Capture ELISA - human Serum - (Mar/02/2006 )
I am working on sandwich ELISA to detect a large human protein. My protocol is as follows:
1. Capture with polyclonal ab (derived from chicken)
2. Block with 5% BSA
3. Wash
4. Add human serum diluted in PBS and Standards (recombinant protein) in PBS
5. Wash
6. Add monoclonal (mouse derived anti human) ab Diluted in 5% BSA
7. wash
8. secondary (goat anti mouse) Diluted in 5% BSA
9. substrate/stop/read
Problem:
1. I get an excellent standard curve
2. The calculated test samples increase as I dilute the human serum.
Am I diluting out an inhibitor?
Dilute out the serum samples and standards in BSA?
I don't think the results you're getting are completely unheard of. Your neat serum sample is obviously more complex matrix than the standards in PBS. As you dilute the serum out you're actually getting closer to the standards in PBS. There must be something interferring with the serum.
We dilute our standards and samples for ELISA in 1% BSA/PBS after blocking in 5% BSA/PBS. You could try diluting the standards normally and in parallel in serum/dilution of serum to see how it effects you standard curve.
Ceri
1. Capture with polyclonal ab (derived from chicken)
2. Block with 5% BSA
3. Wash
4. Add human serum diluted in PBS and Standards (recombinant protein) in PBS
5. Wash
6. Add monoclonal (mouse derived anti human) ab Diluted in 5% BSA
7. wash
8. secondary (goat anti mouse) Diluted in 5% BSA
9. substrate/stop/read
Problem:
1. I get an excellent standard curve
2. The calculated test samples increase as I dilute the human serum.
Am I diluting out an inhibitor?
Dilute out the serum samples and standards in BSA?
I am not too sure but i dont think that your results are unexpected. As you dilute the human serum sample, you are also diluting the albumin content in your serum which i believe would have an effect on your curve. I think by preparing standard curves with different percentages of BSA/PBS would help to give you a better understanding of your curve and how the matrix effect would alter your curve/sensitivity etc.... let me know how it goes..
hi,
try diluting even further too see what is the max dilution u get where you stop seeing increasing conc of your protein of interest, you take that point as starting point and futher dilute your protien, at this point you must see that your protien conc going down (assuming that you max dilution was the highest dilution of your inhibitor) then you can quantify in relation to your standard .
in other point of view, i donot think that you are diluting inhibitor coz in that case you also diluting your protein further, so the ratio between your protien of interest and (assumed) inhibitor is going to be the same.
based on above assumption you should some clue aabout your protein conc in serum.
gud luck
We dilute our standards and samples for ELISA in 1% BSA/PBS after blocking in 5% BSA/PBS. You could try diluting the standards normally and in parallel in serum/dilution of serum to see how it effects you standard curve.
Ceri
I repeated the assay with the standards in PBS and PBS with .1% BSA and finally with 1% PBS with BSA - the slope of the curve decreases with increase in BSA content with good R value. Unfortunately I am still not getting a linear curve with my human serum samples with serial dilutions. My Research mentor suggests using something to eliminate weak hydrophobic interactions such as heparan sulfate or using tween at some point. Any suggestions or ideas.
thanks
I am working on sandwich ELISA to detect a large human protein. My protocol is as follows:
1. Capture with polyclonal ab (derived from chicken)
2. Block with 5% BSA
3. Wash
4. Add human serum diluted in PBS and Standards (recombinant protein) in PBS
5. Wash
6. Add monoclonal (mouse derived anti human) ab Diluted in 5% BSA
7. wash
8. secondary (goat anti mouse) Diluted in 5% BSA
9. substrate/stop/read
Problem:
1. I get an excellent standard curve
2. The calculated test samples increase as I dilute the human serum.
Am I diluting out an inhibitor?
Dilute out the serum samples and standards in BSA?
I am not too sure but i dont think that your results are unexpected. As you dilute the human serum sample, you are also diluting the albumin content in your serum which i believe would have an effect on your curve. I think by preparing standard curves with different percentages of BSA/PBS would help to give you a better understanding of your curve and how the matrix effect would alter your curve/sensitivity etc.... let me know how it goes..
I repeated the assay with the standards in PBS and PBS with .1% BSA and finally with 1% PBS with BSA - the slope of the curve decreases with increase in BSA content with good R value. Unfortunately I am still not getting a linear curve with my human serum samples with serial dilutions. My Research mentor suggests using something to eliminate weak hydrophobic interactions such as heparan sulfate or using tween at some point. Any suggestions or ideas.