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1.
《Vaccine》2019,37(41):6060-6067
BackgroundVaccination provides protection against infection by inducing VNAs mainly against RABV surface GP. The measurement of VNAs to RABV is commonly used to assess the level of immunity in humans and animals after vaccination. A VNA titer of ≥ 0.5 IU/mL of sera indicates adequate response to vaccination. Here, we report the development and validation of a RABV GP serology ELISA kit for semi-quantitative measurement of VNA titers in sera of vaccinated human subjects.MethodsUsing a recombinant RABV GP expressed in mammalian cells as the capture antigen, the ELISA method was established using HuMAb NM57 reference initially and HRIG reference subsequently. The limit of detection (LOD), linear range, reproducibility, and precision of the method were examined. Specificity and sensitivity were established to assess the diagnostic accuracy.ResultsRABV GP for ELISA plate coating and optimal dilution of human serum sample was 1 µg/mL and 1:20, respectively. Multiple assays were carried out by different technicians at different laboratories for assay standardization. Using the HRIG reference, the LOD was found to be 0.02–0.06 IU/mL and the linear range was 0.2–10.0 IU/ mL. The inter-assay CVs were in the range of 6.60–10.79%, indicating the reproducibility. None of the 12 known negative human sera, tested positive by ELISA, highlighting the specificity. A total of 415 unknown positive human sera were double-blind tested by the RFFIT and ELISA. The VNA titer cut-off value of ELISA was set at 1.5 IU/mL to ensure no false-positive. The diagnostic specificity and sensitivity were 100% and 91.1%, respectively.ConclusionsThe validation data characterize this ELISA as a suitable method for semi-quantitative measurement of VNA titers in human serum samples to assess vaccination status. The ELISA kit can offer simplicity, speed, low cost and high throughput, making it a practical tool for monitoring the immune response following vaccination. 相似文献
2.
Characterization of gp 50, a major glycoprotein present in rat brain synaptic membranes, with a monoclonal antibody 总被引:4,自引:0,他引:4
Philip W. Beesley Toni Paladino Claude Gravel Richard A. Hawkes James W. Gurd 《Brain research》1987,408(1-2):65-78
Several cell lines secreting monoclonal antibodies (Mabs) against a major forebrain synaptic membrane (SM) glycoprotein, gp 50, have been raised. Western blots show that the Mabs react with a polypeptide doublet of Mrs 49 and 45 kDa. These polypeptides exist solely in a concanavalin A (Con A) binding form. Removal of the Con A receptors by digestion with endo-beta-N-acetylglucosaminidase H (endo H) lowers the Mrs of the glycoprotein doublet to 36.5 and 34 kDa. Western blots of 2D polyacrylamide gels indicate that gp 50 exists in several isoforms. Solid phase radioimmunoassay (RIA) and Western blots of brain subcellular fractions show the antigenic material to be concentrated in the SM fraction, but to be present in much lower amounts in synaptic junctions and postsynaptic densities. Gp 50 appears to be brain specific. Regional distribution studies show that it is present in all brain regions but is two-fold concentrated in cerebellum, brainstem and midbrain compared to forebrain. Immunocytochemical studies of several brain regions show that gp 50-like immunoreactivity is neuron specific and is concentrated in selected neuronal species, particularly granule cells. In both cerebellar and hippocampal granule cells gp 50-like immunoreactivity is localized in the perikarya and primary dendrites. Though immunocytochemistry did not show staining of synaptic regions this may be due to masking of the reactive epitope. The results are discussed in terms of the molecular properties of gp 50 and its subcellular localization in brain tissue. 相似文献
3.
4.
Bath application of
-fucose and
-fucosyllactose (
F1 increases the potentiation of the population spike amplitude (POP-spike) and the field excitatory postsynaptic potential (fEPSP) after tetanization of the Schaffer collaterals of the rat hippocampus. The ineffective isomers
-fucose and 3-fucosyllactose (3-F1) have no such effect. Since not only the maintenance of long-term potentiation LTP is influenced but also its induction is drastically improved, an effect of the sugars via the formation of glycoproteins but also via different actions on induction mechanisms is discussed. 相似文献
5.
