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Establishment of a double-antigen sandwich ELISA for the detection of antibodies against classical swine fever virus （CSFV） E2 protein. The complete E2 protein sequences of CSFV and bovine viral diarrhea virus （BVDV） were aligned using BioEdit software to identify CSFV-specific epitopes. A recombinant pCDNA3.1-2×rE2-His expression vector was constructed. The 2×rE2-His protein was expressed in CHO cells， followed by thrombin cleavage to remove the His-tag. The purified His-tag-free 2×rE2 protein was used as the coating antigen， while horseradish peroxidase （HRP）-labeled 2×rE2-His protein served as the detection antigen. After systematic optimization of reaction conditions， a double-antigen sandwich ELISA method was successfully established. The specificity， sensitivity， and repeatability of the assay were evaluated， and its concordance rate with a commercial ELISA kit was compared. The optimized assay conditions were as follows： antigen coating concentration at 0.5 μg·mL-1， serum dilution at 1∶4， and enzyme-labeled antigen dilution at 1∶40 000. The method exhibited no cross-reactivity with positive sera against other common swine pathogens， including senecavirus A （SVA）， porcine pseudorabies virus （PRV）， porcine epidemic diarrhea virus （PEDV）， foot-and-mouth disease virus （FMDV）， and porcine reproductive and respiratory syndrome virus （PRRSV）， nor with BVDV sera， which frequently causes serological cross-reactivity， demonstrating high specificity. The maximum detectable dilution ratio for antibody-positive sera reached 1∶2 048. Both intra-assay and inter-assay coefficients of variation were below 10%， indicating excellent repeatability. The concordance rate with the IDEXX CSFV Antibody Test Kit was 97.1%. To further evaluate the assay， both the established double-antigen sandwich ELISA and the IDEXX kit were used to monitor antibody kinetics in two healthy pigs immunized with CSFV C-strain vaccine combined with an E2 subunit vaccine. The developed ELISA detected antibody responses 6 days earlier than the IDEXX kit. Both methods demonstrated that antibody levels peaked at 37 days post-immunization and subsequently stabilized. In this study， a double-antigen sandwich ELISA antibody detection method for CSFV was established with high specificity， sensitivity and reproducibility， which is suitable for the clinical diagnosis and evaluation of immunization effect of swine fever.
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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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