• Preparation of refined and depolymerized capsular polysaccharide of Streptococcus pneumoniae

    LIU Yanli;CAO Xin;ZHAO Yi;GAO Zhixin;ZHANG Dongxu;LI Xuyang;CAI Youyang;LIU Jiankai;LI Yuelong;Beijing Minhai Biotechnology Co.,Ltd.;

    Objective To prepare high-quality refined and depolymerized capsular polysaccharide(CPS) of Streptococcus pneumoniae for preparation of polysaccharide vaccine and polysaccharide conjugate vaccine against Streptococcus pneumoniae.Methods Refined CPS of type 6A was prepared by acid precipitation method from the inactivated fermentation broth of type 6A Streptococcus pneumoniae,and the final concentration of CaCl_2(80,160,240,320 mmol/L) and pH(2.5,3.0,3.5,4.0) were optimized.Three batches of type 6A refined CPSs were prepared by the optimized method,and the applicability of the method to polysaccharides of 5,7F,10A and 19F types was verified.After removing impurities from the inactivated broth of type 6 A Streptococcus pneumoniae by acid precipitation,the type 6 A depolymerized CPS was prepared by trifluoroacetic acid method,and the reaction temperature(8,25 and 40℃),the final concentration of trifluoroacetic acid(0.25,0.50,1.00 mol/L) and the reaction time(1,3,6 h) were optimized.Three batches of depolymerized CPSs were prepared by the optimized method,and compared with the high-pressure homogenization method,and then the applicability of trifluoroacetic acid method to depolymerized CPSs of 5,10A and 19F types was verified.The quality of refined and depolymerized CPSs was tested according to the methods specified in Chinese Pharmacopoeia(Vol Ⅲ,2020 edition).Results The optimum final concentration of CaCl_2 and pH of acid precipitation method were 160 mmol/L and 3.0 respectively.The prepared type 6A,5,7F,10A and 19F refined CPSs were all qualified.The optimum reaction temperature,reaction time and final concentration of trifluoroacetic acid were 25℃,3 h and 0.50 mol/L for trifluoroacetic acid method.The prepared type 6A,5,10A and 19F depolymerized CPSs were all qualified.Conclusion The refined and depolymerized CPSs of Streptococcus pneumoniae prepared in this study meet the requirements of Chinese Pharmacopoeia(Vol Ⅲ,2020 edition),and the preparation process is suitable for the preparation of various types of polysaccharides.

    2025 06 v.38 [Abstract][OnlineView][Download 858K]

  • Identification of stem cell characteristics of human diploid cells 2BS

    WANG Yao;FANG Jiqing;YUAN Ziwei;LI Yaoling;WEI Xiajie;SUN Yunxia;YANG Ying;RAO Chunming;JOINN Drug Quality Research and Testing(Beijing)Co.,Ltd.;

    Objective To identify the stem cell characteristics of human diploid cells 2BS and evaluate the potential as biological function positive cells for quality detection of stem cells.Methods Flow cytometry was applied to detect the expression levels of stem cell surface markers in 2BS cells.The staining with alizarin red S,oil red O and alcian blue was used to evaluate the ability of osteogenic,adipogenic and chondrogenic differentiation,respectively.Co-culture with lymphocytes was used to observe the effects on lymphocyte proliferation.Results Human diploid cells 2BS expressed the surface markers CD73,CD 105 and CD90 of stem cells,and possessed the potential of osteogenic differentiation and inhibiting lymphocyte proliferation.Conclusion 2BS cells have several properties of stem cells and could be the positive control for surface markers detection of stem cells by flow cytometry.

    2025 06 v.38 [Abstract][OnlineView][Download 1064K]

  • Immunogenicity study of Mycobacterium massiliense bacterial protein

    GU Yujie;WANG Ruibai;JIANG Jingwei;ZHAO Xiuqin;LIU Haican;WAN Kanglin;XU Da;National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention;

