We are currently using indirect ELISA to measure human serum levels of anti-troponin-I, anti-troponin-T and anti-tropomyosin antibodies? This indirect ELISA is not sensitive enough for our scope. Does anyone have experience with an alternative protocol for detection of these "anti-heart" antibodies? Thank you Giovanni
Hello Giovanni.
If you have not tried HRP using Biotin-Straptavidin linkers to amplify the signals, try the more specific and stronger detection system. However, most of all it is critical to prepare serum properly before ELISA. Centrifuge the collected serum immediately after the withdrawal at 2,500 rpm for 10 min to remove any cell debris or aggregates. Either store or freeze them for later use. If they are frozen and used immediately, centrifuge again to remove any particles or invisible aggregates to unmask the immunological reactions at 12,000rpm for 20 min. Take the upper parts of supernatant. They are ready for use for ELISA.
Best wishes.
You might consider switching to PLASMA for your sample collection -- not serum. It's possible that you may be losing protein content (even antibodies) due to random protease activity, and of course serum itself (by definition) is the result of extensive protease activity. It's easy enough to test: collect one serum tube and one plasma tube (I would suggest EDTA) from the same subject and see if you get improved assay results.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA