We have bought a detection antibody and used it to run an indirect ELISA. Initially, we were not getting meaningful readings when diluting the recombinant standard sample to the picogram level, so we changed the dilution concentration of the recombinant protein to the nanogram level and got readings.
Should I just get the capture antibody in the matched pair and switch to doing a sandwich ELISA instead and follow the manufacturer's recommended standard dilutions of 1000 pg/ml - 8 pg/ml? Scouring through the literature, everybody uses kits and not manual making of ELISA, even less people use indirect ELISA as a method. So I am here asking for help and advice, any papers appreciated!
For complex samples such as cell extracts, the sandwich ELISA may be more sensitive than the indirect ELISA because it captures all of the antigen onto the surface, no matter how little of the antigen is present, until the capture antibody is saturated, and everything else is washed away.
For the indirect ELISA, the sensitivity is limited by the proportion of the sample that consists of the antigen of interest. There is a limited binding capacity of the surface, and everything in the sample competes for the limited surface.
So, for the indirect ELISA, you probably need to use higher antigen concentrations for the standard curve than you would for the sandwich ELISA.
Indirect ELISA: In an indirect ELISA, the standard curve is created using a purified form of the primary antibody (the one specific to your target analyte). This is because the primary antibody is what binds to the target analyte in your samples. The secondary antibody, which is labeled with an enzyme, is then used to detect the primary antibody bound to the analyte. The standard curve concentrations should still cover the expected range of analyte concentrations in your samples.
- Badges
- Science topic
- Similar topics
- Computer Science
- Program