Preparation of Biotinylated or Avidin-Conjugated Liposomes

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

However, since many of the traditional biotinylation reagents, such as NHS-LC-biotin contain hydrophobic spacers, their use with amphipathic liposomal constructions may not be entirely appropriate. A better choice may be to use a hydrophilic PEG-based biotin compound that creates a water-soluble biotin modification on the outer aqueous surface of the liposome bilayer. Biotinylation reagents of this type are discussed in Chapter 18, Section 3. 

Biotinylation may be done before or after liposome formation, but having a stock supply of biotin-modified PE is an advantage, since it can then be used to test a number of liposomal recipes. In addition, only a very small percent of the total lipid should be biotinylated to prevent avidin-induced aggregation in the absence of antigen. It is difficult to control precisely 

The following method for the formation of a biotinylated liposome is adapted from Plant et al., 1989). It assumes prior production of B-PE. 

Prepare a biotinylated liposome construct by first dissolving in chloroform, the lipids DMPC cholesterol dicetylphosphate (Sigma) at mole ratios of 5 4 1, and adding to this solution 0.1 mol percent B-PE. Larger mole ratios of B-PE to total lipid may result in nonspecific aggregation of liposomes in the presence of avidin. Maintain all lipids under an inert atmosphere to prevent oxidation. 

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