I am using ELISA method for detecting MPO-DNA from blood plasma for my current research purpose. I use 25 micro liter plasma sample and 75 micro liter PBS in each well of microplate as unknown and 100 micro liter of PBS as control. During absorbance measurement, the blank value is automatically subtracted from the sample values and I just manually subtract the control value from the sample values. Now, the problem is, sometimes, I am getting higher control value(PBS) than that of sample values for why, after subtracting, I get an ultimate negative value as result. I use ABTS and measure the absorbance at 405 nm wavelength. What can I do now to get a good control value lesser than unknown sample?
You need to provide sufficient information about your experiment to get relevant answers, comments or suggestions to fix your problem. What did you coat in each wells before adding solution of your MPO-DNA as a test and PBS as a control? Did you block all of your wells prior to adding MPO-DNA solution and PBS to wells? Did you perform washing of wells in between each steps properly? Do you think there will be contamination of wells during washing, which is less likely for binding to occur? Did you use the right secondary Abs? Did you wash the wells extensively after binding the secondary Abs? How long did you perform your color development?
Dear Tekewe,
Thanks for your queries.Actually, we coat the required number of wells with coating buffer (Sodium Carbonate+Sodium Bicarbonate+ distilled water, pH 9.6) before adding sample (plasma) and PBS. I usually block the wells with blocking buffer (1% BSA) and wash the wells 3-4 times with PBS . I think,no contamination occur during washing. We use Anti MPO Ab and finally use anti-DNA-POD (HRP), Source; Roche Diagnostics. Then washing was done 3-4 times with 0.5% Triton X. For color development, we perform a 20 minutes of incubation.If you have any further inquiries, please let me know.
Please go through the profile of
WA Gadalla, NHS Grampian in Research Gate.
For same question, a lot of answers has been already provided. Hope it will be useful to you
Why blank value is higher than sample value in ELISA test? and Is there any way to avoid negative values after substract sample value from blank?
blank 0.208
values of samples ranges from (0.165-2.2080)
a lot of sample values below blank value
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA