ObjectiveTo develop a double antibody sandwich ELISA method for the detection of types Ⅰ, Ⅱ and Ⅲ antigen content of recombinant trivalent poliomyelitis vaccine, and to optimize, verify and preliminarily apply the method, in order to provide a quality control method for vaccine development.MethodsMale New Zealand white rabbits were immunized with types Ⅰ, Ⅱ and Ⅲ recombinant poliomyelitis vaccine antigens, and the corresponding polyclonal antibodies were prepared.The polyclonal antibodies were used as coating antibodies and HRP-labeled antibodies as detection antibodies to establish a double antibody sandwich ELISA method for detecting the content of three types of antigens in recombinant trivalent poliomyelitis vaccine. The checkerboard titration method was used to determine the coating antibody concentration and the detection antibody dilution. The single factor experiments were used to optimize the types of blocking solution, antibody lyophilization time, enzyme-labeled antibody diluents and chromogenic solution formulations. The established method was verified for linear range, accuracy, specificity, precision, lower limit of quantification, robustness and stability, and was used to detect the content of types Ⅰ, Ⅱ and Ⅲ antigens in recombinant trivalent poliovirus vaccine.ResultsThe optimal coating antibody concentration was 5 μg/mL, and the optimal dilutions of enzyme-labeled antibodies were 4 000, 9 000 and 5 000, respectively,for types Ⅰ, Ⅱ and Ⅲ antigens. The optimal conditions were as follows: blocking solution of 1% BSA solution, lyophilization time of 2 h, enzyme-labeled antibody dilution of 1% BSA + 1% sucrose + 1% trehalose + 0. 01% sodium thimerosal, and chromogenic solution of recipe 2 [Solution A: 13. 6 g sodium acetate, 1. 6 g citric acid, 0. 3 mL of 30% hydrogen peroxide,adding distilled water to a total volume of 500 mL. Solution B: 0. 2 g disodium ethylenediaminetetraacetate, 0. 95 g citric acid, 50 mL glycerol, 0. 15 g TMB(dissolved in DMSO before slowly adding to distilled water), adding distilled water to a total volume of 500 mL]. TypesⅠ and Ⅱ antigens showed a good linear relationship with A_(450)in the concentration range of0. 78-25 DU/mL, and type Ⅲ antigen exhibited a good linear relationship with A_(450)in the concentration range of 1. 56-50 DU/mL,each R~2> 0. 99. The recovery rates of spiked samples at high, medium and low concentration of the three types of antigen content detection methods were all between 80% and 120%. All three types of antigen detection methods detected their corresponding specific antigens, and there was no cross-reaction with the other two antigens. The lower limits of quantification of types Ⅰ, Ⅱ and Ⅲ antigen detection methods were 1. 56, 1. 56 and 3. 13 DU/mL, respectively. The CVs of precision and robustness verification were both less than 20%. The antibody-coated plate, detection antibody working solution and chromogenic solution were stored stably at 4 ℃ for six months, and the recovery rates of the three types of antigens were all within the range of 80%-120%. The CVs of harvest solution, clarified solution, concentrated solution, ion exchange chromatography solution, recovery solution and bulk solution samples in the vaccine process were all not more than 15% by the established method.ConclusionThe established double antibody sandwich ELISA quantitative detection method has good specificity,accuracy, precision, robustness and stability, and can be used to detect the content of typesⅠ, Ⅱ and Ⅲ antigens in the development of recombinant trivalent poliovirus vaccines.

