symptom:
1. The ratio of positive to negative cannot reach 2.1, the color of negative is too dark.
2. The ratio of positive to negative cannot reach 2.1, the color of positive is too light.
3. Increase the antigen concentration, the primary antibody concentration remains unchanged, and the color reaction does not show the proper gradient.
4. Increasing the concentration of primary antibody, the color reaction does not show the proper gradient.
5. After adding TMB, the color gradient is obvious, but after adding sulfuric acid to stop the reaction, the color gradient is not so obvious.
Possible problems:
1. The negative control (that is, the other components of the positive control except the target antigen) has a certain component and primary antibody effect.
2. 1) The primary antibody titer is not good.
2) The antigen is too dilute.
3) The closing time is too long.
3. The ratio of primary antibody to antigen is saturated.
4. The ratio of primary antibody to antigen is saturated.
5. Sulfuric acid may carbonize other ingredients.
Solution:
1. Purify the antigen to remove interfering components.
2. 1) Switch to primary antibody or increase primary antibody dosage.
2) Increase antigen concentration.
3) Shorten the closing time, usually within 2 hours.
3. Increase the amount of primary antibody; or decrease the antigen concentration to make a gradient without changing the amount of primary antibody.
4. Increase the amount of antigen; or reduce the concentration of primary antibody to make a gradient when the amount of antigen is unchanged.
5. Add less sulfuric acid, reference value: 50 μl TMB + 35 μl sulfuric acid.
I think that primary antibody and blocking are both important steps. If ELISA still fails to produce satisfactory results, the experimental conditions can be improved from these two aspects to achieve the goal relatively quickly.
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