    Objective To preliminarily analyze the immunogenicity of Mycobacterium massiliense(M. massiliense) whole-cell lysate proteins in order to provide a basis for the application of M. massiliense-based immunology.Methods The whole-cell protein of M. massiliense was extracted by ultrasonic disruption and used to immunize BALB/c mice subcutaneously,either alone or mixed with DP adjuvant [a mixture of dimethyl dioctadecyl ammonium bromide(DDA) and polyinosinic-polycytidylic acid(PolyI:C)],and the control mice were injected with an equal amount of sterile PBS for a total of three times with an interval of 14 d,with eight mice for each group.Two weeks after the final immunization,the blood and spleen samples were collected and the antibody levels of IgG,IgG1,and IgG2a in serum were analyzed by ELISA.The levels of IL-2,IFN-γ,TNF-α,IL-12,granulocyte-macrophage colony stimulating factor(GM-CSF),IL-17A,IL-4,IL-6 and IL-10 secreted by mouse sensitized spleen lymphocytes were detected by Luminex technique,and the effect on TCM(central memory T cells)/IFN-γ~+cells in mouse spleen lymphocytes was measured by flow cytometry.A growth inhibition experiment was performed to evaluate the effect of bacterial protein immunization on the replication of Mycobacterium tuberculosis(M. tuberculosis) H37Rv in mouse spleen lymphocytes.Results IgG,IgG1 and IgG2a serum antibodies in mice were induced by both the bacterial protein and protein-adjuvant complexes.Compared with the control group,IL-12 secretion levels by spleen lymphocytes of mice immunized with bacterial protein and protein-adjuvant complexes had no significant change(0.60≤t≤2.41,each P>0.05).The levels of IFN-γ,TNF-α,IL-2,IL-4,IL-6,IL-10,GM-CSF and IL-17 secreted by spleen lymphocytes of mice immunized with protein-adjuvant complexes increased significantly(3.77≤t≤23.46,each P <0.05) after bacterial protein and purified protein derivative(PPD) stimulation.In mice immunized with bacterial protein,stimulation with bacterial protein and PPD resulted in a significant rise in IL-2 and IL-4 secretion(5.36≤t≤13.26,each P <0.05),whereas TNF-α secretion increased significantly only after PPD stimulation(t=4.15,P <0.05).IL-10 secretion decreased significantly after stimulation(t=3.04 and 4.10,respectively,each P <0.05),and IL-6 secretion increased significantly only after bacterial protein stimulation(t=8.34,P <0.05).Compared with the control group,the proportion of CD4~+TCM/IFN-γ~+ and CD8~+TCM/IFN-γ~+ cells in spleen lymphocytes of mice immunized with protein-adjuvant complexes increased significantly after being stimulated by PPD and bacterial protein(2.63≤t≤5.96,each P<0.05).Compared with the control group,there was no significant difference in the proliferation of M. tuberculosis H37Rv in the spleen lymphocytes of mice immunized with bacterial protein and protein-adjuvant complexes(t=2.29 and 1.10,respectively,each P> 0.05).Conclusion The bacterial protein of M. massiliense has good immunogenicity,which can induce the tendency of Th2-type immune response,and combined with adjuvant to enhance cellular immune response,it can improve the level of induced cellular immune response.

    2025 06 v.38 [Abstract][OnlineView][Download 984K]

  • Establishment of national standard for enterovirus 71 nucleic acid detection kit

    HAO Xiaotian;MA Ning;LI Manyu;ZHOU Haiwei;Division Ⅰ of Diagnostic for Infectious Disease,Institute for In Vitro Diagnostics Control,National Institutes for Food and Drug Control;

    Objective To establish the first national standard for enterovirus 71(EV71) nucleic acid detection kit for quality evaluation of related kits.Methods EV71 virus isolation culture was collected,diluted and lyophilized after inactivation to prepare the national standard for EV71 nucleic acid detection kit.Eleven laboratories were selected to evaluate the standard by digital PCR.The concentration of standard was determined according to the evaluation results,and the homogeneity,stability and applicability were analyzed.Results It was determined that the concentration of the national standard for EV71nucleic acid detection kit was(6.7±0.3) log10U/mL(k=2).The homogeneity of the standard met the requirements.The stability was not affected when the standard was stored at 4℃ for 7 days or at room temperature(25℃) for 3 days.The applicability study results confirmed that the standard could accurately evaluate the limit of detection(LOD) and other performances of the detection kit.Conclusion The first national standard for EV71 nucleic acid detection kit was established,which provides physical standard and basis for quality control and evaluation of performance indicators such as LOD of related kits.

    2025 06 v.38 [Abstract][OnlineView][Download 829K]

  • Preparation of antibody-drug conjugate targeting human carcinoembryonic antigen cell adhesion molecule 5 and its biological activity

    CHEN Zhanhao;ZHANG Kunming;ZHANG Quan;WU Lina;LU Jin;LIANG Hongyuan;QU Aidong;Shanghai Institute of Biological Products Co.,Ltd.;

    Objective To prepare monoclonal antibody against human carcinoembryonic antigen cell adhesion molecule 5(CEACAM5) and its antibody-drug conjugate(ADC),and evaluate the biological activity.Methods Female BALB/c mice were immunized with human CEACAM5 and hFc fusion protein,and hybridomas were prepared by electrofusion technology.Positive hybridoma cell lines were screened by ELISA.The variable region sequences of murine antibodies were obtained by RACE PCR and chimeric antibodies were constructed.The characteristics of chimeric antibodies were evaluated by ELISA and flow cytometry,and the antibody-dependent cell-mediated cytotoxicity(ADCC) activity of chimeric antibodies was detected by reporter gene method.ADCs were prepared by glycosylation conjugation technology and their activity was evaluated.Results A hybridoma cell line with specific binding activity to CERCAM5 was screened using hybridoma technology and named mAb186.The variable region sequence was obtained and chimeric antibody ch186 was constructed,which specifically bound to the B3 domain of human CEACAM5 with no ADCC activity against target cells.The ADC of ch186 conjugated with small molecules of monomethyl auristatin E(MMAE) was prepared,which showed a specific killing effect on target cells and had a certain dose-response effect.Conclusion A monoclonal antibody specifically binding to human CEACAM5 was successfully prepared,and the binding activity and cell-killing activity of chimeric antibody and ADC drug were evaluated.

