Liposomes, lysis

Haga et al. developed another type of immunosensor by combining an enzyme membrane immunoassay and an enzyme sensor using oxygen electrodes (HI). In this assay antigen molecules (theophylline) are attached on the surface of the liposomes and an enzyme (horseradish peroxidase) is encapsulated in the sensitized liposome. When antibody (antitheophylline antibody) and complement are added, the enzyme is released by the liposome lysis. The enzyme activity with the NADH-NAD reaction can be determined by the oxygen electrode. When antigen is added, it competitively binds to antibodies, then liposome lysis and enzyme activity are decreased. The sensitivity of this method for theophylline determination was reported as 0.7 ng/ml. 

Complement induced immune lysis of cells and liposomes to release markers which are then detected electrochemically has been used to detect antibodies and antigens in a homogeneous format at nanomolar levels 252-256) qqjJj amperometric and potentio-metric electrodes have been employed. Unfortunately, major improvements in sensitivity appear unlikely, and instability of liposomes makes development of stable reagents for commercial systems difficult. 

Y. Tatsu, S. Yamamura, and S. Yoshikawa, Fluorescent fibre-optic immunosensing system based on complement lysis of liposome containing carboxyfluorescein, Biosens. Bioelectron. 7(10), 741-745 (1992). 

Figure 14.17. Liposome fluoroimmunoassay depiction, in which antigen-liposome conjugates containing fluorescent dye (concentration quenched) compete with analyte antigen for antibody binding sites, followed by wash and detergent lysis, to release the fluorophore for fluorescence measurement.

M. Umeda, Y. Ishimori, K. Yoshikawa, M. Takada, andT. Yasuda, Liposome immune lysis assay (LILA), J. Immunol. Methods 95, 15-21 (1986). 

Figure 2. Immune lysis of a sensitized liposome. Immobilized antibody-sensitized liposome undergoes complement-induced lysis. Released enzyme catalyzes substrate-product reaction with concomitant reduction of immobilized cofactor. The cofactor is electrochemi-cally reoxidized and the current is related to the analyte concentration. (See text for discussion.) Symbols A, analyte (antigen) Y, antibody S, sub-...

Membrane permeabilization activity of peptides is currently measured by the use of artificial membrane bilayers, such as liposomes or erythrocytes. The hposome leakage assay can be performed by using spectrofluorimetry with a concentration-dependent quenching of a dye (calcein, carboxyfluorescein) encapsulated in liposomes. Disruption of hposomes in the presence of peptide-inducing leakage will lead to an increase in the fluorescence intensity of the liposome solution. Erythrocyte lysis assay is based on the absorption of hemoglobin, which can be measured once released into the extracellular medium upon erythrocyte lysis in the presence of peptide. 

When foreign cells enter the human body they are captured by antibodies, which attach themselves to the cell membrane surface. Complement binds to these antibodies in a specific order, after which the target cells are lysed. Because the liposome bilayer is structurally similar to the cell wall, complement can be used to completely lyse antibody-bound liposomes. Attention has to be paid to the fact that although complement is specific, some liposomes are susceptible to complement lysis, without being bound to an antibody. Most liposome immunoassays are homogeneous complement-based assays. 

The doxorubicin concentration is determined at 495 nm using UV/vis measurement after lysis of the liposomes with Triton X-100 (final concentration of 0.5% V/V) (5). 

The induction of the LCMV gp33 specific CTL tesponse aftet inttadetmal immunization with a liposome fotmulation of pEGFPL-33A DNA is shown by specific lysis of te-stimulated spleen cells 9 days aftet the fitst immunization in Fig. 3. 

Smolarsky, M., Teitelbaum, D Sela, M., and Gitler, C. (1977) A simple fluorescent method to determine complement mediated liposome immune lysis. Immunol. Meth. 15, 255-265. 

L.I. Grossweiner, A.S. Patel, J. Grossweiner (1982). Type I and Type II mechanisms in the photosensitized lysis of phosphatidylcholine liposomes by hematoporphyrin. Photochem. PhotobioL, 36, 159-167. 

Kellogg, E.W. and Fridovich, I. (1977). Liposome oxidation and erythrocyte lysis by enzymatically generated superoxide and hydrogen peroxide. ]. Biol Chem., 752, 6721-6728... 

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