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PubMed | National Institutes for Food and Drug Control NIFDC, CAS Beijing Institute of Genomics, Peking Union Medical College, Beijing Macro & Micro Test Biotech Company and Beijing Institute of Microbiology and Epidemiology
Type: Journal Article | Journal: Clinical biochemistry | Year: 2016

Low-level DNA mutations play important roles in cancer prognosis and treatment. However, most existing methods for the detection of low-level DNA mutations are insufficient for clinical applications because of the high background of wild-type DNA.In this study, a novel assay based on Tm-dependent inhibition of wild type template amplification was developed. The defining characteristic of this assay is an additional annealing step was introduced into the ARMS-blocker PCR. The temperature of this additional annealing step is equal to the Tm of the blocker. Due to this additional annealing step, the blocker can preferentially and specifically bind the wild-type DNA. Thus, the inhibition of wild type template is realized and the mutant DNA is enriched.The sensitivity of this assay was between 10(-4) and 10(-5), which is approximately 5 to 10 times greater than the sensitivity of the assay without the additional annealing step. To evaluate the performance of this assay in detecting K-ras mutation, we analyzed 100 formalin-fixed paraffin-embedded (FFPE) specimens from colorectal cancer patients using this new assay and Sanger sequencing. Of the clinical samples, 27 samples were positive for K-ras mutation by both methods.Our results indicated that this new assay is a highly selective, convenient, and economical method for detecting rare mutations in the presence of higher concentrations of wild-type DNA.


PubMed | National Institutes for Food and Drug Control NIFDC, U.S. Center for Disease Control and Prevention and Sanofi S.A.
Type: Journal Article | Journal: Journal of the Pediatric Infectious Diseases Society | Year: 2016

Two vaccination schedules where inactivated polio vaccine (IPV) was followed by oral polio vaccine (OPV) were compared to an OPV-only schedule.Healthy Chinese infants received a 3-dose primary series of IPV-OPV-OPV (Group A), IPV-IPV-OPV (Group B), or OPV-OPV-OPV (Group C) at 2, 3, and 4 months of age. At pre-Dose 1, 1-month, and 14-months post-Dose 3, polio 1, 2, and 3 antibody titers were assessed by virus-neutralizing antibody assay with Sabin or wild-type strains. Adverse events were monitored.Anti-polio 1, 2, and 3 titers were 8 (1/dil) in >99% of participants, and Group A and Group B were noninferior to Group C at 1-month post-Dose 3 as assessed by Sabin strain-based assay (SSBA). In Group A 1-month post-Dose 3, there was no geometric mean antibody titers (GMT) differences for types 1 and 3; type 2 GMTs were 3-fold higher by wild-type strain-based assay (WTBA) versus SSBA. For Group B, GMTs were 1.7- and 3.6-fold higher for types 1 and 2 via WTBA, while type 3 GMTs were similar. For Group C, GMTs were 6.3- and 2-fold higher for types 1 and 3 with SSBA, and type 2 GMTs were similar. Antibodies persisted in >96.6% of participants. Adverse event incidence in each group was similar.A primary series of 1 or 2 IPV doses followed by 2 or 1 OPV doses was immunogenic and noninferior to an OPV-only arm. SSBA was better at detecting antibodies elicited by OPV with antibody titers correlated to the number of OPV doses (NCT01475539).


Liang C.-G.,National Institutes for Food and Drug Control NIFDC | Wang J.-Z.,National Institutes for Food and Drug Control NIFDC
Chinese Journal of New Drugs | Year: 2012

In order to ensure most Chinese patients, particularly in the population with relatively low incomes, have access to the safe, low-cost, effective and quality-assured medicines, more than thirty of different non-innovator biological products have been marketed in China. After the WHO guidelines on evaluating similar biotherapeutic products (SBPs) had been published, many countries' and regions' regulatory agencies have finished or begin to actively engage in the development of bio-similar guidance and documents. As the major country of new biological product development and non-innovator biological product industry, the regulatory agency and manufacturers in China should collaborate to actively consider and response to it. It is an urgent demand for our country to promote the process of drafting the Chinese guidelines for SBPs considering both the local situation in China and the general WHO framework. This will help our country to face the opportunities and challenges, and may be of benefit for launching the industrial leap of non-innovator biological products again at the new level.