    2025 06 v.38 [Abstract][OnlineView][Download 1042K]

  • Expression and purification of growth differentiation factor 5 in E.coli and its effect on osteoarthritis

    WANG Xuan;FENG Yongjun;CHEN Xuanye;LAI Zhaoli;TIAN Haishan;School of Pharmacy,Wenzhou Medical University;

    Objective To establish a protein preparation process for growth differentiation factor 5(GDF5),and explore its role in cartilage injury through in vivo and in vitro experiments.Methods The gdf5 gene was ligated with the pCZN1 vector to construct the recombinant plasmid pCZN1-hGDF5,which was then transformed into E.coli BL21(DE3) pLysS host bacteria.Single colonies were picked to screen for high-expression strains.The strains were induced by IPTG,and the protein expression form was detected by SDS-PAGE.A method for inclusion body washing,denaturation and dilution renaturation was established,and the protein was purified by Ni affinity column chromatography and DEAE ion exchange column chromatography.ATDC5 cells were used as the detection cell line,and the biological activity of rhGDF5 protein in promoting proliferation was determined by MTT method.A model of cell injury induced by interleukin-1β(IL-1β) was established.The expression of cartilage-related genes and proteins was detected by RT-qPCR and Western blot,and the effect of rhGDF5protein on chondrocyte differentiation was analyzed by Alcian blue staining and alkaline phosphatase(ALP) activity determination.Poloxamer 407(P407) and 188(P188) thermo sensitive gels were prepared by cold dissolution method,and detected for their rheological properties by a rheometer.A male C57BL/6J mouse model of osteoarthritis(OA) induced by destabilization of medial meniscus(DMM) was established.The mice were administered by intra-articular injection once a week for four consecutive weeks.The mice were sacrificed and their knee joint tissues were collected.After decalcification,paraffin embedding and sectioning,they were stained with safranin O and fast green and detected by immunohistochemistry,and the OARSI score was determined.Results The results of double enzyme digestion(Nco Ⅰ/BamH Ⅰ) showed that the recombinant plasmid pCZN1-hGDF5 was correctly constructed.The protein expression level of the selected rhGDF5 high-expression strain accounted for 35% of the total protein.The expressed protein mainly existed in the form of inclusion bodies.After refolding and column chromatography,an rhGDF5 homodimer protein with a relative molecular mass of approximately 25 000and an electrophoretic purity of over 95% was obtained.The rhGDF5 protein exhibited a good proliferation-promoting effect on ATDC5 cells within the dose range of 25 to 800 ng/mL,and no cytotoxicity was observed at high doses.The rhGDF5protein could effectively promote the increase in chondrocyte number and proteoglycan secretion,and significantly inhibit the expression of ALP in ATDC5 cells.The results of RT-qPCR and Western blot demonstrated that compared with the IL-1βgroup,the administration of rhGDF5 protein could effectively reverse the inhibition of collagen type 2(COL-2) and SRY-box transcription factor 9(SOX9)(COL-2:t=4.899 and 9.799,respectively,each P <0.05;SOX9:t=4.950 and 10.73,respectively,each P <0.05),and significantly inhibit the expression of matrix metalloproteinase 13(MMP13)(t=6.500 and23.51,respectively,each P <0.05).The poloxamer thermosensitive hydrogel containing 22% P407 and 5% P188 with rhGDF5 protein prepared by cold dissolution method showed the gelation temperature of(30.06±0.16)℃ and the gelation time of 92.67 s.The results of the DMM mouse OA model experiment showed that compared with the control group(0 ng/mL),the injection of rhGDF5 protein-loaded thermo sensitive hydrogel into the joint cavity effectively repaired the damaged joint,with the high-dose group having the best repair effect,with an OARSI score of 1.The medium-dose group had an OARSI score of 1.67,while the low-dose group had no significant effect,with an OARSI score of 5.33.The immunohistochemical results also showed that compared with the control group,the treatment with rhGDF5 protein increased the distribution of COL-2.Conclusion A simple and efficient preparation process for protein renaturation,dimerization promotion and purification of rhGDF5 inclusion body was established,and its effective effect on cartilage was proved through in vivo and in vitro experiments,which provides technical support for further development of OA treatment products.

