A. Sensitize a 96-well microtiter plate with purified antigen.
1. Prepare a solution of the purified antigen of interest in phosphate buffer (see recipe below) such that a concentration of approximately 10 µg/ml is achieved. Pipette 100 µl of this solution to all wells of the plate with the exception of those in the outermost rows and columns (these areas of the plate are prone to evaporation).
2. Cover the plate with Parafilm and incubate for 2 hours at 37°C, followed by an overnight incubation at 4°C. Cover the plate tightly to avoid evaporation. Store at 4°C. The plate(s) will remain usable for about one month.
B. Block the sensitized plate.
1. When ready to use, discard the antigen solution contained in the wells of the sensitized plate. Pat the inverted plate sharply several times against a pad of paper towels to insure complete removal of the solution.
2. Wash the wells three times with 1x PBS/0.05%-Tween 20. Pat to dry completely after the final wash, as above.
3. Pipette 100 µl of 5% skim milk/PBS/0.005% thimerasol (added to retard bacterial growth) to each well. Incubate the plate, covered with Parafilm, at 37°C for 2 hours to overnight. It is convenient during this incubation step to begin preparation of the samples to be tested, outlined below.
4. Wash the wells three times with PBS/Tween, dry as before after the last wash.
人柠檬酸合酶(CS)酶**测试剂盒(ELISA试剂盒) Homo sapiens (Human) Citrate Synthase (CS)ELISA KIT
双抗体夹心法
1. 包被：用0.05MPH9.牰碳酸盐包被缓冲液将抗体稀释至蛋白质含量为1～10μg/ml。在每个聚苯乙烯板的反应孔中加0.1ml，4℃ 过夜。次日，弃去孔内溶液，用洗涤缓冲液洗3次，每次3分钟。（简称洗涤，下同）。
2. 加样：加一定稀释的待检样品0.1ml于上述已包被之反应孔中，置37℃ 孵育1小时。然后洗涤。（同时做空白孔，阴性对照孔及阳性对照孔）。
3. 加酶标抗体：于各反应孔中，加入新鲜稀释的酶标抗体（经滴定后的稀释度）0.1ml。37℃ 孵育0.5～1小时，洗涤。
4. 加底物液显色：于各反应孔中加入临时配制的TMB底物溶液0.1ml，37℃ 10～30分钟。
5. 终止反应：于各反应孔中加入2M硫酸0.05ml。
6. 结果判定：可于白色背景上，直接用肉眼观察结果：反应孔内颜色越深，阳性程度越强，阴性反应为无色或极浅，依据所呈颜色的深浅，以“+”、“-”号表示。也可测OD值：在ELISA检测仪上，于450nm（若以ABTS显色，则410nm）处，以空白对照孔调零后测各孔OD值，若大于规定的阴性对照OD值的2.1倍，即为阳性。
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