We prepare the gold conjugate with 20 mm gold nanoparticles and 100 mM phosphate buffer pH 8.35, and then we add the mAb previously dialyzed with to a concentration of 16 ug/ml. The conjugate is incubated at room temperature in a rotator and after 1 hour we add 4 mM phosphate buffer with 10 mg/ml BSA. We centrifuge it so the supernatant is discarded and we add 4 mM phosphate buffer with 5 mg/ml BSA. The centrifugation is repeated and we resuspend the pellet with borate buffer 5 mM with 5% sucrose and 10% trehalose.
The conjugation pad is blocked by immersion with 50 mM phosphate buffer with 0.5% PVA, 1% Triton x-100 and 0.5% BSA. We let it dry for 1 hour at 37ºC before adding the conjugate.
We ensamble the complete strip and run the assay with running buffer (10 mM HEPES, 150 mM NaCl, 1% BSA and 0.05% Tween 20) with an excess of the antigen.
With these conditions we achieved the complete release of the conjugate from the pad but we’ve seen false positive signal when we run the tests.
We’ve tried to block the membrane with different concentrations of BSA for 30 min, washing it twice with 5 mM PBS and 0.05% SDS, and it reduced the false positive signal but it remained visible. The antibody for the test line was dialyzed as well, because the original stock have sodium azide but after doing this we have the same results. I appreciate if someone has any idea of what could it be the problem. Thanks!
Often selectivity/cross-reactivity of the antibodies is the reason for problems. General blocking protocols usually cannot resolve these issues. Do you use polyclonal antibodies in any step?
Michael G. Weller No, both are mouse monoclonal antibodies and we actually try to invert these for the conjugation and test line but it didn't work.
Unfortunately, many things can go wrong with antibodies, even monoclonals. A lot of antibodies are not fit for purpose. Short: Try another antibody if possible.
Use 5% sucrose in the drying buffer. Try higher concentrations of detergents in the run buffer as well as different detergents. Sonicate the gold conjugate prior to drying (1-2 10seconds cycles should do).
- Badges
- Science method