    2025 06 v.38 [Abstract][OnlineView][Download 1123K]

  • Epidemiological analysis of breakthrough cases in varicella outbreak during 2007-2023 in Fengxian District of Shanghai

    LI Ruiping;HU Yaqiang ;WAN Kuan;GU Qinmei ;WU Zhengqiang ;WU Xuehong ;SHEN Zhiying;Fengxian District Center for Disease Control and Prevention;

    Objective To analyze the epidemiological characteristics of breakthrough varicella cases in Fengxian District of Shanghai from 2007 to 2023,so as to provide a reference for the effective prevention and control of varicella epidemic.Methods The reported cases of varicella in Fengxian District of Shanghai from 2007 to 2023 were collected by China Information System for Diseases Control and Prevention.The immunization history information of varicella cases was obtained from Shanghai Immunization Program Information System and analyzed by descriptive epidemiological methods.Results A total of 2 175 breakthrough varicella cases were reported in Fengxian District of Shanghai from 2007 to 2023.The proportion of breakthrough cases in each year was from 0.64% to 36.29% with statistically significant difference(χ~2=1 144.60,P <0.01).The varicella breakthrough cases were mainly concentrated in the 3-11 age group,and the primary cases were concentrated in the 5-11 and 15-20 age groups,with breakthrough cases skewed towards younger ages.The varicella breakthrough cases were predominantly preschool children and lower-grade students,while primary cases were more common among lower-grade students and university students,displaying a statistically significant difference(χ~2=242.89,P <0.01).The incidence of varicella showed a peak during the winter season,and the highest incidence of varicella breakthrough cases occurred in 3-7 years after vaccination.Since the adjustment of varicella vaccination program in 2017,the incidence of varicella among children aged 1-6 years showed a downward trend(R=-0.009,P=0.000).Conclusion From 2007 to 2023,the proportion of varicella breakthrough cases in Fengxian District of Shanghai showed a consistent increasing trend.The continuous monitoring of varicella and publicity should be strengthened in schools and kindergartens.It is of substantial significance to strengthen immunization three years after the first dose of vaccine.The adjustment of varicella immunization program has achieved remarkable results.

    2025 06 v.38 [Abstract][OnlineView][Download 872K]

  • Establishment and verification of a quantitative detection ELISA method for IgG antibodies against Norovirus GⅠ.1 and GⅡ.4 in human serum

    HAN Zibo;LEI Zehua;YUAN Runyu ;SUN Zhenlu ;YANG Sensen;TANG Fang;ZHANG Hao;DU Lifang;ZHANG Jing;LI Qiming;LIANG Yu;The Sixth Laboratory,National Vaccine and Serum Institute;

    Objective To establish and verify an ELISA method for quantitative detection of human serum IgG antibodies against Norovirus(NoV) GⅠ.1 and GⅡ.4,so as to provide a more accurate assay for antibody detection in the immunogenicity evaluation and seroepidemiological study of NoV vaccines.Methods High-titer human sera positive for IgG antibodies against NoV GⅠ.1 and GⅡ.4 were mixed,aliquoted,and lyophilized to prepare laboratory reference serum.The EC_(50)values of the reference serum were calibrated by fitting the results of multiple batches of ELISA tests to a four-parameter logistic model.Then a quantitative ELISA method for detection of NoV GⅠ.1 and GⅡ.4 IgG antibodies in human serum was developed with the calibrated laboratory reference serum as working standard.The linearity,determination range,precision and accuracy of the method were verified.The human sera positive and negative for GⅠ.1 and GⅡ.4 IgG antibodies were used as test samples,and based on the specificity and sensitivity of the detection results,the cut-off values used to determine the positive results were determined by using the receiver operating characteristic curve(ROC curve) analysis.Results The calibrated titers of the laboratory reference serum for GⅠ.1 and GⅡ.4 IgG antibodies were 434.2 and 456.5,respectively.A four-parameter logistic-based ELISA method was established for quantifying IgG antibodies against NoV GⅠ.1 and GⅡ.4.The method demonstrated linearity with R2 values greater than 0.99 across nine experimental batches,with the measurement ranges of GⅠ.1 IgG 2.714-0.042 and GⅡ.4 IgG 5.706-0.045.The relative standard deviation(RSDs) of reproducibility tests were not more than 10%,and the RSDs of intermediate precision tests were not more than 25%.The recovery rates from spiked tests ranged from 75% to 125%.The cut-off value of GⅠ.1 IgG was 6.705 with specificity of 96.6% and sensitivity of 96.3%,and that of GⅡ.4 IgG was 12.70 with both specificity and sensitivity of 100%.Conclusion By preparing and calibrating laboratory positive sera for NoV GⅠ.1 and GⅡ.4 IgG antibodies,a quantitative ELISA method based on a fourparameter logistic standard curve for human IgG antibodies against NoV was established,and the cut-off values for GⅠ.1 and GⅡ.4 IgG results were preliminarily determined.The method has a wide determination range with good linearity,precision,accuracy,specificity and sensitivity.

