The sequence encoding an E2 main antigen glycoprotein of the C strain of classical swine fever virus (CSFV) was highly expressed in the host cell E. coli BL21–CodonPlus (DE3)–RIL using the pGEX-4T-1 expression vecto...The sequence encoding an E2 main antigen glycoprotein of the C strain of classical swine fever virus (CSFV) was highly expressed in the host cell E. coli BL21–CodonPlus (DE3)–RIL using the pGEX-4T-1 expression vector and the soluble recombinant product was purified with Glutathione Sepharose TM4B by centrifugation. The soluble recombinant protein showed good immune reactions and was confirmed by Western blot using anti-CSFV-specific antibodies. Then an indirect ELISA with the purified E2 protein as the coating antigen was established to detect antibody against CSFV. The result revealed that the optimal concentration of coated antigen was 0.6 μg/well and the optimal dilution of serum was 1:80. The positive cut-off value of this ELISA assay was OD tested serum / OD negative serum≥2.1. The E2-ELISA method was evaluated by comparison with the indirect hemagglutination test (IHAT). When a total of 100 field serum samples were tested the sensitivity and specificity were 90.3% and 94.7% respectively. Specificity analysis showed that there were no cross-reactions between BVD serum and the purified E2 protein in the E2-ELISA.展开更多
A high affinity polyclonal antibody-based enzyme linked immunosorbent assay （ELISA） was developed for the quantification of zeranol in bovine urine. On the basis of urine matrix studies, the optimized dilution facto...A high affinity polyclonal antibody-based enzyme linked immunosorbent assay （ELISA） was developed for the quantification of zeranol in bovine urine. On the basis of urine matrix studies, the optimized dilution factors producing insignificant matrix interference were selected as 1：5 in pretreatment. In the improved ELISA, the linear response range was between 0.02 and 1 μg/ml, and the detection limit was 0.02 μg/ml for the assay. The overall recoveries and the coefficients of variation （CVs） were in the range of 82%-127% and 3.5%-8.8%, respectively. Thirty-six bovine urine samples spiked with zeranol （ranging from 0.2 to 10 μg/ml） were detected by the ELISA and liquid chromatography （LC） method, and good correlations were obtained between the two methods （R^2=0.9643）. We conclude that this improved ELISA is suitable tool for a mass zeranol screening and can be an altemative for the conventional LC method for zeranol in bovine urine.展开更多
Objective To investigate the main proteinases responsible for CD16b shedding under different stimulators. Methods HEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then ...Objective To investigate the main proteinases responsible for CD16b shedding under different stimulators. Methods HEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then overexpressed with a disintegrin and metalloproteinase 10 （ADAM10） or ADAM17, sup- pressed with short hairpin RNA of ADAM10 or ADAM I 7, and reconstituted with ADAM 10 or ADAM17, respectively. After each treatment, the cell line was stimulated with ionomycin or phorbol 12-myristate- 13-acetate （PMA） for 12 hours. The soluble CD 16b released from cell membrane was detected by immuno- precipition and immunoblot. Quantitation was then implemented to compare the amount of soluble CD 16b in cell supernatant after stimulation. Results HEK293 cell line stably expressing CD16b was successfully established. When CDI6b ex- pressing cell line was overexpressed with ADAM 10, shedding of CD 16b was increased after stimulation with ionomycin but not PMA; when the cell line overexpressed with ADAM I7, shedding of CDI6b was increased after stimulation with PMA but not ionomycin. Similarly, when ADAM10 was suppressed by short hairpin RNA, CD 16b shedding was decreased after stimulation with ionomycin; when ADAM 17 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with PMA. The shedding of CD16b was increased again when CD16b expressing cell line was reconstituted with ADAM10 and stimulated by ionomycin or reconstituted with ADAM 17 and stimulated by PMA. Conclusions Both ADAM10 and ADAM17 could shed CD16b, but they possess differed prefer- ences. ADAM10 is the main sheddase under stimulation of ionomycin, while ADAM17 is the main sheddase under stimulation of PMA.展开更多
