Characteristics of microblotting assay using immunoliposomes

To clarify the applicability of a liposome immunoblotting assay to microassay systems, the effects of the sample volume blotted on polyvinylidiene fluoride (PVDF) membrane on the sensitivity and signal intensity were studied and compared with those in a conventional immunoblotting assay utilizing enzyme-labeled antibody. In the liposome immunoblotting assay, signal intensities per unit area (signal density) were almost the same or increased slightly with decreasing blotted sample volume (from 20 to 2 microl) at the same concentration of analyte (human IgM) adsorbed on PVDF membrane. A substrate, 4-chloro-1-naphtol, for color development formed colored precipitates inside the liposomes. Thus, the colored signal was detected without loss of intensity. On the other hand, in the conventional immunoblotting assay, signal densities decreased with decreasing sample volume at the same concentration of analyte. This is caused by partial losses of the colored signal into the substrate solution by diffusion, because the fraction of loss increases with decreasing sample volume. These results show that the liposome immunoblotting assay is a promising method for the detection of analytes blotted in small areas, because its sensitivity is higher than that of the conventional blotting assay.