    2025 06 v.38 [Abstract][OnlineView][Download 893K]

  • Development and verification of a reversed-phase high-performance liquid chromatography for determination of adipic acid dihydrazide residues in typhoid Vi polysaccharide-protein conjugate vaccine bulk

    REN Zhenyun;ZHOU Haifei;LI Zhengyu;SU Chunyang;LI Huiqing;REN Haofeng;LIU Yueping;TAN Xiaomei;First Department of Research,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research;

    Objective To develop a reversed-phase high-performance liquid chromatography(RP-HPLC) method for the determination of adipic acid dihydrazide(ADH) residues in the bulk of typhoid Vi polysaccharide-protein conjugate vaccine,and to optimize,verify and preliminarily apply the method,so as to use it for the determination of ADH residues in typhoid Vi polysaccharide-protein conjugate vaccine bulk or other conjugate vaccines.Methods RP-HPLC was used to detect ADH residues in the bulk of typhoid Vi polysaccharide-protein conjugate vaccine.The detection wavelength(full wavelength),mobile phase(10 mmol/L PBS(pH 7.0)+10% acetonitrile,10 mmol/L PBS(pH 7.0)+0.8% sodium chloride solution+10%methyl alcohol+5 mmol/L ammonium acetate solution) and flow rate(0.8 and 1.0 mL/min) were optimized.The method was then verified for the specificity,linear range,accuracy and precision,and determined for the limit of detection(LOD) and limit of quantitation(LOQ).The ADH residues in three batches of typhus Vi polysaccharide-protein conjugate vaccine bulks were detected by the established method.Results The optimum detection wavelength was determined to be 202 nm,the mobile phase was 10 mmol/L PBS(pH 7.0)+0.8% sodium chloride solution+10% methyl alcohol+5 mmol/L ammonium acetate solution,and the flow rate was 1.0 mL/min.This method could specifically determine ADH residues in conjugate bulk,and other components in conjugate vaccine bulk exhibited no interference to the detection of ADH peak.At the range of 2-10 μg/mL,ADH concentration in the reference solution showed a good linear correlation with the peak area,with the linear regression equation of y=30 617 x+1 296.5,R2=0.999 7.The spiked recovery rates of ADH in the bulks of typhoid Vi polysaccharide-protein conjugate vaccines were 91.70%-106.00%.The RSDs of precision verification were less than 8%.The LOD and LOQ were 0.2 and 0.5 μg/mL,respectively.ADH residues were not detected in the bulks of three batches of typhoid Vi polysaccharide-protein conjugate vaccines.Conclusion The developed method has good specificity,precision and accuracy with convenience and rapidity,which can be used for the determination of ADH residues in the bulk of typhoid Vi polysaccharide-protein conjugate vaccine.

    2025 06 v.38 [Abstract][OnlineView][Download 839K]

  • Effects of hemolysis, chylemia and repeated freeze-thawing of human serum immunized with pneumococcal vaccine on ELISA clinical evaluation method

    SHI Gang;LU Xu;GUO Lina;LIU Wenbin;MAO Qiqi;LI Hong;Division of Bacterial Polysaccharides and Glycoconjugated Vaccine,National Institutes for Food and Drug Control,Key Laboratory of National Health Commission of PRC for Research on Quality and Standardization of Biotech Products;

    Objective To confirm the effects of hemolysis,chylemia and repeated freeze-thawing of human serum immunized with pneumococcal vaccine on ELISA for the detection of specific IgG antibody against pneumococcal capsular polysaccharide,and to promote the continuous development and improvement of the evaluation methods for clinical serum IgG antibody content of pneumococcal vaccine.Methods The same batch of reconstituted laboratory internal quality control serum 09CS was aliquoted into different volumes and subjected to 1 to 12 freeze-thaw cycles.The IgG antibodies of 24 types were detected respectively with the serum after single freeze-thaw and the repeated freeze-thaw serum.The ELISA test for detecting the content of IgG antibodies specific to pneumococcal capsular polysaccharides was carried out,and the test results were systematically analyzed.The laboratory internal quality control serum 09CS was respectively mixed with hemolyzed and chylous serum after immunization in different volume fractions to prepare hemolyzed and chylous serum,and the IgG antibodies of 24 types were detected respectively.The test was repeated three times,and the recovery rate was calculated.Results The repeated freeze-thaw serum and single freeze-thaw serum were detected for 24 types respectively,and the detection results showed good linear relationship with R~2> 0.99.The detection results were analyzed by Lin's concordance correlation coefficient(r_c),and the r_c was 0.992 3(95% CI:0.990 2-0.993 9),indicating that the test results of single freeze-thaw serum and repeated freeze-thaw serum were highly consistent.The accuracy of serum recovery rate was74.65% for hemolyzed serum and 70.48% for chylous serum,both in line with the acceptance range of accuracy±40%.Conclusion During the ELISA test for the specific IgG antibody content against pneumococcal capsular polysaccharide,hemolyzed and chylous serum and repeated freeze-thawing of serum for 12 times have no effect on the detection,which can satisfy the detection requirements for clinical serum antibody evaluation of pneumococcal vaccine.

