Capture reagents must be applied to the membrane as a distinct line so that the signal can be clearly read (10,11). The concentration of the capture reagent in the application buffer and the dispensing rate determine the mass of capture reagent that is applied per unit of length. If the mass applied remains constant, then the width of the line will determine the intensity of a signal. Intuitively, spreading the same mass of capture reagent across a wider line makes the signal appear weaker. Whereas assessment of strong signals is not adversely affected by wider lines, a weak signal may be impossible to see because the color is too diffuse. In general, the lines should be as thin as possible.
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