ObjectiveTo develop and verify a double antibody sandwich ELISA for the quantitative detection of varicellazoster virus(VZV)glycoprotein E(gE).MethodsHybridoma cell lines secreting antibody against VZV-gE stably were screened by mouse hybridoma fusion technology,using VZV whole virus antigen as immunogen.The antibody titer in mouse ascites was detected by indirect ELISA.After purified by Hi Trap2=0.995 6,P=0.000 1)。结论 建立的双抗体夹心ELISA法具有良好的准确性、精密性和特异性，可用于VZV疫苗中g E抗原含量的快速检测。 ObjectiveTo develop and verify a double antibody sandwich ELISA for the quantitative detection of varicellazoster virus(VZV)glycoprotein E(gE).MethodsHybridoma cell lines secreting antibody against VZV-gE stably were screened by mouse hybridoma fusion technology,using VZV whole virus antigen as immunogen.The antibody titer in mouse ascites was detected by indirect ELISA.After purified by Hi Trap(TM)Mabselect(TM)Mabselect(TM)Su Re and Hi Trap(TM)Su Re and Hi Trap(TM)Desalting,monoclonal antibodies(m Abs)were analyzed for the purity by 12%SDS-PAGE,detected for the specificity by Western blot,and identified for the subtype by mouse monoclonal antibody typing kit.Capture antibody and enzyme-labeled antibody were screened by epitope superposition test,which were determined for the working concentrations by chessboard titration(capture antibody concentrations were 0.25,0.5,1,2,5 and 10μg/m L,enzyme-labeled antibody dilutions were 1∶500,1∶1 000,1∶2 000,1∶5 000 and 1∶10 000),and then a double antibody sandwich ELISA(DAS-ELISA)was developed for the detection of VZV-gE content.In addition,the linear range,accuracy,precision and specificity of the method were verified.The gE content in the supernatant of 3 batches of CHO-VZV-gE cells cultured in 7 L bioreactor for 1～14 d were detected by the developed method.ResultsFour positive hybridoma cell lines secreting specific antibodies against VZV-gE stably were obtained and named as m Ab-B2,m Ab-11,K9C7 and K9F4.The antibodies in mouse ascites showed titers of10(TM)Desalting,monoclonal antibodies(m Abs)were analyzed for the purity by 12%SDS-PAGE,detected for the specificity by Western blot,and identified for the subtype by mouse monoclonal antibody typing kit.Capture antibody and enzyme-labeled antibody were screened by epitope superposition test,which were determined for the working concentrations by chessboard titration(capture antibody concentrations were 0.25,0.5,1,2,5 and 10μg/m L,enzyme-labeled antibody dilutions were 1∶500,1∶1 000,1∶2 000,1∶5 000 and 1∶10 000),and then a double antibody sandwich ELISA(DAS-ELISA)was developed for the detection of VZV-gE content.In addition,the linear range,accuracy,precision and specificity of the method were verified.The gE content in the supernatant of 3 batches of CHO-VZV-gE cells cultured in 7 L bioreactor for 1～14 d were detected by the developed method.ResultsFour positive hybridoma cell lines secreting specific antibodies against VZV-gE stably were obtained and named as m Ab-B2,m Ab-11,K9C7 and K9F4.The antibodies in mouse ascites showed titers of106～106～107with purities of about 97%after purification,which all specifically bound to VZV whole virus protein with light chains ofκchain and heavy chains of Ig G_(2b),Ig G_1,Ig G_(2b)and Ig G_(2a)respectively.m Ab-B2 was determined as capture antibody and HPR-labeled m Ab-11 as enzyme-labeled antibody with the optimum working concentrations of 1.5μg/m L and 1∶5 000respectively.The internal reference concentration of gE antigen was in the range of 1.95～1 000 ng/m L,which showed a good linear relationship with A_(450).The four-parameter equation was Y=(0.15-3.99)/[1+(X/67.4)7with purities of about 97%after purification,which all specifically bound to VZV whole virus protein with light chains ofκchain and heavy chains of Ig G_(2b),Ig G_1,Ig G_(2b)and Ig G_(2a)respectively.m Ab-B2 was determined as capture antibody and HPR-labeled m Ab-11 as enzyme-labeled antibody with the optimum working concentrations of 1.5μg/m L and 1∶5 000respectively.The internal reference concentration of gE antigen was in the range of 1.95～1 000 ng/m L,which showed a good linear relationship with A_(450).The four-parameter equation was Y=(0.15-3.99)/[1+(X/67.4)(1.49)]+3.99,and R(1.49)]+3.99,and R2value was 0.999.The recoveries of accuracy verification were 94.9%～114.0%;The coefficients of variation(CVs)of precision verification were all less than 15%.Except CHO-VZV-gE cell culture supernatant,attenuated live varicella vaccine and attenuated live herpes zoster vaccine,the A_(450)of other samples were all less than 0.15 with no cross reaction.The content of gE in the supernatant of three batches CHO-VZV-gE cells increased gradually with the culture time,and was positively related with culture time within 14 days(R2value was 0.999.The recoveries of accuracy verification were 94.9%～114.0%;The coefficients of variation(CVs)of precision verification were all less than 15%.Except CHO-VZV-gE cell culture supernatant,attenuated live varicella vaccine and attenuated live herpes zoster vaccine,the A_(450)of other samples were all less than 0.15 with no cross reaction.The content of gE in the supernatant of three batches CHO-VZV-gE cells increased gradually with the culture time,and was positively related with culture time within 14 days(R2=0.995 6,P=0.000 1).ConclusionThe developed double antibody sandwich ELISA had good accuracy,precision and specificity,which might be used for rapid detection of gE antigen content in VZV vaccine.

