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Objective To prepare human alpha segment of high affinity IgE receptor(FcεRIα)protein by genetic engineering technology and to identify its biological function for laying the foundation for further researching the role of FcεRIα in allergic disea-ses.Methods The human FcεRIα gene was obtained by the PCR based accurate synthesis(PAS)method and the prokaryotic ex-pression vector pET-28a(+)was constructed.The FcεRIα was expressed at low temperature induction and the recombinant protein was purified by His tag.The biological function of recombinant human FcεRIα protein was identified by ELISA.Results The hu-man FcεRIα gene was amplified by PAS with a size of approximately 560 bp.The pET-FcεRIα plasmid was correct through the double enzyme digestion and sequencing identification.The human FcεRIα with a molecular weight of approximately 22 000 was in-duced and purified.The recombinant human FcεRIα could effectively detect human serum anti-FcεRIα autoantibody and could com-bined with serum IgE antibodies with high efficiency.Conclusion Human FcεRIα protein is successfully prepared,which prelimina-rily has the ability for detecting the human serum anti-FcεRIα autoantibodies and IgE antibodies,and provides a favorable practical base for further study.
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Objective To investigate the infection situation of HIV in patents in hospital ,and to provide basis for the prevention of AIDS in the hospital .Methods Three kinds of enzyme‐linked immunosorbent assay(ELISA)kits(Beijing wantai ,Beijing kewei , French BIO‐RAD)were used for screening HIV antibody and antigen in 240 781 samples from Jan .2011 to Dec .2013 .All HIV re‐peated positive screening samples were confirmed by western blotting in Chongqing shapingba Center for Disease Control and Pre‐vention .Results Among 240 781 samples from 2011 to 2013 ,593 samples(0 .246% ) were HIV positive at the first screening ,558 samples(0 .231% ) were HIV positive in at the repeated screening ,29 samples(0 .012% ) were HIV indeterminate ,6 samples(0 . 002% ) were HIV negative.Male and female ratio was 3 .39:1 .Conclusion Screening in hospital patients could be an important way to discover cases with HIV infection .It is nesscessary to strengthen the promotion and propaganda of HIV detection ,and new technology of HIV detection could be used to strengthen the inspection of HIV .Moreover ,the consciousness of self protection should be promoted in the treatment of HIV patients .
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OBJECTIVE To evaluate primarily the detection limit,specificity and reproducibility of immuno-PCR assay on HIV-1 p24 antigen.METHODS We p24 antigen were detected by established immuno-PCR system,and then the detection limit,specificity and reproducibility were discussed.We quantitatively analyzed the nonspecific amplified bands with fluorescence intensity(FI),and a preliminary determination of the lower specific amplification limit was made.RESULTS We taken(x-+3s)FI of nonspecific amplification as the lower limit of specific amplification signal.The detection limit of immuno-PCR assay was 0.1 ng/L.CONCLUSIONS The detection limit,specificity and reproducibility can meet the needs of HIV-1 p24 antigen detection.
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OBJECTIVE To establishan immuno-PCR assay with the carriers of gold-magnetic particles for detection of HIV-1 p24. METHODS The feasibility of using gold-magnetic particles as the carriers was verified. The gold-magnetic particles were coated with mouse anti-p24 monoclonal antibody as the capture antibody. The reporter DNA was initially generated by PCR amplification using a biotinylated primer, and was bound through streptavidin to biotinylated polyclonal antibody as the detection antibody. HIV-1 p24 sandwiched by two antibodies was detected by amplifying the reporter DNA using PCR. RESULTS The efficiency of gold-magnetic particles coated with mouse anti-p24 monoclonal antibody could reach up to 95%. Furthermore, the amount of antibodies immobilization was consistent among different batches of gold-magnetic particles and there was nearly without nonspecific adsorption. The detection limit of immuno-PCR assay was 0.1 ng/L, an approximately 1.5?104-fold higher compared with an enzyme-linked immunosorbent assay. The linear range of p24 concentration was 0.1-100 ng/L. CONCLUSIONS Gold-magnetic particle is one of the ideal immuno-PCR reaction carriers. The immuno-PCR for detection of HIV-1 p24 reported in this article is indicated to be a promising detection method.
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OBJECTIVE To study the sterilization by gamma radiation on the cytokine levels in manually manipulated platelet concentrates, and its practicability. METHODS Manually manipulated platelet concentrates were irradiated by gamma ray at 25 Gy. Detected the quantity of cytokine levels after irradiation on dd 0, 1, 3, and 5. Platelet counts and pH value were detected on the d0 and d5. RESULTS The contents of cytokines increased with storage time both in irradiation and control groups. But the contents of cytokines in the control group increased more significantly than in the irradiation one (P
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Objective To describe a highly sensitive immuno-polymerase chain reaction (immuno-PCR) assay for the detection of human recombinant HIV-p24 antigen. Methods We used gold-magnetic particles as the carriers,mouse anti-p24 monoclonal antibody as the capture antibody and biotinylated goat anti-p24 polyclonal antibody as the detection antibody. The reporter DNA was initially generated by PCR amplification using a biotinylated primer,and was bound with streptavidin to biotinylated polyclonal antibody. Human recombinant p24 antigen sandwiched by antibodies was detected by amplifying the reporter DNA using PCR. The optimal concentration of sreptavidin and DNA label were determined using square titration. The electrophoresis gels were imaged and analyzed by Quantity One software. Results The optimal concentration of sreptavidin and DNA label were determined to be 0.1 mg/ L and 10 ng/ L,respectively. The detection limit of the immuno-PCR assay was 0.1 ng/L,higher than that of the conventional ELISA. Conclusion A highly sensitive immuno-PCR for human recombinant HIV-p24 antigen was indicated to be an available method for early screening of HIV infectors in blood donors.
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Objective To explore the conditions for construction of human-to-swine hematopoietic chimera and its specific immune tolerance.Methods The human cord blood CD34+ cells (5?105/Kg) were transplanted to neonatal swine through intraperitoneal injections. The experimental swine, injected with human SCF (50ng/Kg.3d) and EPO (100U/Kg. 3d) simultaneously, and swine without injection were defined as transplantation group 1 and group 2, respectively. The swine, injected with saline, were taken as the control group. Peripheral blood (PB) was collected on day 10, 20, 30, 40, 50, and 60 after injection. The human CD71+cells in the chimera PB were quantitated by FACS and the genus-antibodies of PB were detected by blood group gel card. Results The percentage of human CD71+ cells in PB of transplantation group 1 was significantly higher than that of group 2 (P
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Objective To study the bacteriological effect of different dosages of gamma irradiation on Escherichia Coli (E. Coli) in red blood cells (RBC) products,and assess the appropriate dose of gamma irradiation. Methods Prepare RBC samples inoculated with(102-106)/ml of E. Coli. The contaminated RBCs were irradiated by 0 (as control),15,20,25,30 and 35Gy of gamma irradiation. E. Coli in the RBCs were tested on days 0,7, and 14 after irradiation. Results The quantities of E. Coli changed after irradiation. The RBCs inoculated with less than 103cfu/ml of E. Coli were completely sterilized by 25 Gy irradiation. Conclusion 25 Gy gamma irradiation can eradicate less than 103cfu/ml of E. Coli in the RBC products.