LSECtin interacts with filovirus glycoproteins and the spike protein of SARS coronavirus 总被引:9,自引:0,他引:9
Gramberg T Hofmann H Möller P Lalor PF Marzi A Geier M Krumbiegel M Winkler T Kirchhoff F Adams DH Becker S Münch J Pöhlmann S 《Virology》2005,340(2):265-236
Cellular attachment factors like the C-type lectins DC-SIGN and DC-SIGNR (collectively referred to as DC-SIGN/R) can augment viral infection and might promote viral dissemination in and between hosts. The lectin LSECtin is encoded in the same chromosomal locus as DC-SIGN/R and is coexpressed with DC-SIGNR on sinusoidal endothelial cells in liver and lymphnodes. Here, we show that LSECtin enhances infection driven by filovirus glycoproteins (GP) and the S protein of SARS coronavirus, but does not interact with human immunodeficiency virus type-1 and hepatitis C virus envelope proteins. Ligand binding to LSECtin was inhibited by EGTA but not by mannan, suggesting that LSECtin unlike DC-SIGN/R does not recognize high-mannose glycans on viral GPs. Finally, we demonstrate that LSECtin is N-linked glycosylated and that glycosylation is required for cell surface expression. In summary, we identified LSECtin as an attachment factor that in conjunction with DC-SIGNR might concentrate viral pathogens in liver and lymph nodes. 相似文献
6.
Krummenacher C Baribaud F Ponce de Leon M Baribaud I Whitbeck JC Xu R Cohen GH Eisenberg RJ 《Virology》2004,322(2):286-299
The herpesvirus entry mediator A (HVEM/HveA) and nectin-1 (HveC/CD111) are two major receptors for herpes simplex virus (HSV). Although structurally unrelated, both receptors can independently mediate entry of wild-type (wt) HSV-1 and HSV-2 by interacting with the viral envelope glycoprotein D (gD). Laboratory strains with defined mutations in gD (e.g. rid1) do not use HVEM but use nectin-2 (HveB/CD112) for entry. The relative usage of HVEM and nectin-1 during HSV infection in vivo is not known. In the absence of a defined in vivo model, we used in vitro approaches to address this question. First, we screened HSV clinical isolates from various origins for receptor tropism and found that all used both HVEM and nectin-1. Second, we determined the numbers of surface receptors on various susceptible and resistant cell lines as well as on primary fibroblasts derived from an individual with cleft lip/palate ectodermal dysplasia (CLPED1). Although CLPED1 cells can only express a defective form of nectin-1, they allowed entry of wild type and mutant HSV strains by usage of either HVEM or nectin-2. Finally, we compared the ability of HVEM and nectin-1 to mediate entry when expressed at varying cell surface densities. Both receptors showed a direct relationship between the number of receptors and HSV susceptibility. Direct comparison of receptors suggests that nectin-1 is more efficient at promoting entry than HVEM. Overall, our data suggest that both receptors play a role during HSV infection in vivo and that both are highly efficient even at low levels of expression. 相似文献
7.
Retroviruses, such as human and simian immunodeficiency viruses (HIV and SIV), and murine leukemia viruses (MuLV), harbor a tyrosine-based motif in the intracytoplasmic domain of their envelope glycoprotein. This motif can act as an endocytosis signal or as a targeting signal, restricting viral budding at specific cell surface membrane domains. In the present study, proviral DNA of the ecotropic Cas-Br-E strain of MuLV was modified by substitution or deletion of the critical tyrosine residue. Mutant viruses lost basolateral targeting in polarized MDCK epithelial cells while expression level of the glycoprotein at the cell surface was not affected. This suggests that the tyrosine-based motif in MuLV does not act as an endocytosis signal. Only a small delay in the appearance of disease was observed in inoculated mice. In contrast, a striking change in the pathology was observed with enlarged thymus and lymph nodes in animals inoculated with mutant viruses. 相似文献
8.