    2025 06 v.38 [Abstract][OnlineView][Download 905K]

  • Establishment of bacterial endotoxin dynamic chromogenic method and its application in biological products

    GUO Yiyi;SHEN Ailin;LI Juanjuan;JIA Jingbo;ZHANG Juan;LI Siyu;PAN Junjie;CHENG Xiaoling;JIN Yucui;LUO Min;DUAN Kai;MA Huan;Quality Control Department,Wuhan Institute of Biological Products Co.,Ltd.;

    Objective To establish a dynamic chromogenic method for bacterial endotoxin and explore the feasibility of its application in different biological products.Methods The reliability test of the standard curve was carried out using limulus amebocyte lysate by dynamic chromogenic method,the dilution factor was determined by interference pre-experiment,and then the formal interference test was carried out.Five different biological products were measured,and the results were compared with those detected by gel detection.Results The linear range of the standard curve was 0.005-0.5 EU/mL,and the correlation coefficient(r) was 0.999.The non-interference dilution factors of enterovirus 71 inactivated vaccine(Vero cell),Sabin strain inactivated polio vaccine(Vero cell),live attenuated Japanese encephalitis vaccine,adsorbed acellular DPT combined vaccine and group A meningococcal polysaccharide vaccine were at least 800,400,500,1 000 and 10 000times,respectively.The spiked recovery rates of each product under the non-interference dilution factor were between 50% and 200%,in line with the requirements of the pharmacopoeia.The test results were consistent with the gel limit method and met the enterprise limit standards.Conclusion Dynamic chromogenic detection can be used to quantitatively detect the content of bacterial endotoxin in biological products.

    2025 06 v.38 [Abstract][OnlineView][Download 818K]

  • Application of equivalence testing in similarity evaluation of biological activity assay methods for glucagon like peptide-1(GLP-1) receptor agonist drugs

    WANG Mingren;WU Lihong;TIAN Qingya;DUAN Xuhua;SHAO Hong;NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies,Shanghai Institute for Food and Drug Control;

    Objective To explore the feasibility of applying equivalence testing to evaluate the similarity between reference standards and test samples in the biological activity assessment of glucagon like peptide-1(GLP-1) receptor agonist drugs,so as to improve the determination standard of biological activity of various products.Methods Historical release biological activity data from 25 batches of products were collected,and the parameter estimates and standard errors of a four-parameter logistic model were obtained.The slope ratio of test samples to reference standards and dynamic range ratio(ratio of the difference between upper and lower asymptotes of test samples to that of reference standards) were selected as similarity evaluation metrics,and tolerance interval method was used to set equivalence margins for these metrics.Subsequently,biological activity data from 86 additional batches of GLP-1 receptor agonist drugs(57 reference standards with similar biological behaviors to those of test samples and 29 reference standards with non-similar biological behaviors) were analyzed using equivalence testing to determine similarity.For comparison,an F-test was also performed to assess parallelism.The agreement between the two methods was evaluated using Kappa test.Results The equivalence intervals for the slope ratio and dynamic range ratio were 0.64-1.56 and 0.87-1.15,respectively.For the 86 batches,equivalence testing correctly identified 95%(54/57) of truly similar samples as similar and 86%(25/29) of non-similar samples as non-similar.In contrast,the F-test correctly identified 88%(50/57) of parallel samples as parallel and 72%(21/29) of non-parallel samples as non-parallel.Kappa test indicated moderate agreement between the two methods(Kappa=0.417).Conclusion Equivalence testing is suitable for evaluating similarity in the biological activity of GLP-1 receptor agonist drugs and outperforms the F-test in similarity assessment.

