By using this cytokine kit I am getting high absorbance value in my positive controls. Recommended values for positive controls are 1.5-2.5, but I am getting values around 3.0-3.5.
In some of my negative control I have absorbance more than the experimental sample. Eg. negative control value 0.04 and sample value 0.01.
You have to dilute your sample to get the absorbance in reference range afterwards you can calculate you value accordingly.
Thanks Anil,
In experimental samples I am not getting high absorbance. In positive controls I am getting high absorbance, positive controls are provided with kit and I prepared according to the protocol provided with kit.
Should I dilute positive controls?
Though it is not recommended.
A positive control will tell you if your experiment has worked or not. All antigens should give a signal significantly above the background, typically at or above 1.5. In case you get signals for all positive controls your experiment is successful. Even though the values are above 2.5, it’s ok as the Assay is only qualitative.
Negative control values vary between analytes, but are normally less than or equal to 0.5. OD readings largely depend on the pipetting accuracy and the incubation temperature and time. The values are so low for your samples and negative control, so it means there is no antigen detected in your samples or you can give another try with your freshly prepared samples (however sample preparation is another crucial issue for example crushing and homogenisation in case of tissue and centrifugation in case of supernatant to pellet down the cells).
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