Every ELISA test has to do a standard curve, so there are often customer consultations on the specific parameters of the absorbance value of the specific requirements, how much more comprehensive? Some people are in the range of absorbance values ​​in the 0-0.11072, experienced researchers are recommended not to exceed 1, the concentration is too large to be diluted. So how to dilute, dilute it is not the concentration value on the standard?
First, according to Langberbill's law, the absorbance value has a linear relationship with the concentration at 0.2~0.8, as far as possible within this range.
2. In the production of the standard curve, standard samples of different concentrations are selected for testing, and the absorbance is measured. It’s okay until their relevance is not good. The correlation is preferably the best above 0.999. Therefore, under normal circumstances, the absorbance value can reach a level of good correlation at around 1!
Third, it depends on the absorbance value of the different concentrations of your sample to be tested, which is roughly in the range (the method of using the standard curve is not suitable for the sample to be tested, whether there is a sinking or other abnormal phenomenon) The concentration of the sample needs to be explored in advance, and the range is enlarged (eg: 6.25, 12.5, 25, 50, 100, 200, 400, 600, 800, 1000, 2000, etc.) to determine the concentration range required for the experiment, which is the coordinate curve. It is. The best advice is to do 3 sets of parallel samples (including samples) so that the linear relationship is better.
Fourth, dilution is relatively easy. For example, a sample of 1000 μg/ml can be diluted to 800, 600, and 400. Specifically, if the concentration of 1000 is added to 4 ml of a solvent (water, etc.) 1 ml, 800 μg/ml can be obtained, and so on.
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