Liang C.,National Institutes for Food and Drug Control NIFDC | Wang J.,National Institutes for Food and Drug Control NIFDC
Biologicals | Year: 2011

In order to ensure most Chinese patients, particularly in the population with relatively low incomes, have access to safe, low cost, effective and quality-assured medicines, a number of " stand-alone" biological products, which have good quality, safety and efficacy have been marketed in China. Many countries and regions' regulatory agencies are actively engaging in the development of bio-similar guidance and documents, which is being coordinated by WHO. As a major developing country of new drug development, China is now working hard to promote the process of new similar biotherapeutic products (SBPs) approval and also actively involved in developing and updating technical documents. © 2011.


Liu Q.,East China University of Science and Technology | Zhou B.,East China University of Science and Technology | Wang X.,East China University of Science and Technology | Ke Y.,East China University of Science and Technology | And 3 more authors.
Journal of Separation Science | Year: 2012

A search library about benzylisoquinoline alkaloids was established based on preparation of alkaloid fractions from Rhizoma coptidis, Cortex phellodendri, and Rhizoma corydalis. In this work, two alkaloid fractions from each herbal medicine were first prepared based on selective separation on the "click" binaphthyl column. And then these alkaloid fractions were analyzed on C18 column by liquid chromatography coupled with tandem mass spectrometry. Many structure-related compounds were included in these alkaloids fractions, which led to easy separation and good MS response in further work. Therefore, a search library of 52 benzylisoquinoline alkaloids was established, which included eight aporphine, 19 tetrahydroprotoberberine, two protopine, two benzyltetrahydroisoquinoline, and 21 protoberberine alkaloids. The information of the search library contained compound names, structures, retention times, accurate masses, fragmentation pathways of benzylisoquionline alkaloids, and their sources from three herbal medicines. Using such a library, the alkaloids, especially those trace and unknown components in some herbal medicine could be accurately and quickly identified. In addition, the distribution of benzylisoquinoline alkaloids in the herbal medicines could be also summarized by searching the source samples in the library. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Zhu R.,National Institutes for Food and Drug Control NIFDC | Zhu R.,Wuhan Institute of Biological Products | Liu Q.,National Institutes for Food and Drug Control NIFDC | Huang W.,National Institutes for Food and Drug Control NIFDC | And 2 more authors.
Archives of Virology | Year: 2014

Vaccinia virus is widely used as a vector in the development of recombinant vaccines. Vaccinia virus strain Guang 9 (VG9), which was derived from vaccinia virus strain Tian Tan (VTT) by successive plaque-cloning purification, was more attenuated than VTT. In this study, the host cell range and the growth and replication of VG9 were compared with those of VTT. The results showed that both VG9 and VTT could infect permissive cells (Vero, TK-143 and CEF) and semipermissive cells PK (15) and induced a visible cytopathic effect (CPE). Both strains could infect nonpermissive CHO-K1 cells but neither was able to reproduce. The replicative ability of VG9 was a little lower than that of VTT. Additionally, recombinant vaccinia viruses containing a firefly luciferase gene (VG9-L and VTT-L) were constructed, and their expression in vitro and replication and spread in vivo were compared. The expression ability of VG9-L was lower than that of VTT-L. Whole-animal imaging data indicated that VG9-L could reproduce quickly and express the exogenous protein at the site of inoculation, regardless of whether the intramuscular, intracutaneous, subcutaneous or celiac inoculation route was used. VG9-L was better in its ability to express a foreign protein than VTT-L, but the time during which expression occurred was shorter. There was no dissemination of virus in mice inoculated with either strain. In summary, this study demonstrates the possibility of using VG9 for the production of smallpox vaccines or the construction of recombinant vaccinia virus vaccines. © 2014, Springer-Verlag Wien.


Nie J.,National Institutes for Food and Drug Control NIFDC | Liu Y.,National Institutes for Food and Drug Control NIFDC | Huang W.,National Institutes for Food and Drug Control NIFDC | Wang Y.,National Institutes for Food and Drug Control NIFDC
Viruses | Year: 2016

Pseudovirion-based neutralization assay is considered the gold standard method for evaluating the immune response to human papillomavirus (HPV) vaccines. In this study, we developed a multicolor neutralization assay to simultaneously detect the neutralizing antibodies against different HPV types. FluoroSpot was used to interpret the fluorescent protein expression instead of flow cytometry. The results of FluoroSpot and flow cytometry showed good consistency, with R2 > 0.98 for the log-transformed IC50 values. Regardless of the reporter color, the single-, dual-, and triple-color neutralization assays reported identical results for the same samples. In low-titer samples from naturally HPV-infected individuals, there was strong agreement between the singleand triple-color assays, with kappa scores of 0.92, 0.89, and 0.96 for HPV16, HPV18, and HPV58, respectively. Good reproducibility was observed for the triple-color assay, with coefficients of variation of 2.0%–41.5% within the assays and 8.3%–36.2% between the assays. Three triple-color systems, HPV16-18-58, HPV6-33-45, and HPV11-31-52, were developed that could evaluate the immunogenicity of a nonavalent vaccine in three rounds of the assay. With the advantages of an easy-to-use procedure and less sample consumption, the multiple-color assay is more suitable than classical assays for large sero-epidemiological studies and clinical trials and is more amenable to automation. © 2016 by the authors; licensee MDPI, Basel, Switzerland.