Akihiro Umezawa Taketo Yamada Yuuto Ogawa Shigeru Kuramochi Yonosuke Watanabe 《Pathology international》1990,40(9):693-698
An autopsy case of acute megakaryocytic leukemia (AMKL) is presented. The bone marrow was hypercellular with proliferation of three lineages, especially megakaryocytes. Immunohistochemical examination revealed many platelet glycoprotein IIb/IIIa (GP IIb/IIIa)- positive blast cells in bone marrow. The proportion of the blasts was 26.4% by tissue hemogram. GP IIb/IIIa-positive blasts and megakaryoblasts were deposited massively in lymph nodes. lmmunohistochemistry against GP IIb/IIIa and tissue hemograms by paraffin section are needed to diagnose AMKL by postmortem examination, since the identification of ultra-structural platelet peroxidase in autopsy materials is difficult. 相似文献
9.
目的:构建、制备单纯疱疹病毒Ⅰ型(HSV-Ⅰ)糖蛋白BDNA疫苗,检测其诱导机体产生的细胞免疫应答。方法:PCR扩增编码HSV-Ⅰ gB去除N端部分信号肽序列(39bp)的基因片段(2673bp),定向插入pcDNA3载体中,构建pcDNA3-gB基因疫苗并对其进行PCR、酶切及测序鉴定。于BALB/c小鼠注射免疫3次,观察小鼠CD4^ 、CD8^ T细胞亚群的变化,CFSE/PI双标记的流式细胞计数法检测CTL活性。结果:双酶切分析结果为目的基因(2.7kb)和线性质粒pcDNA3(5.4kb);PCR扩增结果为特异产物(2.7kb);测序结果与GenBank gB基因序列同源性达99.5%;CTL,活性增强;BALB/c小鼠脾CD4^ T细胞增加。结论:经鉴定证实了HSV-Ⅰ gB基因疫苗的构建;HSV-Ⅰ gB基因疫苗可以诱导较强的细胞免疫应答,应用于预防HSV-Ⅰ的感染具有良好的前景。 相似文献
10.
T cell epitope spreading to myelin oligodendrocyte glycoprotein in HLA-DR4 transgenic mice during experimental autoimmune encephalomyelitis 总被引:2,自引:0,他引:2
Klehmet J Shive C Guardia-Wolff R Petersen I Spack EG Boehm BO Weissert R Forsthuber TG 《Clinical immunology (Orlando, Fla.)》2004,111(1):53-60
Epitope spreading has been implicated in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and human multiple sclerosis (MS). T cell epitope spreading has been demonstrated in rodents for myelin basic protein (MBP) and proteolipid protein (PLP) determinants, but not for myelin oligodendrocyte glycoprotein (MOG), another important myelin antigen. Moreover, the role of human autoimmunity-associated MHC molecules in epitope spreading, including HLA-DR2 and DR4, has not been formally examined. To address these questions, we investigated epitope spreading to MOG determinants in HLA-DR4 (DRB1*0401) transgenic mice during EAE. The data show that upon induction of EAE in HLA-DR4 transgenic mice with the immunodominant HLA-DR4-restricted MOG peptide 97-108 (MOG(97-108); TCFFRDHSYQEE), the T cell response diversifies over time to MOG(181-200) (core: MOG(183-191); FVIVPVLGP) and MBP. The spreading epitope MOG(181-200) binds with high affinity to HLA-DRB1*0401 and is presented by human HLA-DRB1*0401+antigen presenting cells. Moreover, this epitope is encephalitogenic in HLA-DRB1*0401 transgenic mice. This study demonstrates intra- and intermolecular epitope spreading to MOG and MBP in "humanized" HLA-DR4 transgenic mice. 相似文献