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.