Hello,
I am using an in-house competitive ELISA for the detection of a small peptide in the various samples (plasma, cell culture supernatants etc.). For the method's needs, polyclonal rabbit Abs are used and after optimization, the LOD is 0.1 ng/ mL. The sample is co-incubated overnight with biotynilated peptide and the detection Ab. Recently, a problem has been observed, regarding the standard curve (Absorbance after the addition of the substrate). Specifically, the absorbance value of the minimum concentration (0,1ng/mL) is lower than the value of the previous one (0,5ng/mL). I have tried using freshy dilluted reagents, however the problem has not been solved yet. Have you ever experienced a similar situation? Could you kindly help me with finding a solution?
Thank you.
I would suggest you read the samples in a different plate reader and the one you were using before for your next experiment. This could be an error with the instrument especially if it has fixed detectors. If the same issue persists you need to recheck your dilutions.
How old is the lamp? Can you check, if it is still working properly?
The ELISA reader is only 3 years old, however I have tried analysing the plate in a different reader and the results were similar. I double checked the dillutions, everything is correct. I tried adjusting the quantity of the primary Abs (2x concentration), with no result, as I got the same inversed standard curve again. Would it be possible that the quantity of the biotynilated peptide needs adjustment?
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA