10 results on '"Kozue Hagiwara"'
Search Results
2. LM-GlycomeAtlas Ver. 1.0: A Novel Visualization Tool for Lectin Microarray-Based Glycomic Profiles of Mouse Tissue Sections
- Author
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Kozue Hagiwara, Yoshinori Suzuki, Chiaki Nagai-Okatani, Atsushi Kuno, Misugi Nagai, Kiyoko F. Aoki-Kinoshita, Kiyohiko Angata, Noriaki Fujita, Shuichi Kakuda, Katsue Kiyohara, and Takashi Sato
- Subjects
Male ,Protein glycosylation ,formalin-fixed paraffin-embedded tissue section ,Glycan ,Glycosylation ,Microarray ,Protein Array Analysis ,Pharmaceutical Science ,Mouse tissue ,Computational biology ,Biology ,Article ,Analytical Chemistry ,lcsh:QD241-441 ,Mice ,User-Computer Interface ,03 medical and health sciences ,0302 clinical medicine ,lectin microarray ,lcsh:Organic chemistry ,Lectins ,Drug Discovery ,Animals ,Physical and Theoretical Chemistry ,Glycomics ,030304 developmental biology ,Laser capture microdissection ,0303 health sciences ,Organic Chemistry ,Lectin ,mouse tissue glycome mapping ,Glycome ,Visualization ,Organ Specificity ,Chemistry (miscellaneous) ,030220 oncology & carcinogenesis ,biology.protein ,Molecular Medicine ,laser microdissection ,Microdissection ,Software - Abstract
For the effective discovery of the biological roles and disease-specific alterations concerning protein glycosylation in tissue samples, it is important to know beforehand the quantitative and qualitative variations of glycan structures expressed in various types of cells, sites, and tissues. To this end, we used laser microdissection-assisted lectin microarray (LMA) to establish a simple and reproducible method for high-throughput and in-depth glycomic profiling of formalin-fixed paraffin-embedded tissue sections. Using this &ldquo, tissue glycome mapping&rdquo, approach, we present 234 glycomic profiling data obtained from nine tissue sections (pancreas, heart, lung, thymus, gallbladder, stomach, small intestine, colon, and skin) of two 8-week-old male C57BL/6J mice. We provided this LMA-based dataset in the similar interface as that of GlycomeAtlas, a previously developed tool for mass spectrometry-based tissue glycomic profiling, allowing easy comparison of the two types of data. This online tool, called &ldquo, LM-GlycomeAtlas&rdquo, allows users to visualize the LMA-based tissue glycomic profiling data associated with the sample information as an atlas. Since the present dataset allows the comparison of glycomic profiles, it will facilitate the evaluation of site- and tissue-specific glycosylation patterns. Taking advantage of its extensibility, this tool will continue to be updated with the expansion of deposited data.
