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Beijing, China

Objective: To prepare monoclonal antibodies (McAbs) against capsular polysaccharides of Neisseria meningitides serogroups A, C, W135 and Y (shortened as GAMP, GCMP, GWMP and GYMP respectively), and apply to immunoturbidimetric assay. Methods: BALB/c mice were immunized with the conjugates of N. meningitides capsular polysaccharides to avirulent variant CRM197 of diphtheria toxin, i. e. GAMP-CRM197, GCMP-CRM197, GWMP-CRM197 and GYMP-CRM197 respectively, of which the spleen cells were fused with myeloma SP2/0 cells. Hybridoma cell strains were screened by indirect ELISA, tested for specificity and determined for the titer of purified antibodies. One clone of each group was selected for immunoturbidimetric assay. Results: Twenty, eighteen, nine and thirteen strains secreting McAbs against GAMP, GCMP, GWMP and GYMP were obtained respectively, of which five against GAMP, five against GCMP, three against GWMP and five aginst GYMP showed high specificities, without cross reactions with CRM197 or polysaccharides of other three serogroups. The titers of purified antibodies against GAMP, GCMP, GWMP and GYMP were more than 1:4 000 000, more than 1:200 000, 1:40 000 and 1:40 000 respectively. McAbs 6B7 against GAMP and 2H4 against GCMP were suitable for immunoturbidimetric assay within a standard concentration range of 2 ∼ 10 μg/ml,. with R2 values of 0. 990 9 and 0. 991 9 respectively. However, McAbs 5F1 against GWMP and 7C6 against GYMP were suitable for the assay within standard concentration ranges of 4. 33 ∼ 21. 7 and 1 ∼8 μg/ml, with R2 values of 0. 851 8 and 0. 998 7, respectively. Conclusion: Monoclonal antibodies against GAMP, GCMP, GWMP and GYWP were successfully prepared and applied to immunoturbidimetric assay. Source


Zhu Y.,Beijing Normal University | Lei Y.,Beijing Normal University | Du B.,Beijing Normal University | Zheng Y.,Chinese Academy of Sciences | And 5 more authors.
PLoS ONE | Year: 2015

INMAP is a spindle protein that plays essential role for mitosis, by ensuring spindle and centromere integrality. The aim of this study was to investigate the relevant functions of INMAP for genomic stability and its functional pathway. We overexpressed INMAP in HeLa cells, resulting in growth inhibition in monolayer cell cultures, anchorage-independent growth in soft agar and xenograft growth in nude mice. In this system caused micronuclei (MNi) formation, chromosome distortion and γH2AX expression upregulation, suggesting DNA damage induction and genomic stability impairment. As a tumour biochemical marker, lactate dehydrogenase (LDH) isoenzymes were detected to evaluate cell metabolic activity, the results confirming that total activity of LDH, as well as that of its LDH5 isoform, is significantly decreased in INMAP-overexpressing HeLa cells. The levels of p53 and p21 were upregulated, and however, that of PCNA and Bcl-2, downregulated. Indirect immunofluorescence (IIF) and coimmunoprecipitation (CoIP) analyses revealed the interaction between INMAP and p21. These results suggest that INMAP might function through p53/p21 pathways. © 2015 Zhu et al. Source


Shen L.,Chinese National Institute for Viral Disease Control and Prevention | Gu Y.,Xiamen University | Sun L.,AbMax Biotechnology Co. | Yang Y.,Chinese National Institute for Viral Disease Control and Prevention | And 3 more authors.
Journal of Medical Virology | Year: 2012

Co-infection with hepatitis delta virus (HDV) and hepatitis B virus (HBV) has been shown to be associated with a more severe form of acute and chronic hepatitis. Cloning and expression of recombinant HDV antigen (rHDAg) in Escherichiacoli are described. Using purified rHDAg, a cost-effective indirect anti-HDV enzyme-linked immunosorbent assay (ELISA) kit was developed. Direct comparison of 15 known HDV-positive sera and 15 HDV-negative sera showed concordance agreement between the new assay kit and the Abbott Murex Anti-Delta (total) kit. In addition, 1,486 hepatitis B surface antigen (HBsAg) positive blood samples collected from various areas of China were tested using this indirect anti-HDV ELISA. It was found that 1.2% (95% CI: 0.7-1.9%) of the samples were anti-HDAg positive. It is suggested that the prevalence of HDV and HBV co-infection in China is relatively low. © 2011 Wiley Periodicals, Inc. Source