Objective To compare the immunogenicity of varicella-zoster virus(VZV)Oka-7S strain and VZV Oka strain.Methods Female BALB/c mice were subcutaneously immunized with VZV Oka-7S and Oka strains at high,medium and low doses(1 000,250 and 62.5 PFU/dose)for total two doses at an interval of 14 d,separately,with 12 mice in each group.The VZV-specific humoral immunity was evaluated based on fluorescent antibody to membrane antigen assay(FAMA),and the VZV-specific cellular immunity was evaluated by enzyme-linked immunospot assay(ELISPOT).Results Both VZV Oka-7S and VZV Oka strains induced specific humoral and cellular immune responses,and showed considerable immune effects;The level of anti-VZV specific IgG in serum of mice in Oka-7S-2.2 group increased significantly with the increase of immunization times and immunization doses.Conclusion The VZV Oka-7S strain had comparative immunogenicity with VZV Oka strain.

OBJECTIVE:To establish a RP-HPLC method to determine the content of ofloxacin in compound ofloxacin eye drops METHODS:Using Shim-pack ODS as fixed phase,a mixture to citric acid(0 05mol/L)-acetonitrile-ammonium acetate(0 5mol/L)-0 3% phosphoric acid(78∶22∶1∶3)as mobile phase,and detecting wavelength:293nm,flow rate:1 0ml/min RESULTS:The linear range of ofloxacin was 8 12?g～40 6?g(r=0 9 996,n=5) The average recovery was 99 29% and RSD(n=6)was 0 57% CONCLUSION:The method is rapid,simple and accurate

Objective: To study comparatively HPLC-FPS of three kinds of Radix Acanthopanacis Senticosi from different sources. Methods: HPLC fingerprint analysis method of Radix Acanthopanacis Senticosi from Ningan was developed, and the HPlC-FPS of three samples were established. Results: The methodological evaluation showed that this method had a good repeatability, and the ratio of common peaks' area of different samples were different. Conclusion: This method can be used to differentiate Radix Acanthopanacis Senticosi from different sources conveniently.

[Objective] To evaluate the biomechanical stability of lumbar intact specimen,anterior lumbar interbody fixation(ALIF)specimen,ALIF specimen fixed with pedicle screw and ALIF specimen fixed with translaminar facets screw.[Method]Each of eight adult fresh cadaver specimen of lumbarsacral segmcnts was analyzed in four states(groups):intact,L5S1 ALIF,L5S1 ALIF fixed with pedicle screw or fixed with translaminar facets screw,test and compare its range of motion(ROM)in flexion,extension,lateral bending and axial rotation.[Result]The ROM of ALIF specimen in extension was larger than that of intact specimen(P

Objectives To help nursing students to adapt to the environment of the job interviews by simulation interview, train the students'psychological quality and the adaptability, give the necessary guidance for seeking job after graduation. Method Five interview groups were composed by 15 nurse executives and interpersonal communication teachers from 4 hospitals, after the simulation interview, the students were surveyed using the self-desigued questionnaire. Results There are 10 items having the most significant influence, 10 items having more obvious influence the last 7items.Conclusion It can improve all aspects'ability of the students effectively through carrying out the simulation interview pertinence, and can help the students find job successfully.

