Liposome Immune Lysis Assay Research Articles

Immunologic Aspects of Liposomes for Medical Applications

Recent progress of immunologic aspects of liposomes for medical applications are reviewed. Liposomal application for immunoassay, targeting and adjuvant activity for vaccination are discussed. Development of complement dependent liposome immune lysis assay enabled to measure small amount of serum proteins, such as cancer markers, and extended to various microbial toxins. New technology of targeting for malignant tumors have been developed. The use of liposomes as an safe, versatile, universal adjuvant that can induce humoral and cellmediated immunity to antigen have been investigateted increasingly.

An automated micromethod for detection of lytic antimycobacterial antibodies by immune lysis of liposomes sensitized to tuberculin.

Calcein was concentrated inside multilamellar liposomes (MLV) by encapsulation through an osmotic gradient in order to increase its quenching. Immune lysis of these MLV sensitized to PPD by direct encapsulation or by exposure to preformed vesicles was studied in the presence of rabbit anti-BCG serum and of a non-immune rabbit serum as a source of complement, successively added in the wells of a microplate to a final volume of 0.1 ml. A 40 to 50% release of encapsulated calcein was observed after 5 min using a Titertek Fluoroskan II. Preformed liposomes exposed to PPD were more sensitive to anti-BCG serum than liposomes formed in PPD. Trials with human sera revealed a significantly higher level of specific lytic immunoglobulins in tuberculous patients than in non-tuberculous subjects. