Hello!
Our lab has developed a standard curve with known concentration that we use to determine our ELISA concentrations. We then multiply by the dilution factor for our final concentration.
Recently, I had really high binding samples, some which fell outside the standard range so I diluted the samples from 1:100 to 1:1000. Expectedly, the absorbances decreased, for example from 0.4 OD to 0.04 OD. However, the conversion using the standard curve equation gave us concentrations of ~9000 ng/ml [1:100] vs.~7 ng/ml [1:1000]. After multiplying for dilution factor, the 1:1000 concentration seems really low. I can't understand the explanation for this.
I have discounted calculation errors, standard curve error [R2=0.999 for both plates] + my plate's blank wells and positive controls are consistent in their absorbance values.
Is there another way to approach my thinking about the standard curve?
Thank you all for your insights!
For ELISA, you can prepare 4PL logistic curve. You can analyze your data by online software tool.
https://myassays.com/
You will directly get your unknown sample concentration, when you enter all the required plate set-up, dilution factors, name of samples/ standards. You will get concentration of your sample based on the standard curve along with %CV, SEM and SD.
what was the solution you used for dilution of your sample? It must be the same with the solution you primarily used to make your samples and standard solutions. Additionally, every kit has a certain detection range. Check whether the concentration of your sample falls within the range or not.
If you are able measure your samples again, more suitable would be if you could use the sample matrix as diluent, so the influence of the matrix will be the same, in your standards and in your samples.
The possibly ideal method includes the extraction of your analyte. What are you after and what is your matrix? How is your actual experimental setup?
Thank you for your answers.
Vajaria-I have not used "myassays" before, but it sounds very useful. We have been using https://elisaanalysis.com/.
Rahimmi- we used the same solution -Non Fat Dry Milk- to make the samples and the standard solutions. We do not use a kit but are using an house made standard curve.
Schechinger-Apologies I am unclear what you mean when asking about the matrix? As for the experimental set up- Our sample serums are prepared in 5% NFDM in 0.2%Tween. Our purified peptide standard is diluted the same solution to make a stock 1000ng/ml which is then diluted 1:3 serial dilutions. We use HRP conjugated antibody and TMB step. The absorption is then analyzed using https://elisaanalysis.com/ to get our values for our 4-Parameter Logistic Regression formula.
I hope the new information helps!
Janna Bashar , "Matrix" is understood as the material your analyte is in. As I understand your experiment, you're diluting serum samples with NFDM/Tween (diluent) and you're seeing problems when you're diluting some samples further. Is the serum/diluent ratio the same in all samples (also after further dilution) and is (negative) serum also present in the standards?
Are you using homemade plates? How are you coating them? Could you describe your setup more detailed?
Thank you everyone for your interest in my question.
Our lab concluded that the relationship between the absorbance and converted ng/ml values broke down when the absorbance reached the upper limits of the standard curve. I appreciate all your guidance!
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