Hi everyone,
I'm gonna try to detect the IL-10, IFN-y, TGF-beta1 level in the human serum by ELISA.
I bought the ELISA set from the BD contain Capture Ab, Detection Ab, Enzyme reagent, Standards.
I checked the datasheet and 3 kinds of cytokine have different standard (for example TGF-beta1 standard start from 8000pg/ml to 125pg/ml, IL-10 is from 500pg/ml to 7.8pg/ml, IFN is from 300pg/ml to 4.7pg/ml)
The serum was collected by serum-separating tube from the patient whole blood.
I have two questions about the experiment:
1) Which dilution fold should I dilute the serum for the experiment? Or just use 100µL of serum for each test?
2) I bought the ELISA set from BD but I did not know that they did not provide other buffers, solutions like: coating buffer, assay diluent, washing buffer, substrate solution, stop solution.
Can I use the ELISA accessory pack from another company (Abcam)? Because I need to finish the test within this year, not enough time to order Reagent set B from BD.
Thank you for any suggestion.
Trang Phan
A couple of comments: serum concentration of IFNg in human serum is around the detection treshold. Any variation is of doubtful significance. IFNg effects are local ( paracrine) and not systemic. TGFb1 detection should be performed in platelet poor plasma, not in serum. During the clot formation, platelets release a lot of TGFb making variations in the concentrations detected more related to clotting than to the underlying condition to be investigated.
There are indeed publications detecting these cytokines in serum but usually not in high quality journals with stringent peer review.
I hope you find this useful
Hello, Phan Thi!
You can use reagents from another manufacturer for ELISA, if they have the same composition and functionality (you will also need to check the concentration).
To increase the serum concentration, you do a trial ELISA test: titrate the serum from the initial concentration (for example, in 2-fold dilution step) for each standard IL-10, IFN-y, TGF-beta1
You need to do a set of dilutions of your experimental sera to make sure you have a linear ascending curve followed by a plateau. Then you can read the values values from the linear part of the curve from the linear part of the standard curve.
If you test just one concentration of serum you will not get accurate values.
A couple of comments: serum concentration of IFNg in human serum is around the detection treshold. Any variation is of doubtful significance. IFNg effects are local ( paracrine) and not systemic. TGFb1 detection should be performed in platelet poor plasma, not in serum. During the clot formation, platelets release a lot of TGFb making variations in the concentrations detected more related to clotting than to the underlying condition to be investigated.
There are indeed publications detecting these cytokines in serum but usually not in high quality journals with stringent peer review.
I hope you find this useful
Other the points mentioned above, your dilution factor depends on what kind of experiment or stimulus you are using ( what were the mean concentrations reported by previous literatures). So look into that. Usually other than IFN gamma, I have used 10 times dilution so as to negate any effect of the color of the serum.
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