A rapid, efficient and convenient method to detect one or more biological entities on a blotting membrane is provided. The detection can relate to the position, nature or amount of the biological substance on one or more membranes. The invention method involves a pressure assisted regiment (such as vacuum or positive
gas pressure) for the supply and removal of reagents and permits washing of the contaminants from substances embedded in the membrane to be detected using very low volumes of liquid. This method enables completion of the blocking, washing and
antibody binding steps in about 30 minutes without comprising
blot quality. In another aspect, the invention is directed to an apparatus useful in conducting the method of the invention. The device is comprised of several
layers including a porous support layer below the blotting membrane(s), a flow
distributor above the blotting membrane(s) and a well on the flow
distributor to contain the liquid to the desired area and to allow for lower starting volumes of such liquid. Preferably, the flow
distributor is a non-binding or low binding hydrophilic
porous membrane such as a 0.22 micron membrane.