Competitive ELISA
1. Coat Nunc immuno-module plates overnight, in usual manner.
2. Set up competition assay between antibody and competing substance :-
Prepare a 1:2 serial dilution of the competitive substance from 480 to 0.4 ng/ml. Add 50ul of each of the above dilutions to 25ul of 3x strength titre of antibody (i.e. a solution 3 x the titre giving 50% inhibition on direct ELISA), in sealed tubes.
3. Incubate overnight at room temperature.
4. Set up competition between unknown concentration of competitive substance and antibody as above.
DAY 2
1. Wash plate with 4x 200ul/well PBS/Tween.
2. Add 50ul/well of antibody and incubate for 2hrs.
3. Wash 4x with 200ul/well PBS/Tween.
4. Add 50ul/well secondary antibody and incubate for 2hrs.
5. Wash 4x with 200ul PBS/Tween.
6. Add 50 ul peroxidase substrate (TMB) as for direct ELISA.
7. Read OD @ 450nm.
8. Plot standards against absorbance and determine unknown concentrations.
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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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