Preparation of Antibody—Liposome Conjugates

Dissolve the hapten or antigen to be encapsulated at a concentration of 21 pmol/ml in degassed, nitrogen-purged lOmM HEPES, 0.15M NaCl, pH 6.5. 

Protocol for the Coupling of Peptide Haptens Containing Sulfhydryl Groups to Liposomal Vesicles 

Dissolve a sulfhydryl-containing peptide hapten at a concentration of 25 pmol/ml in degassed, nitrogen-purged lOmM HEPES, 0.15M NaCl, pH 7.0. Add the peptide solution to the liposome suspension at a molar ratio necessary to obtain at least a 5 1 excess of thiol groups to the amount of maleimide groups present (as MPB-DPPE). 

React overnight at room temperature. Maintain an inert-gas blanket over the vessel to prevent lipid oxidation. 

However, there are problems associated with the use of antibody—liposome conjugates for drug delivery in vivo. Particularly, since lipid vesicles are huge compared to 

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 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 13) (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 8, Section 3.1). The NHS ester reacts with the primary amine of PE residues, forming an amide bond linkage (Fig. 348). Since the modification occurs at the hydrophilic end of the phospholipid molecule, after vesicle formation the biotin component protrudes from the liposomal surface. In this configuration, the surface-immobilized biotins are able to bind 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 

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Reliable lipid and protein analysis of the prepared antibody-liposome conjugate is essential for proper characterization and subsequent interpretation of results obtained in the application of the conjugates. In addition, we strongly advise that the liposome conjugate size be determined with a particle sizer, if one is available, since liposome size plays such an important role in the pharmacokinetics of the system in vivo. 

Mix lipids (as showing on Table 2 for preparing 30 mg of appropriate sample) in a 250 mL round bottom flask according to the desired lipid molar ratio. For liposomal conjugation with an antibody or similar targeting agent, MPB-PE or PEG-PE is to be added. 

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