間接ELisa的一般方案
Indirect ELISA Protocol
1. Dilute antigen to a final concentration of 1-20 μg/ml using PBS or Coating buffer. Coat the wells of a PVC microtiter plate with the antigen by pipeting 50μl of the antigen dilution in the top wells of the plate. Dilute down the plate as required. Seal the plate and incubate overnight at 4°C or 2 h at room temperature.
2. Wash plate 3 times with PBS.
3. Block the remaining protein-binding sites in the coated wells by adding 200 μl blocking buffer, 5% non fat dry milk/PBS, per well. Alternative blocking reagents include BlockACE or BSA.
4. Cover the plate with an adhesive plastic and incubate for at least 2h at room temperature or, if more convenient, overnight at 4°C.
5. Wash the plate 3 times with PBS.
6. Add 100 μl of diluted primary antibody to each well.
7. Cover the plate with an adhesive plastic and incubate for 2 h at room temperature.
8. Wash the plate 4 times with PBS.
9. Add 100 μl of conjugated secondary antibody, diluted at the optimal concentration (according to the manufacturer) in blocking buffer immediately before use.
10. Cover the plate with an adhesive plastic and incubate for 1-2 h at room temperature.
11. Wash the plate 5 times with PBS.
12. Dispense 100 μl (or 50 μl) of the substrate solution per well with a multichannel pipet or a multipipet.
13. After sufficient color development (if it is necessary) add 50-100μl of stop solution to the wells.
14. Record the absorbance at 450 nm on a plate reader within 30 minutes of stopping the reaction.
1. 使用 PBS 或包被緩沖液將抗原稀釋至終濃度 1-20 μg/ml。通過在板的頂部孔中吸取 50μl 的抗原稀釋液，將抗原涂在 PVC 微量滴定板的孔中。根據需要稀釋板。密封板并在 4°C 下孵育過夜或在室溫下孵育 2 小時。
2. 用 PBS 洗板 3 次。
3. 每孔加入 200 μl 封閉緩沖液、5% 脫脂奶粉（或者BSA）/PBS，封閉包被孔中剩余的蛋白質結合位點。
4. 用封板膠密封Elisa板并在室溫下孵育至少 2 小時，或者，如果更方便，在 4°C 下孵育過夜。
5. 用 PBS 洗板 3 次。
6. 每孔加入 100 μl 稀釋的一抗。
7. 用封板膠密封Elisa板并在室溫下孵育 2 小時。
8. 用 PBS 洗板 4 次。
9. 使用前立即加入 100 μl 偶聯二抗，在封閉緩沖液中稀釋至最佳濃度（根據制造商）。
10. 用封板膠密封Elisa板并在室溫下孵育 1-2 小時。
11. 用 PBS 洗板 5 次。
12. 用多道移液管或多管移液管在每孔中分配 100 μl（或 50 μl）底物溶液。
13. 充分顯色后（如果需要）向孔中加入 50-100μl 終止液。
14. 在反應停止后 30 分鐘內，在酶標儀上記錄 450 nm 處的吸光度。
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