Overthelastseveraldecades,harmfulalgalblooms(HABs)havebecomeaseriousenvironmental problem in many parts of the world. A rapid and accurate detection process for HAB algae has yet to be developed. Heterosigma akashiwo ...Overthelastseveraldecades,harmfulalgalblooms(HABs)havebecomeaseriousenvironmental problem in many parts of the world. A rapid and accurate detection process for HAB algae has yet to be developed. Heterosigma akashiwo is one of the most important HABs species in China. The objective of this study was to develop an immunologic technique that can rapidly and sensitively count H. akashiwo cells. Five HABs species (Alexandrium catenella, Thalassiosira sp., Cryptomonas sp., Alexandrium tamarense and Symbiodinium sp.,) were used in this study to evaluate the analysis process we developed. A polyclonal antibody with high titers against H. akashiwo was obtained by injecting H. akashiwo cells into rabbits. Immuno-magnetic beads (IMB) were produced via conjugated polyclonal antibodies with magnetic beads and applied to isolate and count H. akashiwo cells from the culture. Results show that 66.7%-91.6% of the cells were captured from unialgal culture by IMBs, and only 5.3%-12.5% of the four other HAB microalgae species were captured, indicating that the constructed IMBs combined specifically with the H. akashiwo cells. At the same time, flow cytometry (FCM) sorting was exploited to screen H. akashiwo cells after labeling with FITC conjugated polyclonal antibodies. Using the FCM technique, 91.7% of the targeted cells were sorted out from mixed microalgae samples in just a few minutes. These results indicate that both antibody-involved IMB and antibody-based FCM techniques are highly effective at detecting and quantifying HAB species. These techniques, especially immuno-magnetic separation, have low associated cost, and are fast and simple processes compared with other techniques currently in use.展开更多
Objective: To observe the effectiveness of highly active antiret-rovirus therapy (HAART) on HIV/AIDS patients. Methods: Using HIV-1 quantitative methods and immu-nological function inspection, we monitored 4 HIV/AIDS ...Objective: To observe the effectiveness of highly active antiret-rovirus therapy (HAART) on HIV/AIDS patients. Methods: Using HIV-1 quantitative methods and immu-nological function inspection, we monitored 4 HIV/AIDS pa-tients who were suffering from immunological deficiency andwere treated with HAART Results: The reproduction of HIV in all 4 patients was effi-ciently controlled at the 4th week of the treatment. The averageviral load decreased by 1.99 Log/ml (0.73-2.46 Log/ml). Thenumber of CD^+_4 and CD^+_8 showed a steady continuous increase4 to 12 weeks after the treatment, with an increase of 67.2%and 103.0% respectively. Correlative study among differentvariables after the treatment revealed that positive correlationexisted between the number of CD^+_4 and CD^+_3 as well as CD^+_8,while negative correlation existed between the number of CD^+_4and plasma viral load. Conclusion: HIV-1 quantitative method (plasma viral load)and the number of CD^+_4 in peripheral blood can be used asimportant reference indicators in evaluating HAART.展开更多
A series of human tissue samples and cultured cell lines were formalin-fixed and paraffin-embedded. Specimen cylinder （1.2 -1.8ram） were punched by a modified bone marrow biopsy needle and arrayed on a recipient par...A series of human tissue samples and cultured cell lines were formalin-fixed and paraffin-embedded. Specimen cylinder （1.2 -1.8ram） were punched by a modified bone marrow biopsy needle and arrayed on a recipient paraffin block. Microscopic analysis on the sections from this tissue microarray （ TMA ） block demonstrated that the spots of tissues and cells were well preserved, and the cultured cell samples were successfully embedded from 5 × 104 to 2 × 105 in number. These TMA sections were also suitable for immunohistochemistry and RNA in situ hybridization.展开更多
