Objective To develop and verify a sandwich ELISA method with bovine polyclonal antibody against rabbit polyclonal antibody for the determination of D antigen content of Sabin strain inactivated poliovirus vaccine(sIPV).Methods The rabbit polyclonal antibodies were prepared with sIPV vaccine bulks of type Ⅰ,Ⅱ and Ⅲ as antigens and detected for the titer and specificity by indirect ELISA.A double antibody sandwich ELISA with bovine polyclonal antibody as coating antibody and rabbit polyclonal antibody as detection antibody was developed to determine D antigen content,and the accuracy,precision and specificity to D antigen of the method were verified.sIPV vaccine samples from five domestic enterprises were detected by the developed method.Results The rabbit polyclonal antibodies for type Ⅰ,Ⅱ and Ⅲ sIPV with good specificity and high titer were prepared,and the double antibody sandwich ELISA method was successfully developed.Using four-parameter fitting,all three standard curves showed good linear relationship,and R~2 values were more than 0.99.The spike recoveries of type Ⅰ,Ⅱ and Ⅲ D antigens were all within 80%～120%,with average values of98.11%,97.41% and 98.66%,respectively.The CVs of repeatability and intermediate precision were all below 10%.The method also distinguished D antigens from C antigens.The developed method determined the D antigen contents of sIPV vaccines from five enterprises.Conclusion A sandwich ELISA method for determination of D antigen content in sIPV vaccine was successfully developed with satisfying accuracy,precision and certain D antigen specificity,which can be used to detect vaccines produced by different manufacturers.

SARS-CoV-2;Inactivated vaccine;IgG antibody;Neutralizing antibody Objective To evaluate the immunogenicity of prototype strain,Beta strain,Gamma strain and Delta strain of SARS-CoV-2 inactivated vaccines in rats.Methods Five female Wistar rats were immunized with SARS-CoV-2 inactivated vaccines of prototype,Beta,Gamma and Delta strains through thigh muscle twice at an interval of 14 d,with an immunization dose of 3 μg virus protein/(0.5 mL per rat).Serum samples were collected and isolated by vein 14,28 and 42 d after the first immunization.The serum IgG antibody levels were detected by indirect ELISA,the titers of serum neutralizing antibody were measured by microneutralization,and the antigenic ratios of the serum neutralizing antibody titers were calculated to evaluate the antigenicity difference between different strains.Results at 14 d after the first immunization,IgG antibodies against four strains of virus were detected in all immunized serum samples.The levels of IgG antibodies increased by more than 10 times at 28 d compared with those at 14 d,and decreased slightly at 42 d.At 14 d after the first immunization,all the neutralizing antibodies against the four strains were positive in the serum of rats immunized with prototype strain or Delta strain vaccine;In the serum samples of rats immunized with Beta and Gamma strains,all the neutralizing antibodies against Beta and Gamma strains were positive,while some neutralizing antibodies against prototype or Delta strains were positive.At 28 d after the first immunization,the neutralizing antibodies in the immune serum of the four strains were positive,and the titers of neutralizing antibodies were significantly higher than those at 14 d;The neutralizing antibody titers were slightly lower at 42 d after the first immunization than 28 d.There was small difference in the antigenicity between Beta and Gamma,prototype and Gamma,but significant difference in the antigenicity between prototype and Beta strains.Conclusion The prototype strain,Beta strain,Gamma strain and Delta strain of SARS-CoV-2 inactivated vaccines can stimulate rats to produce neutralizing antibodies with high titer,while the immunogenicity has difference.

Objective To evaluate the immunogenicity of prototype strain,Beta strain,Gamma strain and Delta strain of SARS-CoV-2 inactivated vaccines in rats.Methods Five female Wistar rats were immunized with SARS-CoV-2 inactivated vaccines of prototype,Beta,Gamma and Delta strains through thigh muscle twice at an interval of 14 d,with an immunization dose of 3 μg virus protein/(0.5 mL per rat).Serum samples were collected and isolated by vein 14,28 and 42 d after the first immunization.The serum IgG antibody levels were detected by indirect ELISA,the titers of serum neutralizing antibody were measured by microneutralization,and the antigenic ratios of the serum neutralizing antibody titers were calculated to evaluate the antigenicity difference between different strains.Results at 14 d after the first immunization,IgG antibodies against four strains of virus were detected in all immunized serum samples.The levels of IgG antibodies increased by more than 10 times at 28 d compared with those at 14 d,and decreased slightly at 42 d.At 14 d after the first immunization,all the neutralizing antibodies against the four strains were positive in the serum of rats immunized with prototype strain or Delta strain vaccine;In the serum samples of rats immunized with Beta and Gamma strains,all the neutralizing antibodies against Beta and Gamma strains were positive,while some neutralizing antibodies against prototype or Delta strains were positive.At 28 d after the first immunization,the neutralizing antibodies in the immune serum of the four strains were positive,and the titers of neutralizing antibodies were significantly higher than those at 14 d;The neutralizing antibody titers were slightly lower at 42 d after the first immunization than 28 d.There was small difference in the antigenicity between Beta and Gamma,prototype and Gamma,but significant difference in the antigenicity between prototype and Beta strains.Conclusion The prototype strain,Beta strain,Gamma strain and Delta strain of SARS-CoV-2 inactivated vaccines can stimulate rats to produce neutralizing antibodies with high titer,while the immunogenicity has difference.

