Preparation of Antibody-Coupled Liposomes Containing Horseradish Peroxidase as a Marker Molecule

Abstract

Horseradish peroxidase (HRP) was encapsulated in liposomes prepared by an extrusion technique. The liposomes were coupled covalently to anti-rabbit IgG using N-hydroxysuccinimide ester palmitic acid as a component of liposomes. The number of encapsulated HRP molecules per liposome was about 800. A large portion of HRP was encapsulated inside the liposomes for about one week at 4˚C. The catalytic activity of HRP was measured by a luminol chemiluminescence (CL) method and was found to be almost constant during storage. The CL intensity per antibody in the detection of HRP encapsulated in the antibody-coupled liposomes was 125-times greater than that of HRP conjugated directly to the antibody.

Application of Horseradish Peroxidase-Encapsulated Liposomes as Labels for Immunodotblotting

“…6 The procedure preparing for multilamellar vesicles (MLVs) containing HRP was the same as that previously reported. 5 Next, biotinylated MLVs containing HRP were extruded through polycarbonate filters with 1000 nm pore size. The filters were mounted in LiposoFast-Basic (Avestin Inc.) fitted with two 0.50 ml Hamilton syringes.…”

“…4 In addition, the HRP-encapsulated VETs were coupled covalently to anti-rabbit IgG (antibodytagged VETs). 5 The detection of HRP encapsulated in the antibody-tagged VETs was made by a luminol chemiluminescent (CL) method. On the other hand, biotin-tagged molecules are currently more universal as a marker in immunoassays compared to antibody-tagged marker molecules.…”

Luminol Encapsulated Liposome as a Signal Generator for the Detection of Specific Antigen-antibody Reactions and Nucleotide Hybridization

“…14,15 The supposed emitter of luminol is the excited 3-aminophthalate anion with maximum emission at 425 nm. 16 There are two methods for coupling biomolecules to a liposome surface, which include covalent 17,18 and noncovalent reactions. 19,20 The noncovalent methods, which may be based on the reaction of Protein A (for binding to antibodies) 20 or of streptavidin (for binding to biotinylated molecules), 19,21 provide a milder reaction condition.…”