- Published
- 2019
3. Incomplete clearance of apoptotic cells by core 1-derived O-glycan-deficient resident peritoneal macrophages
- Author
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Satoru Takahashi, Takashi Kudo, Hiromasa Wakui, Riku Suzuki, Sayaka Fuseya, Risa Okada, Kozue Hagiwara, Hisashi Narimatsu, Takashi Sato, Miki Shimbo, Mitchito Hamada, and Akihiro Kuno
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0301 basic medicine ,Male ,Transgene ,Biophysics ,Apoptosis ,Mice, Transgenic ,Biochemistry ,03 medical and health sciences ,Mice ,Animals ,Receptor ,Efferocytosis ,Molecular Biology ,ITGAV ,Cells, Cultured ,Chemistry ,Macrophages ,Cell Biology ,MERTK ,Fusion protein ,Cell biology ,Mice, Inbred C57BL ,030104 developmental biology ,Cytophagocytosis ,Knockout mouse ,Female ,Peritoneum ,Molecular Chaperones - Abstract
The core 1 β1,3-galactosyltransferase-specific molecular chaperon (Cosmc) is essential for the synthesis of the core 1 structure of mucin-type O-glycans. To clarify the physiological role of core 1-derived O-glycans in macrophages, we exploited the LysM-Cre transgene to generate a conditional Cosmc mutant allele (conditional Cosmc knockout; cKO) in myeloid cells. cKO mice developed normally with no gross phenotypic abnormalities or abnormal peripheral blood counts. Resident peritoneal macrophages (rpMacs) of cKO mice exhibited impaired engulfment of apoptotic cells but showed normal macrophage differentiation and counts. T-cell immunoglobulin and mucin domain-containing molecule 4 (Tim4) is a phosphatidylserine (PS) receptor expressed on rpMacs and possesses a heavily O-glycosylated domain. Tim4 tethers apoptotic cells through PS binding. Expression of the Tim4 transcript was unchanged in cKO rpMacs, whereas flow cytometric, Western and dot blot analyses revealed that Tim4 protein expression in cKO rpMacs was significantly lower than that in wild-type (WT) rpMacs. Moreover, the expression levels of other efferocytosis-related molecules, Mertk, Itgav and Itgb3, were normal in rpMacs. In addition, hypoglycosylated Tim4-FLAG fusion protein sufficiently recognized PS. These results demonstrated that core 1-derived O-glycan is required for Tim4-dependent normal efferocytosis and may contribute to the stable expression of the Tim4 glycoprotein.
- Published
- 2017
4. A standardized method for lectin microarray-based tissue glycome mapping
- Author
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Atsushi Kuno, Hisashi Narimatsu, Yoko Itakura, Kozue Hagiwara, Xia Zou, Binbin Tan, Atsushi Matsuda, Jun Iwaki, Yan Zhang, Chiaki Nagai-Okatani, Masashi Toyoda, Hiroyuki Kaji, Takashi Sato, Maki Yoshida, and Erika Noro
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Male ,0301 basic medicine ,Glycan ,Proteome ,Protein Array Analysis ,Computational biology ,Kidney ,Article ,Glycomics ,Mice ,03 medical and health sciences ,Lectins ,Testis ,Animals ,Biomarker discovery ,Glycoproteins ,Laser capture microdissection ,Multidisciplinary ,biology ,Microarray analysis techniques ,Kidney metabolism ,Glycome ,Molecular biology ,030104 developmental biology ,Organ Specificity ,biology.protein ,Biomarkers - Abstract
The significance of glycomic profiling has been highlighted by recent findings that structural changes of glycans are observed in many diseases, including cancer. Therefore, glycomic profiling of the whole body (glycome mapping) under different physiopathological states may contribute to the discovery of reliable biomarkers with disease-specific alterations. To achieve this, standardization of high-throughput and in-depth analysis of tissue glycome mapping is needed. However, this is a great challenge due to the lack of analytical methodology for glycans on small amounts of endogenous glycoproteins. Here, we established a standardized method of lectin-assisted tissue glycome mapping. Formalin-fixed, paraffin-embedded tissue sections were prepared from brain, liver, kidney, spleen, and testis of two C57BL/6J mice. In total, 190 size-adjusted fragments with different morphology were serially collected from each tissue by laser microdissection and subjected to lectin microarray analysis. The results and subsequent histochemical analysis with selected lectins were highly consistent with previous reports of mass spectrometry-based N- and/or O-glycome analyses and histochemistry. This is the first report to look at both N- and O-glycome profiles of various regions within tissue sections of five different organs. This simple and reproducible mapping approach is also applicable to various disease model mice to facilitate disease-related biomarker discovery.
