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Chen Y.,Lanzhou Institute of Biological Products Co.
Advances in Experimental Medicine and Biology | Year: 2015

The study on dynamic analysis of human urinary proteome is the foundation that we discriminate certain various urinary proteins as potential biomarker derived from the disease itself or normal physiological change. In our results, based on RPLC-MS/MS and spectral count to study pooled and individual urine samples and other researchers’ studies, it can be known that the content of many urinary proteins maintain relatively stable. We have reason to believe that the relatively stable urinary protein is a very valuable resource as biomarkers. Many similar proteins such as prostaglandin-H2 D-isomerase and apolipoprotein D proteins have been proved our hypothesis. The following field, the number, preservation and treatment methods of urine sample, the standardization of analysis method and data processing, and suitable quantitative method, is ought to the focus of future study. © Springer Science+Business Media Dordrecht 2015.


Chang Y.-L.,Lanzhou Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2013

Objective: To develop and verify a double antibody sandwich ELISA diagnostic kit for F1 antigen of Yersinia pestis. Methods: Antisera were prepared by immunizing rabbits with F1 antigen of Y. pestis, subjected to salting out with 50% ammonium sulfate, the purified by Sephacryl S-300 gle column chromatography and used as the coating antibody. The murine ascites containing monoclonal antibody (McAb) against F1 antigen was prepared, purified, labled with HRP and used as the enzyme-labeled antibody. Three batches of double antibody sandwich ELISA kits were prepared, and verified for precsion, sensitivity, linear range, specificity, reproducibility, accuracy and stability by intra- and inter-assays using internal reference. Results: The CVs of results of intra- and inter-assays on reference for precision by the prepared kit were 5.2% and 8.23% respectively. The linear range and minimum detection limit of the kit were 3.91-62.5 and 3.0 ng/ml respectively. The detection result of Y. pestis was positive, and no cross reaction with Y. pseudotuberculosis was observed. The CVs of F1 antigen at high, moderate and low contents determined by the kit were 4.54%-8.4%, while the recovery rates were 104%-108%. The kit showed high stability, of which the validity period was not less than 12 months. Conclusion: A double antibody sandwich ELISA diagnostic kit for F1 antigen of Y. pestis was successfully prepared, which provided a rapid method for clinical diagnosis and epidemiological surveillance of plaque, and a tool for determination of F1 antigen content in novel plaque component vaccine.


Chang Y.-L.,Lanzhou Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2012

Objective: To determine the F1 and rV antigen contents in plague component vaccine by double antibody sandwich ELISA. Methods: The antigens in bulk of plague component vaccine were identified with monoclonal antibodies (McAbs) against F1 rV antigens, and determined quantitatively by double antibody sandwich ELISA. The completeness of antigen adsorbed onto aluminium hydroxide adjuvant was verified. The final product of vaccine was stored at 4°C for 18 months, from which samples were taken 1 and 18 months after storage respectively and determined for F1 and rV antigen contents by the developed double antibody sandwich ELISA method to analyze the stability of antigen. The three-step purification procedure for three batches of bulk rV antigen of Yersiria pestis were verified. Results: The F1 and rV antigens in bulk of plague component vaccine showed good reactogenicity. The F1 and rV antigen contents in four batches of bulks were determined by the developed method, of which the results showed an error of not more than 20% with those determined by Lowry method. No F1 or rV antigen was detected in the centrifuge supernatant of four batches vaccine, indicating that the antigens were completely adsorbed by aluminium hydroxide adjuvant. The F1 and rV antigen contents in vaccine were stable after storage at 4°C for 18 months. After three batches of bulk were purified by anion exchange, hydrophobic and gel filtration chromatography, both the purity and content of rV antigen increased. Conclusion: The developed double antibody sandwich ELISA method may be used for the quantitative determination of F1 and rV antigens in plague component vaccine.


He X.,Lanzhou Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2012

Objective: To analyze the property of active pharmaceutical ingredient (API) of botulinum toxin type A (BTXA) for injection, as well as the specific activities of the preparations manufactured in 2009-2011. Methods: Various components were separated from the harvested BTXA by anion exchange chromatography. The properties of BTXA complex and its components were analyzed by hemagglutination test, HPLC, SDS-PAGE, isoelectric focusing electrophoresis and amino acid sequencing at N-terminus. The data on specific activity of bulks of BTXA manufactured in 2009-2011 were analyzed, based on which the stability of production procedure and the reproducibility of quality were evaluated. Results: The harvested BTXA was dissociated in basic environment. The obtained neurotoxin showed no hemagglutination titer, of which the relative molecular mass was about 150 000. The API of BTXA was an intact monomer, of which the purity was more than 99.5%. The isoelectric points of BTXA complex and neurotoxin were 4.97 and 4.91 respectively, of which the amino acid sequences at N-terminus were basically consistent with those reported in GenBank. The mean specific activity of bulks of BTXA manufactured in 2009-2011 was 3.0 × 107 LD50/mg protein. The load of API in final product of BTXA was about 5 ng per container. Conclusion: The API of BTXA was a specific complex which was dissociated in basic environment to obtain neurotoxin. The specific activities of bulk BTXA manufactured in 2009-2011 were stable, indicating good persistency of quality, which provided a basis for safety of BTXA in clinic.


Liang X.,Chinese National Institute for Communicable Disease Control and Prevention | Zhang H.,Chinese National Institute for Communicable Disease Control and Prevention | Zhang E.,Chinese National Institute for Communicable Disease Control and Prevention | Wei J.,Chinese National Institute for Communicable Disease Control and Prevention | And 4 more authors.
Virulence | Year: 2016

Anthrax toxins and capsule are the major virulence factors of Bacillus anthracis. They are encoded by genes located on the plasmids pXO1 and pXO2, respectively. The vaccine strain Pasteur II was produced from high temperature subcultures of B. anthracis, which resulted in virulence attenuation through the loss of the plasmid pXO1. However, it is unclear whether the high temperature culture completely abolishes the plasmid DNA or affects the replication of the plasmid pXO1. In this study, we tested 3 B. anthracis vaccine strains, including Pasteur II from France, Qiankefusiji II from Russia, and Rentian II from Japan, which were all generated from subcultures at high temperatures. Surprisingly, we detected the presence of pXO1 plasmid DNA using overlap PCR in all these vaccine strains. DNA sequencing analysis of overlap PCR products further confirmed the presence of pXO1. Moreover, the expression of the protective antigen (PA) encoded on pXO1 was determined by using SDS-PAGE and western blotting. In addition, we mimicked Pasteur's method and exposed the A16R vaccine strain, which lacks the pXO2 plasmid, to high temperature, and identified the pXO1 plasmid in the subcultures at high temperatures. This indicated that the high temperature treatment at 42.5°C was unable to eliminate pXO1 plasmid DNA from B. anthracis. Our results suggest that the attenuation of the Pasteur II vaccine strain is likely due to the impact of high temperature stress on plasmid replication, which in turn limits the copy number of pXO1. Our data provide new insights into the mechanisms of the remaining immunogenicity and toxicity of the vaccine strains. © 2016 Taylor & Francis

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