    2025 06 v.38 [Abstract][OnlineView][Download 839K]

  • Evaluation of growth-promoting effect of PL-NBCS instead of FBS on BHK-21, Vero and HEK293T cells

    WANG Limei;WANG Xin ;HU Wei ;ZHAN Jianping ;LV Shumin;ZHANG Yongming;College of Life Science and Technology,Inner Mongolia Normal University;

    Objective To compare the growth-promoting effects of platelet lysate-containing newborn calf serum(PL-NBCS) and fetal bovine serum(FBS) on cultured BHK-21,Vero,HEK293T cells,and to provide experimental basis for exploring the feasibility of culturing cells with PL-NBCS instead of FBS.Methods BHK21 cells,Vero cells and HEK293T cells were cultured with PL-NBCS and FBS,respectively.The growth-promoting effect of PL-NBCS on cells was detected by kinetic statistical analysis of morphological observation,average cell proliferation density,viability and doubling time of continuously subcultured cells.Results There was no significant difference in morphological characteristics between three kinds of cells cultured in PL-NBCS and those cultured in FBS.Under the two culture conditions,the cells grew in an "S" shape.The average proliferation density,viability and doubling time of BHK-21 cells cultured in PL-NBCS showed no significant difference with the BHK-21 cells cultured in FBS(t=0.734,0.432 and 0.029,P=0.472,0.671 and 0.977,respectively).The average proliferation density and viability of Vero cells cultured in PL-NBCS were higher than those in FBS group,and the average doubling time was significantly higher than that in FBS group(t=2.228,P=0.042).The average proliferation density,viability and doubling time of HEK293T cells cultured in PL-NBCS were significantly higher than those in FBS group(t=2.309,2.848 and 2.682,P=0.033,0.011 and 0.015,respectively).Conclusion PL-NBCS has better growth-promoting effect on cells,and can replace FBS to subculture cells.

    2025 06 v.38 [Abstract][OnlineView][Download 984K]

  • Development and verification of a double-antibody sandwich ELISA for detection of varicella-zoster virus-ORF7 protein

    WANG Menghan;HOU Anqi;TAN Shiming;DONG Xiang;LI Shuang;ZHANG Fukun;Changchun Keygen Biological Products Co.,Ltd.;

    Objective To develop a double-antibody sandwich ELISA for the detection of varicella-zoster virus(VZV)-ORF7 protein,and verify the method for the detection of ORF7 antigen across manufacturing stages of live attenuated varicella vaccine Oka-7S strain.Methods Recombinant plasmid pET-28a-ORF7 was used to induce prokaryotic expression of VZVORF7 protein,which was then purified by Ni~(2+) affinity chromatography column.Using ORF7 prokaryotic protein as the immunogen,the hybridoma cell lines were screened by mouse hybridoma fusion technique,and the monoclonal antibodies(mAbs)were prepared.The antibody titer was detected by indirect ELISA,and the antibody specificity was identified by Western blot.The antigenic epitopes of mAbs were analyzed by ELISA,and the double-antibody sandwich ELISA for VZV-ORF7antigen was developed by antibody pairing screening.The sensitivity,specificity and repeatability of the method were verified,and three batches of VZV Oka-7S harvested samples were detected by using the developed method.Results Four hybridoma cell lines steadily secreting anti-VZV-ORF7 specific antibodies,named M2B5,M2G5,8C2 and 1C4,were produced.The antibody titers of culture supernatant and mouse ascites were 10~4-10~5 and 10~6-10~7,respectively.The purity of purified mAbs was both about 97% in reduced and non-reduced states.All the four mAbs exhibited specific binding to VZV whole virus protein and purified ORF7 prokaryotic protein.M2G5 was selected as the capture antibody and M2B5-HRP as the enzyme-labeled antibody,with the optimum working concentrations of 4 μg/mL and 1:8 000,respectively.The cutoff value was 0.111,and when A_(450)≥0.111,it was determined as positive and <0.111 as negative.The linear range of this method was 3.9-250 ng/mL with a four-parameter fit,and the standard curve equation was y=(0.049 5-2.24)/(1+x/97.8)~(1.27)+2.24,R~2=0.999,with the limit of detection of 2.2 ng/mL.There was no cross-reaction with tetravalent influenza virus split vaccine,mumps virus and measles virus.In addition,the intra-assay and inter-assay CVs of repeatability were all less than 10%.Three batches of VZV Oka-7S strains were all detected to be negative,and the coincidence rate was 100%.Conclusion The developed double-antibody sandwich ELISA against VZV-ORF7 has high sensitivity,good specificity and high repeatability,and can be used for the rapid detection of VZV-ORF7 protein and VZV Oka and Oka-7S strains.

    2025 06 v.38 [Abstract][OnlineView][Download 877K]

  • Research progress on candidate vaccine of human Norovirus

    MIAO Shuidi;ZHANG Jvmei ;WU Qingping ;XUE Liang;College of Food Science,South China Agricultural University;

    Human Norovirus(HuNoV) is the main pathogen of acute gastroenteritis worldwide, which is highly contagious, especially for children, the elderly and immunodeficient people. As an RNA virus, HuNoV is highly variable and rich in genetic diversity, which makes it difficult to develop HuNoV vaccine. At present, there is still no related product approved for marketing. With the in-depth understanding of the evolutionary mechanism of HuNoV, the establishment of various antigen expression systems and the development of different immune adjuvants, breakthrough progress has been made in the research and development of HuNoV vaccine. Currently, a number of HuNoV candidate vaccines have been put into clinical trials,and a series of clinical evaluation data in terms of effectiveness, safety, stability and immune persistence have been obtained.In this paper, the antigen expression systems, immune adjuvants and clinical trials of HuNoV candidate vaccines are summarized, so as to provide a reference for the follow-up development of HuNoV vaccines.