- Published
- 2017
5. C1galt1-deficient mice exhibit thrombocytopenia due to abnormal terminal differentiation of megakaryocytes
- Author
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Nobuhiro Ohkohchi, Yuzuru Ikehara, Michito Hamada, Hisashi Narimatsu, Takashi Sato, Soichiro Murata, Takashi Kudo, Satoru Takahashi, Takashi Yamaguchi, Ken Matsumoto, Kozue Hagiwara, Masatsugu Ema, and Yukinori Kozuma
- Subjects
Male ,Pathology ,medicine.medical_specialty ,Megakaryocyte differentiation ,Transgene ,Immunology ,Mice, Transgenic ,Biology ,Biochemistry ,Thrombopoiesis ,Mice ,Western blot ,medicine ,Animals ,Platelet ,Cells, Cultured ,Megakaryocyte Progenitor Cells ,Messenger RNA ,medicine.diagnostic_test ,Gene Transfer Techniques ,Cell Differentiation ,Cell Biology ,Hematology ,Galactosyltransferases ,Thrombocytopenia ,Molecular biology ,Mice, Inbred C57BL ,Haematopoiesis ,Giant platelets ,medicine.anatomical_structure ,Female ,Bone marrow ,Megakaryocytes - Abstract
C1galt1 is essential for synthesis of the core 1 structure of mucin-type O-glycans. To clarify the physiological role of O-glycans in adult hematopoiesis, we exploited the interferon-inducible Mx1-Cre transgene to conditionally ablate the C1galt(flox) allele (Mx1-C1). Mx1-C1 mice exhibit severe thrombocytopenia, giant platelets, and prolonged bleeding times. Both the number and DNA ploidy of megakaryocytes in Mx1-C1 bone marrow were similar to those in wild-type (WT) mice. However, there were few proplatelets in Mx1-C1 primary megakaryocytes. Conversely, bone marrow transplanted from Mx1-C1 to WT and splenectomized Mx1-C1 mice gave rise to observations similar to those described above. The expression of GPIbα messenger RNA was unchanged in Mx1-C1 bone marrow, whereas flow cytometric and western blot analyses using megakaryocytes and platelets revealed that the expression of GPIbα protein was significantly reduced in Mx1-C1 mice. Moreover, circulating Mx1-C1 platelets exhibited an increase in the number of microtubule coils, despite normal levels of α- and β-tubulin. Our observations suggest that O-glycan is required for terminal megakaryocyte differentiation and platelet production and that the decrease in GPIbα in cells lacking O-glycan might be caused by increased proteolysis.
- Published
- 2013
6. Simultaneous Detection of Antibodies to Mouse Hepatitis Virus Recombinant Structural Proteins by a Microsphere-Based Multiplex Fluorescence Immunoassay
- Author
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Satoshi Kunita, Miyuki Ishida, Tomoko Ishida, Shuko Kameda, Fumihiro Sugiyama, Akira Takakura, Kazuo Goto, Kozue Hagiwara, Ken-ichi Yagami, and Kanako Kato
- Subjects
Microbiology (medical) ,viruses ,Molecular Sequence Data ,Clinical Biochemistry ,Immunology ,Antibodies, Viral ,Sensitivity and Specificity ,Veterinary Immunology ,Fluorescence ,law.invention ,Serology ,Rodent Diseases ,Mice ,Mouse hepatitis virus ,Antigen ,law ,medicine ,Animals ,Immunology and Allergy ,Multiplex ,Immunoassay ,Antiserum ,Murine hepatitis virus ,biology ,medicine.diagnostic_test ,Clinical Laboratory Techniques ,Sequence Analysis, DNA ,biology.organism_classification ,Virology ,Molecular biology ,Microspheres ,Rats ,Recombinant DNA ,biology.protein ,RNA, Viral ,Female ,Antibody ,Coronavirus Infections - Abstract
We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.
