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OBJECTIVE To develop the methods for biological activity assay and immunological characteristics analysis of anti-SARS-CoV-2 neutralizing antibodies. METHODS The binding affinity of 9MW3311 Fab and S1-RBD were analyzed by biolayer interferometry. Enzyme linked immunosorbent assay (ELSA) and fluorescence activated cell sorter (FACS) were used to evaluate the relative binding activity to S protein and blocking activity to angiotensin converting enzyme 2(ACE2) of 9MW3311 antigenbinding fragments (Fab). In vitro cytological activity of neutralizing antibody was evaluated by pseudovirus system. The binding affinities of neutralizing antibody Fc with Fc receptor (Fcγ) and Fc receptor neonatal (FcRn) receptor were determined by surface plasmon resonance (SPR). The binding activity of Fc and complement component 1 (C1q) receptor was determined by ELISA. The antibody-dependent cell-mediated cytotoxicity(ADCC) and complement dependent cytotoxicity(CDC) of neutralizing antibodies were determined by peripheral blood mononuclear cell (PBMC) method. The antibody-dependent enhancement (ADE) effect was evaluated using pseudovirus system. RESULTS The affinity constants (KD) of 9MW3311 and reference to S1-RBD were 1.15×10-9, 1.01×10-9, 1.15×10-9 and 9.45×10-10, respectively. ELISA and FACS showed that the binding activities of neutralizing antibodies were 101%, 96%, 100% and 98%, 113%, 108%, respectively. ELISA and FACS showed that the blocking activities of neutralizing antibodies against ACE2 were 100%, 95%, 91% and 94%, 101%, 94%, respectively. The neutralizing activities of the three batches of neutralizing antibodies against pseudovirus were 91%, 93% and 108%, respectively. The three batches of 9MW3311 and reference showed the same affinity constants (KD) with Fcγ and FcRn. 9MW3311 showed no ADCC and CDC activity. L234A/L235A (LALA) mutant of 9MW3311 could effectively avoid ADE effect. CONCLUSION The methods for analysis of the biological activity and immunological characteristics of anti-SARS-CoV-2 neutralizing antibodies are preliminarily established and can be used for routine quality control and release.
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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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