Shi K.,Shenyang Pharmaceutical University | Liu Y.,Shenyang Pharmaceutical University | Ke L.,Shenyang Pharmaceutical University | Fang Y.,Shenyang Pharmaceutical University | And 2 more authors.
International Journal of Biological Macromolecules | Year: 2015

This work presents new spherical nanoparticles that are fabricated from supramolecular self-assembly of therapeutic proteins for inhalation treatment. The formation involved self-assembly of insulin into nanospheres (INS) by a novel thermal induced phase separation method. Surface functional modification of INS with ε-poly-. l-lysine (EPL), a homopolymerized cationic peptide, was followed to form a core-shell structure (INS@EPL). Both INS and INS@EPL were characterized as spherical particles with mean diameter size of 150-250. nm. The process of transient thermal treatment did not change their biological potency retention significantly. FTIR and CD characterizations indicated that their secondary structures and biological potencies were not changed significantly after self-assembly. The in vivo investigation after pulmonary administration, including lung deposition, alveoli distribution, pharmacological effects and serum pharmacokinetics were investigated. Compared to that of INS, intratracheal administration of INS@EPL offered a pronounced and prolonged lung distribution, as well as pharmacological effects which were indicated by the 23.4% vs 11.7% of relative bioavailability. Accordingly, the work described here demonstrates the possibility of spherical supramolecular self-assembly of therapeutic proteins in nano-scale for pulmonary delivery application. © 2014 Elsevier B.V.


PubMed | National Institutes for Food and Drug Control NIFDC
Type: Journal Article | Journal: Viruses | Year: 2016

Pseudovirion-based neutralization assay is considered the gold standard method for evaluating the immune response to human papillomavirus (HPV) vaccines. In this study, we developed a multicolor neutralization assay to simultaneously detect the neutralizing antibodies against different HPV types. FluoroSpot was used to interpret the fluorescent protein expression instead of flow cytometry. The results of FluoroSpot and flow cytometry showed good consistency, with R > 0.98 for the log-transformed IC50 values. Regardless of the reporter color, the single-, dual-, and triple-color neutralization assays reported identical results for the same samples. In low-titer samples from naturally HPV-infected individuals, there was strong agreement between the single- and triple-color assays, with kappa scores of 0.92, 0.89, and 0.96 for HPV16, HPV18, and HPV58, respectively. Good reproducibility was observed for the triple-color assay, with coefficients of variation of 2.0%-41.5% within the assays and 8.3%-36.2% between the assays. Three triple-color systems, HPV16-18-58, HPV6-33-45, and HPV11-31-52, were developed that could evaluate the immunogenicity of a nonavalent vaccine in three rounds of the assay. With the advantages of an easy-to-use procedure and less sample consumption, the multiple-color assay is more suitable than classical assays for large sero-epidemiological studies and clinical trials and is more amenable to automation.


PubMed | National Institutes for Food and Drug Control NIFDC and Capital Medical University
Type: | Journal: Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases | Year: 2016

Delineating the course of NAb (neutralizing antibody) development in natural infection may provide clues for NAb-targeted HIV-1 vaccine design. Two subjects, A (non-neutralizer) and E (neutralizer), were chosen from 75 HIV-1 positive subjects of a MSM (men who have sex with men) cohort to investigate the key events of virus evolution in the development course of neutralizing antibodies. Pseudovirus quasispecies (at least 10 strains) were generated for each time points of the infection course. The diversity and divergence of the env quasispecies per time point for subject E were significantly higher than those for subject A (p<0.05). Compared with subject A, the gp160 derived from subject E acquired longer V1V2 region and more N-glycans during the development of neutralizing antibodies. The developing course of neutralizing antibody lagged behind the virus evolution, of which the pseudoviruses could only been neutralized by the latter time-point sera. The neutralization-driven evolution of the virus for subject E was mostly mapped to the C1-C3 region of gp160. Through site-directed mutagenesis, some key sites and region were identified to be associated with the virus escape, including: Q85P, H183P, K340E, L365S, L369I, I372V and insertions of 355N in C3 and NITDEVKIG in V1 region.

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