- Published
- 2011
7. Chondroitin Sulfate N-Acetylgalactosaminyltransferase 1 Is Necessary for Normal Endochondral Ossification and Aggrecan Metabolism
- Author
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Hisashi Narimatsu, Yuzuru Ikehara, Masatsugu Ema, Takashi Sato, Koji Kimata, Akira Togayachi, Satoru Takahashi, Kozue Hagiwara, Hiroyasu Ogawa, Katsue Kiyohara, Takashi Kudo, Tomoko Hirano, and Hideto Watanabe
- Subjects
Glycobiology and Extracellular Matrices ,Biochemistry ,Chondrocyte ,Extracellular matrix ,Glycosaminoglycan ,Mice ,chemistry.chemical_compound ,Chondrocytes ,Osteogenesis ,medicine ,Animals ,Chondroitin ,Aggrecans ,Growth Plate ,Chondroitin sulfate ,Molecular Biology ,Endochondral ossification ,Aggrecan ,Mice, Knockout ,Extracellular Matrix Proteins ,Cartilage ,Chondroitin Sulfates ,Cell Biology ,Molecular biology ,medicine.anatomical_structure ,chemistry ,N-Acetylgalactosaminyltransferases ,Proteoglycans - Abstract
Chondroitin sulfate (CS) is a glycosaminoglycan, consisting of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues, and plays important roles in development and homeostasis of organs and tissues. Here, we generated and analyzed mice lacking chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNAcT-1). Csgalnact1(-/-) mice were viable and fertile but exhibited slight dwarfism. Biochemically, the level of CS in Csgalnact1(-/-) cartilage was reduced to ∼50% that of wild-type cartilage, whereas its chain length was similar to wild-type mice, indicating that CSGalNAcT-1 participates in the CS chain initiation as suggested in the previous study (Sakai, K., Kimata, K., Sato, T., Gotoh, M., Narimatsu, H., Shinomiya, K., and Watanabe, H. (2007) J. Biol. Chem. 282, 4152-4161). Histologically, the growth plate of Csgalnact1(-/-) mice contained shorter and slightly disorganized chondrocyte columns with a reduced volume of the extracellular matrix principally in the proliferative layer. Immunohistochemical analysis revealed that the level of both aggrecan and link protein 1 were decreased in Csgalnact1(-/-) cartilage. Western blot analysis demonstrated an increase in processed forms of aggrecan core protein. These results suggest that CSGalNAcT-1 is required for normal levels of CS biosynthesis in cartilage. Our observations suggest that CSGalNAcT-1 is necessary for normal levels of endochondral ossification, and the decrease in CS amount in the growth plate by its absence causes a rapid catabolism of aggrecan.
- Published
- 2011
8. Postnatal lethality and chondrodysplasia in mice lacking both chondroitin sulfate N-acetylgalactosaminyltransferase-1 and -2
- Author
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Miki Shimbo, Hiromasa Wakui, Riku Suzuki, Katsue Kiyohara, Hisashi Narimatsu, Hideto Watanabe, Risa Okada, Yuki Tsunakawa, Satoru Takahashi, Takashi Sato, Koji Kimata, Takashi Kudo, Kozue Hagiwara, and Sayaka Fuseya
- Subjects