Objective To study the possibility of using heterogeneous antiserum in single radial immunodiffusion (SRID) for quantitative detection of HA in H7N9 influenza vaccine product when H7N9-specific antiserum is not available in order to establish a testing method for the detection of H 7N9 antigen in any urgent situation.Methods Antisera specific for H7N1, H7N2, H7N3 and H7N7 were obtained from NIBSC and used for SRID assay .Amino acid sequences of hemagglutinins were comparatively analyzed be-tween H7N9 virus and other viruses used to prepare heterogeneous antiserum .The titers of antisera against H7N9 and their homogenous antigens were detected by double immunodiffusion method .Based on the results of homology analysis and cross-reaction, a suitable antiserum was selected and its applicability was further validated by the SRID assay using H7N9 antigen.Results Influenza A virus subtype H7N3 that used for preparation of 07/278 antiserum showed the highest HA homology with H7N9 (97.14%).The titer of 07/278 antiserum against H7N9 antigen was 1 ∶8 as detected by double immunodiffusion assay .The H7N9 anti-gen and the 07/278 antiserum could form a clear precipitation line in SRID assay .The detection of H7N9 antigen in the range of 10 to 40μg/ml showed a good linearity in the standard curve .Conclusion The 07/278 antiserum from NIBSC can be used as an alternative reagent for the quantitative detection of hemaggluti -nin in H7N9 influenza virus vaccine .

Objective To establish a method for rapid preparation of antiserum against influenza virus (H7N9) hemagglutinin,and to study the possibility of using it in single radial immunodiffusion (SRID) assay for quantitative detection of antigen in H7N9 influenza vaccine.Methods Hemagglutinin proteins expressed in eukaryotic cells were used to immunize sheep.Serum samples were collected to detect antibody titers by ELISA and double immunodiffusion assay.Different concentrations of antiserum were used in SRID assay to get the optimized concentration.Results After 4 times of immunization,the antiserum titers achieved 1 ∶ 1 000 000 and 1 ∶ 32 as indicated by ELISA and double immunodiffusion assay,respectively.The antiserum could form a clear precipitation line in SRID assay.The detection of antigen in the range of 10 to 40 μg/ml showed good linearity in the standard curve.The antigen titers in six batches of H7N9 vaccine detected by this SRID assay were identical with those by SDS-PAGE assay.Conclusion The antiserum against H7N9 hemagglutinin for SRID assay was developed successfully,and could be used as a reagent for the quantitative detection of antigen in H7N9 influenza vaccine.

Background and purpose:Diffusion-weighted imaging (DWI) is a non-invasive technique of breast magnetic resonance imaging (MRI). DWI is an alternative to dynamic contrast-enhanced (DCE) MRI for differentiating malignant from benign lesions in breast screening or not. This study aimed to evaluate the potential role of DWI in differentiating malignant breast lesions from benign lesions.Methods:Seventy-four patients underwent digital mammography, DCE and DWI (49 patients’b-value of 0, 400, 600 and 800 s/mm2). The detectability, sensitivity and speciifcity of DWI and DCE were compared. Absolute apparent diffusion coefifcient (ADC) was compared with standardized ADC for quantitative analysis.Results:Sixty-four of 74 patients had positive pathologic findings (38 malignant, 26 benign). All of the malignant lesions were detected on DWI and DCE. The sensitivity of DWI was 83.33%, 90.00% and 93.33%, and the specificity was 85.91%, 76.19% and 72.72%, forb-value of 400, 600 and 800 s/mm2, respectively. The sensitivity and speciifcity of DCE were 86.61% and 90.48%. There was no signiifcant difference between absolute and standardized ADC in detecting breast cancer (P>0.05).Conclusion:DWI is an important complemented technique to DCE-MRI for differentiating malignant from benign lesions in breast MRI.

Objective:To develop a new method based on neuraminidase (NA) activity for detec-ting virus titers of cell culture-based influenza vaccines and preliminarily analyze its application.Methods:Reaction conditions including the substrate concentration for enzymatic reaction, stop solution, the number of initially infected target cells and cell lysis buffer were optimized. The titers of cell culture-based influenza vaccine strains were detected by the established method and the results were compared with those by the traditional viral titration test.Results:The optimal substrate concentration for enzymatic reaction was 25 μmol/L, and the optimal stop solution was 0.2 mol/L Na 2CO 3. In the detection of NA activity in infected cells, the maximum relative fluorescence value was obtained by infecting 4×10 4 cells/well with influenza virus for 48 h and using 0.5% TritonX-100 for lysis. The developed method showed no significant differences with the traditional virus titration test in detecting the titers of four batches of influenza vaccine virus strains ( P>0.05), indicating that the two methods had a good consistency. Conclusions:This study established a new method based on NA activity to detect virus titers of cell culture-based influenza vaccines. The method could be used for the detection of virus strains used in the production of cell cultured-based influenza vaccines.