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To develop a highly sensitive, efficient, and stable cell-based platform for detection of anti-adeno-associated virus 8 (AAV8) neutralizing antibody titer in monkey plasma and perform preliminary validation of the relevant performance of the method.
In vitro, an equal volume of diluted monkey plasma was incubated with AAV8-Luciferase and then the mixture was added to a 96-well plate lined with HEK293 cells and incubated overnight to infect HEK293 cells; after cell lysis, the cells were added to a firefly luciferase reporter gene assay and the fluorescence signal (RLU) was detected using ELISA to assess the plasma levels of anti-AAV8 neutralising antibodies and subsequently obtain the corresponding antibody titres. The assay's sensitivity (LOD) and titer reproducibility, precision, method robustness and sample stability were preliminary were preliminary validated.
A highly sensitive, efficient and stable method for the detection of anti-AAV8 neutralizing antibody titer in monkey plasma at the cellular level was developed and preliminarily validated. The LOD value of the method was 10.36 ng·mL-1. The repeatability, precision and method robustness of the titer assay were good, and the positive control samples were stable after being placed at room temperature and 2 ℃~8 ℃ for 24 h and after freeze-thawing for 6 times at -65 ℃~-90 ℃/room temperature.
It has been preliminarily validated that the method can be used for the analysis and detection of anti-AAV8 neutralizing antibody titer in monkey plasma, which is useful for grasping the process of drug treatment and adjusting the treatment protocol in time and is suggestive for the development of AAV8 serotype gene therapy drugs and individualized treatment.
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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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