Objective To explore the concentration of required residual available chlorine dioxide(ClO 2 )and optimun contact time for ClO 2 disinfection in order to ensure the microbiological indexes of waste water of hospital disinfected by ClO 2 qualified.Meth ods The waste water samples collected from hospital,to which were aritificially added with predetermined con-centrations of bacteria,were disinfected by various concentrations of ClO 2 (2?4?6?8?10?12mg /L)with various contact time (5?10?30?60?90min).After disinfection,the concentration of residual ClO 2 and the contents of microorganism in waste water samples were determined.Re sults After30-min disinfection by10.0mg /L ClO 2 ,the waste water sample originally containing1.52?10 2 /L E.coli showed a bactericidal rate of99.99%and a concentration of residual ClO 2 at2.5mg /L.After10-min disin-fection by10.0mg /L ClO 2 ,the waste water samples originally containing1.12?10 10 /L Salmonella showed a bactericidal rate of100%and a concentration of residual ClO 2 at2mg /L.After5-min disinfection by10.0mg /L ClO 2 ,the waste water samples o-riginally containing M.tu berculosis at concentration of++++showed a negative result and a concentration of residual ClO 2 at3.5mg/L.Conclusion After the primary treatment,ClO 2 disinfection with contact time of30minutes and a residual concentration of ClO 2 at2.5mg /L could ensure the germicidal effect for E.coli,Salmonella and M.tu berculosis in waste water of hospital qualified.

Objective:To explore the apoptosis inducing effect of matrine on C6 glioma cells and the related mechanisms.Methods:MTT assay was used to examine the inhibition of C6 glioma cell line by matrine at various concentrations and the IC50 was calculated.Inverted microscope and TEM(transmission electron microscope) were employed to observe the morphological alterations of C6 glioma cells after exposure to matrine;FCM(Flow cytometry) was used to detect the apoptosis rate of C6 glioma cells;and real-time PCR was used to examine the differential expression of related genes.ICC and Western blotting was used to detect the expression of caspase-3.Results:MTT showed that the cell inhibition effect of matrine increased with its concentration(0.1-1.0 mg/ml)(P

Objective To study the effects and the mechanism of astragalus menbranaceus (AM) on neonatal lymphocyte apoptosis in cord blood. Methods Cord blood mononuclear cells (CBMC) of 30 full-term neonates were cultured with pure phytohemagglutinin (PHA), PHA combined with IL-6 (PHA + IL-6) or AM (PHA + AM) respectively for 48 hours. The apoptosis index (AI) of lymphocytes of CBMC after cultivation were determined by acridine orange-ethidium bromide dying method. The positive expression of CD38 antigen and CD25 antigen were detected by indirect immunofluorescence procedure. The levels of IL-6 in the supernatants of CBMC were detected by ELISA . Results (1) The AI of the PHA + AM group (16. 5?3. 5)% and PHA+IL-6 group (16. 9?4. 0)% was lower than that of the pure PHA group (32.4?2.8)% (P0. 05). (2) The positive expression rates of CD38 of the PHA+AM group and the PHA+IL-6 group were lower than those of the pure PHA group (P0. 05). The positive expression rates of CD25 of the PHA + AM group and the PHA + IL-6 group were higher than those of the pure PHA group (P0. 05). The positive expression of CD38 of CBMC had positive correlation, but CD25 had negative correlation with the AI of CBMC(r=0. 68, -0. 65,P

Objective To establish a quality standard for Aiweixin oral liquid. Methods TLC was applied to the identification of Caryophylli Flos and Crocin in Aiweixin oral liquid. Eugenol was analyzed by GC. Results TLC of Caryophylli Flos and Crocin had distinct separation of characteristic spots and there was no interference in negative comparison. The linear response ranges of Eugenol were between 0.052 17-2.086 8μg (r=0.999 9). The samples were steady within 24 h. The average recovery of eugenol was 95.7%, RSD=1.4% (n=6). Conclusion The method is simple, accurate and reliable, and can be used for the quality control of Aiweixin oral liquid.