four alternative promoters, which exhibit cell-type-specific activity. The CIITA promoter III (PIII) is constitutively active in B cells, whereas promoter IV (PIV) becomes activated upon interferon-γ activation. The aim of this study was to investigate whether these two promoters exhibit a sequence variability like the MHC class II promoters do. We isolated PIII and PIV fragments from healthy individuals and rheumatoid arthritis patients and screened them for sequence polymorphisms. Single base pair substitutions within the CIITA PIV were found in 9% of the individuals analyzed. The majority of the substitutions were located upstream of the known cis-acting elements of the promoter. PIII was non-polymorphic. To evaluate the functional relevance of the detected polymorphism we cloned variable PIV upstream of the luciferase reporter gene. Such prepared constructs were transfected into monocytes, melanoma and HeLa cells, which were subsequently stimulated with interferon-γ. The analysis of promoter activities did not reveal significant differences in all three cell types. We conclude that the level of CIITA expression does not vary within the population. Thus the differences in the level of MHC class II expression, which are observed between individuals, stem for the polymorphisms of the MHC class II promoters themselves.
{"title":"Analysis of the Sequence Polymorphism within Class II Transactivator Gene Promoters","authors":"M. Janitz, L. Reiners-Schramm, A. Muhlethaler‐Mottet, Mark Rosowski, R. Lauster","doi":"10.1159/000049198","DOIUrl":"https://doi.org/10.1159/000049198","url":null,"abstract":"The class II transactivator is a major transcriptional factor acting on the promoters of MHC class II genes. Transcription of the CIITA gene is driven by four alternative promoters, which exhibit cell-type-specific activity. The CIITA promoter III (PIII) is constitutively active in B cells, whereas promoter IV (PIV) becomes activated upon interferon-γ activation. The aim of this study was to investigate whether these two promoters exhibit a sequence variability like the MHC class II promoters do. We isolated PIII and PIV fragments from healthy individuals and rheumatoid arthritis patients and screened them for sequence polymorphisms. Single base pair substitutions within the CIITA PIV were found in 9% of the individuals analyzed. The majority of the substitutions were located upstream of the known cis-acting elements of the promoter. PIII was non-polymorphic. To evaluate the functional relevance of the detected polymorphism we cloned variable PIV upstream of the luciferase reporter gene. Such prepared constructs were transfected into monocytes, melanoma and HeLa cells, which were subsequently stimulated with interferon-γ. The analysis of promoter activities did not reveal significant differences in all three cell types. We conclude that the level of CIITA expression does not vary within the population. Thus the differences in the level of MHC class II expression, which are observed between individuals, stem for the polymorphisms of the MHC class II promoters themselves.","PeriodicalId":77124,"journal":{"name":"Experimental and clinical immunogenetics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000049198","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64622049","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
K. Bauer, M. Janitz, L. Reiners-Schramm, A. Mühlethaler-Mottet, Mark Rosowski, R. Lauster, J. Agnholt, K. Kaltoft, K. W. Eriksen, M. Nielsen, K. Kaltoft, A. Svejgaard, M. Nissen, C. Röpke, N. Ødum, Christèle Martinez-Jean, G. Folch, M. Lefranc, P. Ramsland, A. Kaushik, J. Marchalonis, A. Edmundson, A. Császár, J. Duba, B. Melegh, J. Kramer, C. Szalai, Z. Prohászka, I. Karádi, M. Kovács, K. Méhes, L. Romics, G. Füst, H. Takeshita, T. Yasuda, E. Nakazato, T. Nakajima, Shinjiro Mori, K. Mogi, Y. Kaneko, R. Iida, K. Kishi
{"title":"Contents Vol. 18, 2001","authors":"K. Bauer, M. Janitz, L. Reiners-Schramm, A. Mühlethaler-Mottet, Mark Rosowski, R. Lauster, J. Agnholt, K. Kaltoft, K. W. Eriksen, M. Nielsen, K. Kaltoft, A. Svejgaard, M. Nissen, C. Röpke, N. Ødum, Christèle Martinez-Jean, G. Folch, M. Lefranc, P. Ramsland, A. Kaushik, J. Marchalonis, A. Edmundson, A. Császár, J. Duba, B. Melegh, J. Kramer, C. Szalai, Z. Prohászka, I. Karádi, M. Kovács, K. Méhes, L. Romics, G. Füst, H. Takeshita, T. Yasuda, E. Nakazato, T. Nakajima, Shinjiro Mori, K. Mogi, Y. Kaneko, R. Iida, K. Kishi","doi":"10.1159/000049207","DOIUrl":"https://doi.org/10.1159/000049207","url":null,"abstract":"","PeriodicalId":77124,"journal":{"name":"Experimental and clinical immunogenetics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000049207","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64622508","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
H. Takeshita, T. Yasuda, E. Nakazato, T. Nakajima, S. Mori, K. Mogi, Y. Kaneko, R. Iida, K. Kishi
To obtain human deoxyribonuclease I (DNase I) as an immunogen, we have developed a procedure that is more useful and effective than the conventional procedure, which uses human urine as a starting material. In the new procedure, we culture COS-7 cells transfected with expression vector carrying human DNase I cDNA, and then purify the enzyme from the culture medium. The enzyme can be easily isolated to apparent homogeneity by passage through only three chromatography columns. The rabbit antiserum that we used against the recombinant DNase I was not inferior to that used against DNase I from human urine, in terms of both its ability to discriminate DNase I phenotypes and its ability to neutralize enzyme activity. Therefore, our procedure may be useful for producing an antibody specific for human DNase I.
{"title":"Use of Human Recombinant DNase I Expressed in COS-7 Cells as an Immunogen to Produce a Specific Anti-DNase I Antibody","authors":"H. Takeshita, T. Yasuda, E. Nakazato, T. Nakajima, S. Mori, K. Mogi, Y. Kaneko, R. Iida, K. Kishi","doi":"10.1159/000049201","DOIUrl":"https://doi.org/10.1159/000049201","url":null,"abstract":"To obtain human deoxyribonuclease I (DNase I) as an immunogen, we have developed a procedure that is more useful and effective than the conventional procedure, which uses human urine as a starting material. In the new procedure, we culture COS-7 cells transfected with expression vector carrying human DNase I cDNA, and then purify the enzyme from the culture medium. The enzyme can be easily isolated to apparent homogeneity by passage through only three chromatography columns. The rabbit antiserum that we used against the recombinant DNase I was not inferior to that used against DNase I from human urine, in terms of both its ability to discriminate DNase I phenotypes and its ability to neutralize enzyme activity. Therefore, our procedure may be useful for producing an antibody specific for human DNase I.","PeriodicalId":77124,"journal":{"name":"Experimental and clinical immunogenetics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000049201","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64621830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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