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Objective: To prepare monoclonal antibodies (MAbs) that can specifically recognize genistein(Gen), and to establish a specific and rapid quantitative method for Gen using indirect competitive enzyme-linked immunosorbent assay (ELISA). Methods: Gen was coupled with carrier protein by the Mannich method to synthesize artificial antigens. Femal Balb/c mice aged 6-8 weeks old were immunized by Gen-BSA. After the booster immunization, splenocytes of the mice were collected and fused with SP2/0 myeloma cells under the action of 50% polyethylene glycol (PEG). Subclones were screened by the limiting dilution method to obtain specific MAbs and an indirect competitive ELISA method was established. Results: Two hybridoma cell lines (C1 and C3)were isolated successfully with the titers of 1 ： 32 000, 1 ： 40 000, respectively. Under the optimized conditions,the indirect competitive ELISA based on C3 for Gen showed a half maximum inhibition concentration (IC50) of 16.15 ng · mL-1 and detection ranges of 3.78-55.85 ng · mL-1 with cross-reactivity for biochanin A and irisolidone of 4.96% and 2.40%, respectively, negligible cross-reactivity with other Gen analogs including apigenin and 4-oxo-4H-1-benzopyran-2-carboxylic acid. The recovery test showed that the recovery rate was between 85% and 110%, precision tests showed RSD of inter-assay and inter-assay was less than 10%. Conclusion: In this study,Gen-specific MAbs are prepared successfully. An sensitive and accurate indirect competitive ELISA based on MAb for Gen is developed. The developed method can meet the requirements for immunoassay.
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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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