In order to quantitate the bovine immunodeficiency virus line （BIVL） was established by transfecting baby hamster kidney （BIV） cells infection in vitro, a BIV indicator cell with reporter plasmids containing the f...In order to quantitate the bovine immunodeficiency virus line （BIVL） was established by transfecting baby hamster kidney （BIV） cells infection in vitro, a BIV indicator cell with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.展开更多
Objective: The aim of our study was to investigate the expression of Survivin in lung adenocarcinoma of Xuanwei and Kunming patients. Methods: Twenty-five specimens of Xuanwei patients and 28 specimens of Kunming pati...Objective: The aim of our study was to investigate the expression of Survivin in lung adenocarcinoma of Xuanwei and Kunming patients. Methods: Twenty-five specimens of Xuanwei patients and 28 specimens of Kunming patients were observed and analyzed. The Survivin expression was detected by immunohistochemistry. The results were quantitatively analyzed by image analysis system. Results: There were significant differences in Survivin expression (P < 0.01) between Xuanwei patients and Kunming patients. Conclusion: Survivin expression in lung adenocarcinoma of Xuanwei patients was significantly higher than that of Kunming patients. The pathogenesis of lung adenocarcinoma might be different between Xuanwei patients and Kunming patients. High Survivin expression might be one of the reasons to explain high incidence of lung cancer in Xuanwei.展开更多
The sera of 180 human samples were tested for the presence of antibodies against Borrelia burgdorferi using the ELISA （enzyme-linked immunosorbent assay） technique. Out of 180 sero-samples, 46 （25.55%） were positi...The sera of 180 human samples were tested for the presence of antibodies against Borrelia burgdorferi using the ELISA （enzyme-linked immunosorbent assay） technique. Out of 180 sero-samples, 46 （25.55%） were positive. Females of the age range 18-35 years had the highest rate of sero-positive samples 14 （38.88%）, while the highest percentage of sero-negative samples was found in males of the age range 50-80 years. The other sero-positive samples were： 6 （26.08%）, 6 （25%） and 3 （11.53%） in males of ages between 18-35, 35-50 and 50-80 years, respectively, and 11 （29.72%） and 6 （17.64%） in females in the age ranges 35-50 and 50-80 years, respectively. The mean concentration of Anti- B. burgdorferi antibody was higher （16.7 U/mL） when compared with mean concentration of normal value （5.5 U/mL）, P 〈 0.001.展开更多
The existing methods of detecting pesticide residue include gas chromatography, high performance liquid chromatography, gas chromatograph-mass, liquid chromatograph-mass, capillary electrophoresis, radioimmunoassay, b...The existing methods of detecting pesticide residue include gas chromatography, high performance liquid chromatography, gas chromatograph-mass, liquid chromatograph-mass, capillary electrophoresis, radioimmunoassay, biosensor and rapid detection on the spot. The paper analyzes the comparison of gas chromatography and liquid chromatogram detecting pesticide residue, for achieving the development tendency and the future goal of analyzing pesticide residue.展开更多
基金Society Commonweal Study of China (2001DIA10006)
文摘The sequence encoding an E2 main antigen glycoprotein of the C strain of classical swine fever virus (CSFV) was highly expressed in the host cell E. coli BL21–CodonPlus (DE3)–RIL using the pGEX-4T-1 expression vector and the soluble recombinant product was purified with Glutathione Sepharose TM4B by centrifugation. The soluble recombinant protein showed good immune reactions and was confirmed by Western blot using anti-CSFV-specific antibodies. Then an indirect ELISA with the purified E2 protein as the coating antigen was established to detect antibody against CSFV. The result revealed that the optimal concentration of coated antigen was 0.6 μg/well and the optimal dilution of serum was 1:80. The positive cut-off value of this ELISA assay was OD tested serum / OD negative serum≥2.1. The E2-ELISA method was evaluated by comparison with the indirect hemagglutination test (IHAT). When a total of 100 field serum samples were tested the sensitivity and specificity were 90.3% and 94.7% respectively. Specificity analysis showed that there were no cross-reactions between BVD serum and the purified E2 protein in the E2-ELISA.