0301 basic medicine ,Embryology ,Physiology ,lcsh:Medicine ,Apoptosis ,Ossification ,Biochemistry ,Extracellular matrix ,Mice ,chemistry.chemical_compound ,Animal Cells ,Medicine and Health Sciences ,lcsh:Science ,Cells, Cultured ,Connective Tissue Cells ,Mice, Knockout ,Staining ,Multidisciplinary ,biology ,Sulfates ,Enzymes ,Cell biology ,Chemistry ,medicine.anatomical_structure ,Connective Tissue ,Physical Sciences ,N-Acetylgalactosaminyltransferases ,Bone Remodeling ,Anatomy ,Cellular Types ,medicine.symptom ,Chondroitin ,Research Article ,Osteochondrodysplasias ,Real-Time Polymerase Chain Reaction ,Research and Analysis Methods ,Chondrocyte ,03 medical and health sciences ,Chondrocytes ,Transferases ,medicine ,Animals ,RNA, Messenger ,Chondroitin sulfate ,Cytoplasmic Staining ,Endochondral ossification ,Aggrecan ,Cell Proliferation ,Cartilage ,Embryos ,lcsh:R ,Chemical Compounds ,Biology and Life Sciences ,Glycosyltransferases ,Proteins ,Cell Biology ,Biological Tissue ,030104 developmental biology ,chemistry ,Proteoglycan ,Specimen Preparation and Treatment ,Enzymology ,biology.protein ,Genes, Lethal ,Safranin Staining ,Salts ,lcsh:Q ,Physiological Processes ,Developmental Biology - Abstract
Chondroitin sulfate (CS) is a sulfated glycosaminoglycan (GAG) chain. In cartilage, CS plays important roles as the main component of the extracellular matrix (ECM), existing as side chains of the major cartilage proteoglycan, aggrecan. Six glycosyltransferases are known to coordinately synthesize the backbone structure of CS; however, their in vivo synthetic mechanism remains unknown. Previous studies have suggested that two glycosyltransferases, Csgalnact1 (t1) and Csgalnact2 (t2), are critical for initiation of CS synthesis in vitro. Indeed, t1 single knockout mice (t1 KO) exhibit slight dwarfism and a reduction in CS content in cartilage compared with wild-type (WT) mice. To reveal the synergetic roles of t1 and t2 in CS synthesis in vivo, we generated systemic single and double knockout (DKO) mice and cartilage-specific t1 and t2 double knockout (Col2-DKO) mice. DKO mice exhibited postnatal lethality, whereas t2 KO mice showed normal size and skeletal development. Col2-DKO mice survived to adulthood and showed severe dwarfism compared with t1 KO mice. Histological analysis of epiphyseal cartilage from Col2-DKO mice revealed disrupted endochondral ossification, characterized by drastic GAG reduction in the ECM. Moreover, DKO cartilage had reduced chondrocyte proliferation and an increased number of apoptotic chondrocytes compared with WT cartilage. Conversely, primary chondrocyte cultures from Col2-DKO knee cartilage had the same proliferation rate as WT chondrocytes and low GAG expression levels, indicating that the chondrocytes themselves had an intact proliferative ability. Quantitative RT-PCR analysis of E18.5 cartilage showed that the expression levels of Col2a1 and Ptch1 transcripts tended to decrease in DKO compared with those in WT mice. The CS content in DKO cartilage was decreased compared with that in t1 KO cartilage but was not completely absent. These results suggest that aberrant ECM caused by CS reduction disrupted endochondral ossification. Overall, we propose that both t1 and t2 are necessary for CS synthesis and normal chondrocyte differentiation but are not sufficient for all CS synthesis in cartilage.