Yunlei Z.,Beijing Normal University | Zhe C.,Beijing Normal University | Yan L.,Beijing Normal University | Pengcheng W.,Beijing Normal University | And 3 more authors.
Molecular and Cellular Biochemistry | Year: 2013

INMAP was first identified as an interphase nucleus and mitotic apparatus-associated protein that plays essential roles in the formation of the spindle and cell-cycle progression. Here, we report that INMAP might be conserved from prokaryotes to humans, is a truncated version of the RNA polymerase III subunit B POLR3B, and is up-regulated in several human cancer cell lines including HeLa, Bel-7402, HepG2 and BGC-823. Deletion analysis revealed that the 209-290 amino-acid region is necessary for the punctate distribution of INMAP in the nucleus. Furthermore, over-expression of INMAP inhibited the transcriptional activities of p53 and AP-1 in a dose-dependent manner. These results suggest that INMAP may function through the p53 and AP-1 pathways, thus providing a possible link of its activity with tumourigenesis. Integrating our data and those in previous studies, it can be concluded that INMAP plays dual functional roles in the coordination of mitotic kinetics with gene expression as well as in cell-fate determination and proliferation. © 2012 Springer Science+Business Media New York. Source


Mi L.,Pacific Meinoke Biopharmaceutical Co | Li W.,National Center for Safety Evaluation of Drugs | Li M.,AbMax Biotechnology Co. | Chen T.,Pacific Meinoke Biopharmaceutical Co | And 3 more authors.
Journal of Immunological Methods | Year: 2016

The clinical effect of patient immune responses to therapeutic antibodies affect product safety and efficacy, which makes the development of valid, sensitive immune assays a key aspect of antibody drug development. In this paper, we reported the generations of mouse monoclonal and Cynomolgus monkey polyclonal antibodies against the anti-CD147 antibody (Metuzumab) as the internal standards and the positive controls. Seven mouse monoclonal antibodies were shown to recognize both (Fab)2 and full length of Metuzumab, but not the control normal human IgGs, and monoclonal anti-Metuzumab, Clone 2D9 was chosen to be used as the internal standard for anti-Metuzumab study. A Bridging ELISA assay was developed by coating the wells with the antibody drug, and the anti-drug antibody (ADA) in the animal sera were detected by enzyme-labeled antibody. Its limit of detection (LOD) was determined to be 0.39 ng/ml of anti-Metuzumab antibody (ADA) with linear range between 0.39-50 ng/ml and R2 = 0.994. For normal monkey sera, a minimal dilution was determined to be 1:80. However, very different from peptide or other protein drugs, strong interferences from the residual antibody drugs were observed from most of the testing monkey sera in the preclinical study. It was experimentally determined that the concentration of the residual antibody drug in the assay have to be lower than 1 μg/ml, so the assays were carried out at 1:100 dilution of the monkey sera. In the pre-clinical study, 32 monkeys were treated with escalating doses of Metuzumab between 0, 10, 50, 200 mg/kg for 13 times over 13 weeks of time period. 16 of them were terminated right after the last injection, while the other 16 were rested for additional 4 weeks before termination. Afraid to miss any positive response to antibody drug, sera samples were collected at six time points, including 2-, 6- and 10-weeks post 1st dose, prior to last dose, and 2-, 4-weeks into recovery. The highest positive rates were seen with the Medium- and High-dose group 2-weeks post the first injection, 6 out 8 monkeys in the High-dose were positive for free ADA. However, no significant pathologic and clinic adversary effect was observed in those monkeys. © 2016 Elsevier B.V. Source

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