基金Project supported by the National Natural Science Foundation of China (No. 30471155) the Agriculture Key Technologies R & D Program of Shanghai (No. (2003) 9-4), China
文摘A high affinity polyclonal antibody-based enzyme linked immunosorbent assay （ELISA） was developed for the quantification of zeranol in bovine urine. On the basis of urine matrix studies, the optimized dilution factors producing insignificant matrix interference were selected as 1：5 in pretreatment. In the improved ELISA, the linear response range was between 0.02 and 1 μg/ml, and the detection limit was 0.02 μg/ml for the assay. The overall recoveries and the coefficients of variation （CVs） were in the range of 82%-127% and 3.5%-8.8%, respectively. Thirty-six bovine urine samples spiked with zeranol （ranging from 0.2 to 10 μg/ml） were detected by the ELISA and liquid chromatography （LC） method, and good correlations were obtained between the two methods （R^2=0.9643）. We conclude that this improved ELISA is suitable tool for a mass zeranol screening and can be an altemative for the conventional LC method for zeranol in bovine urine.
基金Supported by the National Natural Science Foundation of China (30872287)
文摘Objective To investigate the main proteinases responsible for CD16b shedding under different stimulators. Methods HEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then overexpressed with a disintegrin and metalloproteinase 10 （ADAM10） or ADAM17, sup- pressed with short hairpin RNA of ADAM10 or ADAM I 7, and reconstituted with ADAM 10 or ADAM17, respectively. After each treatment, the cell line was stimulated with ionomycin or phorbol 12-myristate- 13-acetate （PMA） for 12 hours. The soluble CD 16b released from cell membrane was detected by immuno- precipition and immunoblot. Quantitation was then implemented to compare the amount of soluble CD 16b in cell supernatant after stimulation. Results HEK293 cell line stably expressing CD16b was successfully established. When CDI6b ex- pressing cell line was overexpressed with ADAM 10, shedding of CD 16b was increased after stimulation with ionomycin but not PMA; when the cell line overexpressed with ADAM I7, shedding of CDI6b was increased after stimulation with PMA but not ionomycin. Similarly, when ADAM10 was suppressed by short hairpin RNA, CD 16b shedding was decreased after stimulation with ionomycin; when ADAM 17 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with PMA. The shedding of CD16b was increased again when CD16b expressing cell line was reconstituted with ADAM10 and stimulated by ionomycin or reconstituted with ADAM 17 and stimulated by PMA. Conclusions Both ADAM10 and ADAM17 could shed CD16b, but they possess differed prefer- ences. ADAM10 is the main sheddase under stimulation of ionomycin, while ADAM17 is the main sheddase under stimulation of PMA.
基金Supported by Specialized Research Fund for the Doctoral Program of Higher Education of China (No. SRFDP 20100132110007)Shandong Provincial Natural Science Foundation, China (No. ZR2011DZ002)
文摘Overthelastseveraldecades,harmfulalgalblooms(HABs)havebecomeaseriousenvironmental problem in many parts of the world. A rapid and accurate detection process for HAB algae has yet to be developed. Heterosigma akashiwo is one of the most important HABs species in China. The objective of this study was to develop an immunologic technique that can rapidly and sensitively count H. akashiwo cells. Five HABs species (Alexandrium catenella, Thalassiosira sp., Cryptomonas sp., Alexandrium tamarense and Symbiodinium sp.,) were used in this study to evaluate the analysis process we developed. A polyclonal antibody with high titers against H. akashiwo was obtained by injecting H. akashiwo cells into rabbits. Immuno-magnetic beads (IMB) were produced via conjugated polyclonal antibodies with magnetic beads and applied to isolate and count H. akashiwo cells from the culture. Results show that 66.7%-91.6% of the cells were captured from unialgal culture by IMBs, and only 5.3%-12.5% of the four other HAB microalgae species were captured, indicating that the constructed IMBs combined specifically with the H. akashiwo cells. At the same time, flow cytometry (FCM) sorting was exploited to screen H. akashiwo cells after labeling with FITC conjugated polyclonal antibodies. Using the FCM technique, 91.7% of the targeted cells were sorted out from mixed microalgae samples in just a few minutes. These results indicate that both antibody-involved IMB and antibody-based FCM techniques are highly effective at detecting and quantifying HAB species. These techniques, especially immuno-magnetic separation, have low associated cost, and are fast and simple processes compared with other techniques currently in use.
