I striped three test lines. The top and bottom ones turn purple the full size of where the antibody was spread, but the middle one only appears as a very thin line at the bottom of where it was spread. I even doubled the width of the spreading of the middle line and it still shows up thin (see pictures). What might be the issue and how do I correct it?
Hi Thomas,
this may be a reflection of antibody activity and affinity.The Antigen contacts the bottom of the middle test line first and directly binds there. It is concentrated there, forming a sharp thin line. I think you don't need to correct this.
Tell us more about the reagents you are using and the how exactly you have done the experiment, then we may come up with additional explanations.
Cheers Markus
Markus Sack Thanks for the feedback. The goal is that all the lines look the same width when positive. The three lines are for different analytes so having one that always shows up thin is not ideal. We are diluting an antigen sample (recombinant and native show the same results) in our running buffer (Tris, NaCl, BSA, Detergent, Urea) and adding 100uL to the sample pad and running for 20mins.
I agree with Markus that this is sounds like it is caused by the high affinity of the antibody in the middle line. Have you tried diluting the concentration of the capture antibody in this line? Perhaps this would allow the antigen to penetrate further into the zone and thus broaden the line.
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