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Objective: To establish and validate a double antibody sandwich ELISA method for the in vitro relative potencytestof severe acute respiratory syndrome coronavirus 2, (SARS-CoV-2) recombinant protein vaccine(CHO cells). Methods: Human monoclonal antibodies GH4 and CB6 against the receptor binding domain (RBD)of SARS-CoV-2 spike protein were prepared by genetic recombinant technology. A double antibody sandwich ELISA method was established using GH4 as coating antibody and CB6 labeled with HRP enzyme (CB6-HRP) as detection antibody. The working concentrations of GH4 and CB6-HRP were determined. Methodological validation was carried out,including linearity and range, specificity, precision (repeatability, intermediate precision), accuracy, and robustness. The established method was used to detect the in vitro relative potencies of three batches of process-validated and twenty-two batches of continuous process verification of SARS-CoV-2 recombinant protein vaccines (CHO cells). Results: The optimal working concentrations of GH4 and CB6-HRP were 1 000 ng · mL-1 and 31.25 ng · mL-1, respectively. The vaccine reference had a good log-linear relationship with A450 in the concentration range of 0.312 5-5 ng · mL-1,and the slopes of the regression equations were all in the range of 0.80-1.25 and the R2 values were all>0.99.The established method could specifically detect the SARS-CoV-2 recombinant protein vaccine (CHO cells), and there was no cross-reactivity with the MERS Vaccine (CHO cells), influenza virus vaccine, rabies virus vaccine and CHO host cell proteins. For precision validation, the geometric coefficient of variation (GCV) of repeatability was in the rang of 1.6% to 2.4%, and the GCV of intermediate precision was in the range of 1.2% to 2.6%. The R2 value of the regression equation was 0.994 5, and the slope was 0.999 5 which was in the range of 0.80-1.25. For accuracy validation, the relative bias (RB) was in the range of -4.04% to 7.36%. In the robustness validation, the in vitro relative potency under different test conditions ranged from 0.96 to 1.08. The geometric mean in vitro relative potency of the three batches of process-validated SARS-CoV-2 recombinant protein vaccines (CHO cell) was 1.02, with an GCV of 4.8%; the geometric mean in vitro relative potency of twenty-two batches of continuous process verification SARS-CoV-2 recombinant protein vaccines (CHO cells) was 1.04, with an GCV of 4.4%. Conclusion: The established double antibody sandwich ELISA method has good specificity, precision, accuracy and robustness. It can be used for the detection of the in vitro relative potency and the quality control of SARS-CoV-2 recombinant protein vaccine (CHO cells).
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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