Liposomes avidin

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). 

Biotinylated liposomes usually are created by modification of PE components with an amine-reactive biotin derivative, for example NHS-LC-Biotin (Chapter 11, Section 1). The NHS ester reacts with the primary amine of PE residues, forming an amide bond linkage (Figure 22.19). A better choice of biotinylation agent may be to use the NHS-PEG -biotin compounds (Chapter 18), because the hydrophilic PEG spacer provides better accessibility in the aqueous environment than a hydrophobic biotin spacer. Since the modification occurs at the hydrophilic end of the phospholipid molecule, after vesicle formation the biotin component protrudes out from the liposomal surface. In this configuration, the surface-immobilized biotins are able to bind (strept)avidin molecules present in the outer aqueous medium. 

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... 

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. 

Figure 22.21 Antibodies may be conjugated to liposomes using an indirect approach incorporating a (strept)avidin-biotin system. Biotinylated liposomes may be complexed with biotinylated antibodies using (strept)avidin as a bridging molecule or may be complexed with an antibody-(strept)avidin conjugate.

Hashimoto, K., Loader, J.E., and Kinsky, S.C. (1986) Iodoacetylated and biotinylated liposomes Effect of spacer length on sulfbydryl ligand binding and avidin precipitability. Biochim. Biophys. Acta 856, 556-565. 

Kaasgaard T, Mouritsen O, Jorgensen K. Screening effect of PEG on avidin binding to liposome surface receptors. Int J Pharm 2001 214 63. 

Fig. 20. Targeting of the endothelial integrin a P3 as a specific angiogenesis marker. The receptor is recognized by a biotinylated antibody that is then bound by an avidin moiety bearing a Gd(III) loaded liposome.

Q Yang, X-Y Liu, M Hara, P Lundahl, J Miyake. Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin-biotin binding. Anal Chem 280 94-102, 2000. 

X-Y Liu, Q Yang, C Nakamura, J Miyake. Avidin-biotin-immobilized liposome column for chromatographic fluorescence on-line analysis of solute-membrane interactions. J Chromatogr B 750 51-60, 2001. 

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 13) (Savage et al., 1992). 

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