- Published
- 2017
9. Polylactosamine on glycoproteins influences basal levels of lymphocyte and macrophage activation
- Author
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Atsushi Kuno, Nami Suzuki, Akira Togayachi, Yuzuru Ikehara, Yuko Kozono, Hisashi Narimatsu, Kozue Hagiwara, Jun Hirabayashi, Hiroyasu Ishida, Yuki Tsunoda, Kouichi Tachibana, Sumie Abe, Nobuo Sato, Takashi Ohkura, and Takashi Sato
- Subjects
T-Lymphocytes ,CD14 ,Lymphocyte ,Antigens, CD19 ,Receptors, Antigen, T-Cell ,Biology ,Lymphocyte Activation ,N-Acetylglucosaminyltransferases ,Calcium in biology ,CD19 ,Mice ,Immune system ,CD28 Antigens ,Solanum lycopersicum ,Polysaccharides ,medicine ,Animals ,Macrophage ,Mice, Knockout ,chemistry.chemical_classification ,Multidisciplinary ,CD28 ,Amino Sugars ,Macrophage Activation ,Biological Sciences ,Molecular biology ,medicine.anatomical_structure ,chemistry ,biology.protein ,Plant Lectins ,Glycoprotein - Abstract
β1,3- N -acetylglucosaminyltransferase 2 (β3GnT2) is a polylactosamine synthase that synthesizes a backbone structure of carbohydrate structures onto glycoproteins. Here we generated β3GnT2-deficient (β3GnT2 −/− ) mice and showed that polylactosamine on N -glycans was markedly reduced in their immunological tissues. In WT mice, polylactosamine was present on CD28 and CD19, both known immune costimulatory molecules. However, polylactosamine levels on these molecules were reduced in β3GnT2 −/− mice. β3GnT2 −/− T cells lacking polylactosamine were more sensitive to the induction of intracellular calcium flux on stimulation with anti-CD3ε/CD28 and proliferated more strongly than T cells from WT mice. β3GnT2 −/− B cells also showed hyperproliferation on BCR stimulation. Macrophages from β3GnT2 −/− mice had higher cell surface CD14 levels and enhanced responses to endotoxin. These results indicate that polylactosamine on N -glycans is a putative immune regulatory factor presumably suppressing excessive responses during immune reactions.
- Published
- 2007
10. Development of ELISA Using Recombinant Antigens for Specific Detection of Mouse Parvovirus Infection
- Author
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Ken-ichi Yagami, Hiroyoshi Iseki, Satoshi Kunita, Tomoko Ishida, Kozue Hagiwara, Kumiko Fuke, Akira Takakura, Miyuki Chaya, Fumihiro Sugiyama, and Tatsuya Sugimoto
- Subjects
Enzyme-Linked Immunosorbent Assay ,Viral Nonstructural Proteins ,General Biochemistry, Genetics and Molecular Biology ,Virus ,law.invention ,Serology ,Parvoviridae Infections ,Parvovirus ,Rodent Diseases ,Mice ,Antigen ,law ,medicine ,Animals ,Seroconversion ,Antigens, Viral ,Antiserum ,General Veterinary ,biology ,Parvovirus infection ,General Medicine ,medicine.disease ,biology.organism_classification ,Virology ,Recombinant Proteins ,Minute Virus of Mice ,Recombinant DNA ,Capsid Proteins ,Animal Science and Zoology - Abstract
Nucleotide sequences of mouse parvovirus (MPV) isolate, named MPV/UT, and mouse minute virus (MMV) were analyzed and used for expressing recombinant proteins in E. coli. ELISA tests using recombinant major capsid protein (rVP2) and recombinant major non-structural protein (rNS1) as antigens were developed and their performance in serologic detection of rodent parvovirus infection was assessed. MPV-rVP2 and MMV-rVP2 ELISAs reacted specifically with anti-MPV and anti-MMV mouse sera, respectively. MMV-rNS1 antigen had a wide reaction range with antisera to rodent parvoviruses including MPV, MMV, Kilham rat virus (KRV) and H-1 virus. All mice oronasally infected with MPV were seropositive at 4 weeks post-infection in screening by ELISAs using MPV-rVP2 and MMV-rNS1 antigens, but were negative by conventional ELISA using whole MMV antigen. A contact transmission experiment revealed that transmission of MPV occurred up to 4 weeks post-infection, and all cage mates were seropositive in screening with MPV-rVP2 and MMV-rNS1 ELISAs. These results indicate that MPV-rVP2 and MMV-rVP2 are specific ELISA antigens which distinguish between MPV and MVM infection, while MMV-rNS1 antigen can be used in generic ELISA for a variety of rodent parvoviruses. The higher sensitivity of MPV-rVP2 ELISA than conventional ELISA for detecting seroconversion to MPV in oronasally infected mice as well as in cage mates suggests the usefulness of MPV-rVP2 ELISA in quarantine and microbiological monitoring of MPV infection in laboratory mice.
- Published
- 2006
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