    2025 06 v.38 [Abstract][OnlineView][Download 880K]

  • Recent advances and challenges of CpG ODN adjuvants

    ZHANG Linyu;DONG Chunyan;TAN Dejiang;HE Qing;National Institutes for Food and Drug Control;

    Adjuvants are an important part of vaccines. In order to overcome the weaknesses of traditional adjuvants, it is essential to develop new adjuvants. At present, CpG ODN has been used as vaccine adjuvants in clinical and preclinical studies of preventive antiviral, anti-parasitic and therapeutic tumor vaccines. Hepatitis B vaccine and COVID-19 vaccine with CpG ODN 1018 as adjuvant have been approved for marketing. In addition to single application, it is one of the most important research directions to develop new delivery systems of anti-tumor vaccines(such as nanoparticles and liposomes)with CpG ODN as adjuvant, and the delivery efficiency and targeting of CpG ODN need to be improved. In addition to the traditional subcutaneous and intramuscular immunization routes, CpG ODN is also being applied to the development of intranasal mucosal immune adjuvants, and its efficacy and mechanism as mucosal immune adjuvants have not been fully clarified.CpG ODN is an agonist of Toll-like receptor 9(TLR9), which can activate natural immunity and further mediate adaptive immune response. Its characteristic is that it can cause strong Th1 immune response. Although many clinical studies showed that CpG ODN adjuvants only cause slight and short-term adverse reactions, some studies also showed that CpG ODN may cause or aggravate autoimmune diseases and inflammatory syndrome by promoting Th1 response. How to overcome this shortcoming of CpG ODN adjuvants may be a challenge for the development of CpG ODN adjuvants in the future. In this paper,the latest research progress and challenges of CpG ODN adjuvants are reviewed.

    2025 06 v.38 [Abstract][OnlineView][Download 873K]

  • Research progress on mRNA influenza vaccine

    DU Jingni;LI Zhuang;WU Yehong;Changchun Institute of Biological Products Co.,Ltd.;

    Influenza virus exhibits high genetic variability, which represents the primary cause of the relatively low efficacy of conventional influenza vaccines, only 20%-60%. This limitation underscores the critical need for developing novel vaccines capable of addressing rapid viral evolution. mRNA-based influenza vaccines, manufactured through a cell-free production system based on in vitro transcription(IVT) technology, offer distinct advantages such as significantly reduced biosafety risks, enhanced immunogenicity and short production cycles, enabling prompt response to emerging variants, which have become one of the research and development hotspots of novel influenza vaccines. In this paper, the history and current status of mRNA vaccines and the development progress of mRNA influenza vaccines are summarized, in order to provide scientific basis for upgrading and broad-spectrum protection strategy of influenza vaccines.

    2025 06 v.38 [Abstract][OnlineView][Download 819K]

  • Research progress on CGRP/CGRPR targeted monoclonal antibody and its application in treatment of migraine

    DU Jialiang;WANG Lan;Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Biological Products,Division of Monoclonal Antibody Products,National Institutes for Food and Drug Control;

    With the deepening of the research on the mechanism of migraine,the role of calcitonin gene-related peptide(CGRP) in the trigeminal neurovascular system in the pathogenesis of migraine has attracted much attention,and has become the most promising target for the treatment and prevention of migraine. By 2020,four kinds of CGRP/CGRP receptor(CGRPR) targeted monoclonal antibody drugs have been approved by the Food and Drug Administration(FDA) and/or the European Medicines Agency(EMA). This paper reviewed the research progress of CGRP/CGRPR targeted monoclonal antibody in the treatment of migraine.

    2025 06 v.38 [Abstract][OnlineView][Download 841K]

  • Inspiration on China's national biological reference standards from WHO's guidelines and practices on stability of biological reference standards

    WANG Yiping;ZHANG Xuanxuan ;MAO Qunying ;LIANG Zhenglun;The Center for Reference Material and Standardization,National Institutes for Food and Drug Control;

    Stability represents a critical quality attribute of reference materials. To ensure the stability of biological reference standards, World Health Organization(WHO) has continuously refined and elaborated the stability requirements in guidelines for both international(primary) and national(secondary) biological reference materials, aiming to enhance the scientificity and operability of stability studies and monitoring. Based on a summarization of the WHO guidelines on stability studies for biological reference standards and case studies of international reference standards development documented in the literature, this paper proposes considerations and recommendations for improving stability studies and stability monitoring of national biological reference standards in China.

    2025 06 v.38 [Abstract][OnlineView][Download 848K]
  • 下载本期数据