I use 1% BSA in 10 mM phosphate buffer (pH 7.4) solution when striping my nitrocellulose membranes with antigens and antibodies. However, I always observe deformed flow when my sample or running buffer approaches the test and control lines regardless of the antigen and antibodies used. I also read MERCK's guideline (Rapid Lateral Flow Test Strips) and it said that 0.05% SDS or 0.005% Tritonx-100 should be added to the capture reagent solution to promote rewetting of the membrane. So, I tried both surfactants separately with the usual 1% BSA in the aforementioned phosphate buffer, however, the results were the same. I am considering increasing the concentration of the surfactants and testing the system again.
- Has anyone found a capture reagent solution formula that prevents deformed flow and air-locked regions?
- Is it possible that 1% BSA is too high of a concentration that it might be blocking the membrane pores? If so, what concentration is recommended?
Could be too much protein. Are the 1% BSA required by your protocol? What happens if you leave it out or use less, say 0.1%?
I haven't tried leaving BSA out or using a lower concentration. I plan on testing both conditions to see the effects on the flow. I have noticed that using BSA at higher concentrations in pretreatment solutions often renders the conjugate pad hydrophobic. Therefore, it might be having the same effect on the membrane reaction lines. I'll provide an update here once I have conducted the experiments.
Update:
I conducted two sets of experiments as follows:
- The test and control line reagents were diluted in 10 mM phosphate buffer (pH 7.4) containing 0.1% BSA and striped onto the nitrocellulose membrane
- The test and control line reagents were diluted in 10 mM phosphate buffer (pH 7.4) without any BSA and striped onto the nitrocellulose membrane
In both cases, the test and control lines appeared with significantly less flow deformation and air-entrapment at the T and C lines. However, in the case where no BSA was used, the T and C lines were not smooth at the upper and lower edges and were rather zigzagged. So, a small amount of BSA is necessary for the formation of sharp T and C lines. In order to further enhance flow, 0.005% TritonX-100 or 0.05% SDS can be added into the striping buffer. Another update will follow after performing these tests.
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