文摘Objective: To observe the effectiveness of highly active antiret-rovirus therapy (HAART) on HIV/AIDS patients. Methods: Using HIV-1 quantitative methods and immu-nological function inspection, we monitored 4 HIV/AIDS pa-tients who were suffering from immunological deficiency andwere treated with HAART Results: The reproduction of HIV in all 4 patients was effi-ciently controlled at the 4th week of the treatment. The averageviral load decreased by 1.99 Log/ml (0.73-2.46 Log/ml). Thenumber of CD^+_4 and CD^+_8 showed a steady continuous increase4 to 12 weeks after the treatment, with an increase of 67.2%and 103.0% respectively. Correlative study among differentvariables after the treatment revealed that positive correlationexisted between the number of CD^+_4 and CD^+_3 as well as CD^+_8,while negative correlation existed between the number of CD^+_4and plasma viral load. Conclusion: HIV-1 quantitative method (plasma viral load)and the number of CD^+_4 in peripheral blood can be used asimportant reference indicators in evaluating HAART.
文摘A series of human tissue samples and cultured cell lines were formalin-fixed and paraffin-embedded. Specimen cylinder （1.2 -1.8ram） were punched by a modified bone marrow biopsy needle and arrayed on a recipient paraffin block. Microscopic analysis on the sections from this tissue microarray （ TMA ） block demonstrated that the spots of tissues and cells were well preserved, and the cultured cell samples were successfully embedded from 5 × 104 to 2 × 105 in number. These TMA sections were also suitable for immunohistochemistry and RNA in situ hybridization.
基金The General Foundation of Tianjin Science Committee for Applied Basic Research (08JCZDJC21000)Chinese Ministry of Education (30770081)
文摘In order to quantitate the bovine immunodeficiency virus line （BIVL） was established by transfecting baby hamster kidney （BIV） cells infection in vitro, a BIV indicator cell with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.
基金Supported by grants from Basic Project of Social Development and Science Practice Plan of Yunnan Provincial Department of Science (No. 2009ZC120M)Practice Fund of Yunnan Provincial Education Department (No. 09Y0173)
文摘Objective: The aim of our study was to investigate the expression of Survivin in lung adenocarcinoma of Xuanwei and Kunming patients. Methods: Twenty-five specimens of Xuanwei patients and 28 specimens of Kunming patients were observed and analyzed. The Survivin expression was detected by immunohistochemistry. The results were quantitatively analyzed by image analysis system. Results: There were significant differences in Survivin expression (P < 0.01) between Xuanwei patients and Kunming patients. Conclusion: Survivin expression in lung adenocarcinoma of Xuanwei patients was significantly higher than that of Kunming patients. The pathogenesis of lung adenocarcinoma might be different between Xuanwei patients and Kunming patients. High Survivin expression might be one of the reasons to explain high incidence of lung cancer in Xuanwei.
文摘The sera of 180 human samples were tested for the presence of antibodies against Borrelia burgdorferi using the ELISA （enzyme-linked immunosorbent assay） technique. Out of 180 sero-samples, 46 （25.55%） were positive. Females of the age range 18-35 years had the highest rate of sero-positive samples 14 （38.88%）, while the highest percentage of sero-negative samples was found in males of the age range 50-80 years. The other sero-positive samples were： 6 （26.08%）, 6 （25%） and 3 （11.53%） in males of ages between 18-35, 35-50 and 50-80 years, respectively, and 11 （29.72%） and 6 （17.64%） in females in the age ranges 35-50 and 50-80 years, respectively. The mean concentration of Anti- B. burgdorferi antibody was higher （16.7 U/mL） when compared with mean concentration of normal value （5.5 U/mL）, P 〈 0.001.
文摘The existing methods of detecting pesticide residue include gas chromatography, high performance liquid chromatography, gas chromatograph-mass, liquid chromatograph-mass, capillary electrophoresis, radioimmunoassay, biosensor and rapid detection on the spot. The paper analyzes the comparison of gas chromatography and liquid chromatogram detecting pesticide residue, for achieving the development tendency and the future goal of analyzing pesticide residue.
