Actually when I perform my ELISA sandwich assay using commercially purchased antigen it works well but when I used positive serum sample it did not work. I assumed that serum protein interfered with the assay. So, any one could let me know how to remove unnecessary protein like albumin, globulin, etc. from the serum. Please suggest solution without using kit.
I don’t think these serum proteins should make any problems.
How sure are you that your serums are actual positive? Have you tried WB?
To verify you can make a dilution series of your commercial Antigen in serum and compare to dilution in your dilution buffer check to see if there is interference.
You might be below detection level or your protein could be unavailable for the capture antibodies in sera.
I tested serum sample first using commercially available kit and later I chose positive sample for my assay. I also spiked my commercial antigen with negative serum sample it also did not work.
Dear Om,
In order to rule out nonspecific reactions between your antibodies and your serum, my suggestion for you is to use your negative serum as antigen in the ELISA and test your antibodies for staining with the appropriate secondary antibodies (both the capture and detection). If the problem is with the kit antibodies, take it up to the vendor who sold you this kit.
If the problem is your antigen I would seek other method of detection such as Western blotting since it is probably possible to purify your antigen from other proteins in the serum but in most cases it will not be worthwhile.
If you need some help, please give more details about the assay.
If the antigen you are using is a common antigen, it's could be possible that humans have already antibodies which are blocking the antibodies you are looking for.
The are to many different possibilities ...
yes, I need some help. No, it is not a common antigen it is more specific. Moreover, I am looking for antigen in the serum not for antibodies.First I immobilize the anti-antigen antibodies on the surface . after blocking with BSA I add serum in order to get the target antigen. Later, I used HRP labelled anti-antigen antibody(different clone). I hope this will help you to analyze the situation.
You can try removing the most abondant protein such as albumin by solting out approach using amonium sulfate. You can find the protocol in the "protein protocols".
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