Objective To develop double antibody sandwich ELISA methods for the determination of P3296, P5668 and PRX1 antigen components in recombinant pneumococcal protein vaccine based on pneumococcal surface protein A(PspA), and to optimize,verify and preliminary apply it, in order to provide a reliable detection method for the quality monitoring of the vaccine.Methods The male New Zealand white rabbits were immunized with P5668, P3296 and PRX1 purified proteins. The immunized serum was purified by Protein A-Sephaorse 4B affinity chromatography, and P5668, P3296 and PRX1 polyclonal antibodies were obtained. Using the polyclonal antibodies as the coating antibodies and the corresponding HRP-labeled monoclonal antibodies as the enzyme-labeled antibodies, the double antibody sandwich ELISA methods were developed. The concentration of coating antibodies(all three polyclonal antibodies diluted to 2, 4 and 8 μg/mL), the dilution of enzyme-labeled antibodies(HRP-labeled P5668 monoclonal antibody diluted at 1∶4 000 and 1∶8 000, HRP-labeled P3296 monoclonal antibody diluted at 1∶40 000 and 1∶60 000, HRP-labeled PRX1 monoclonal antibody diluted at 1∶12 000 and 1∶24 000),the sealing liquid type(1% BSA, 1% fish skin gelatin, 1% skimmed milk powder and 1% casein), the diluent type(purified water, 1 × PBS, 2 × PBS), and the diluent pH(6. 4, 7. 4, 8. 4) were optimized. The linear range, specificity, accuracy, precision, and robustness of the methods were verified. The developed methods and Lowry method were used to detect purified proteins of P5668, P3296 and PRX1(20 batches each), and the correlation between the results of the methods was analyzed. Three batches of P5668, P3296 and PRX1 mixed vaccines were desorbed by propanesulfonic acid internal salt, and the antigen contents of P3296, P5668 and PRX1 were detected by the developed methods. Results The protein concentrations of purified P5668,P3296 and PRX1 polyclonal antibodies were 1. 27, 2. 20 and 1. 53 mg/mL, respectively. The optimal coating concentration of P3296, P5668 and PRX1 polyclonal antibodies was all 4 μg/mL, and the optimal dilution of HRP-labeled P5668, P3296,and PRX1 monoclonal antibodies was 1∶4 000, 1∶60 000, and 1∶12 000, respectively. The optimal sealing liquid was 1% BSA, the diluent was 1 × PBS, and the diluent pH was 7. 4. Three reference materials of P5668, P3296 and PRX1 in the range of 3. 125-100 ng/mL showed a good linear relationship with A_(450), with R~2 values more than 0. 99. The spike recovery rates of three antigens with high, medium and low concentration were all in the range of 80%-120%. All the three methods detected the corresponding specific antigen, with no cross-reaction to the other two antigen proteins. The CVs of repeatability and intermediate precision verification were less than 20%, and the CVs of detection results of the same sample under different conditions were also less than 20%. The R of ELISA and Lowry methods for the determination of P5668, P3296 and PRX1 antigens was 0. 984 6, 0. 997 0 and 0. 990 9(each P < 0. 000 1), respectively, and the Lowry method exhibited a positive correlation with the developed methods. Three batches of P3296, P5668 and PRX1 mixed vaccines were detected for the antigen contents by the developed method, and the coincidence rate between the results and theoretical values was 87%-114%.Conclusion The developed double antibody sandwich ELISA methods have good specificity, precision, accuracy and robustness, and can be used for the determination of P3296, P5668 and PRX1 antigens in recombinant pneumococcal protein vaccines.

Objective: To detect the expression of CRLF2 in adult Ph negative acute B lymphocytic leukemia (B-ALL) in newly diagnosed cases, and to investigate the relationship between CRLF2 and the general clinical characteristics, efficacy and prognosis. Methods: 103 cases of newly diagnosed adult B-ALL patients were investigated from Apr 2016 to Dec 2017 in the Department of Hematology, Henan Cancer Hospital. Bone marrow samples was used to detect the expression of CRLF2 in leukemic cells. The expression of CRLF2 ≥20% was defined as CRLF2-high group and <20% was defined as CRLF2-low group. The clinical characteristics and prognosis of the two groups were compared. Results: The Median overall survival (OS) and disease free survial (DFS) in CRLF2-high group were 9.0 months and 4.25 months, respectively. CRLF2-low group were 15.5 months and 10.25 months, respectively. There was a statistically significant difference in median OS and DFS between the two groups (P=0.007, P=0.000) . The 18-month OS and DFS in CRLF2-high group were 38.6% and 25.1%, respectively. CRLF2-low group were 57.8% and 42.3%, respectively. Multivariate analysis showed high expression of CRLF2 was an independent risk factor for OS (HR=2.991, 95% CI 1.429-6.261, P=0.004) and DFS (HR=2.374, 95%CI 1.146-4.960, P=0.041) in patients. Conclusion Patients with high expression of CRLF2 had poor prognosis.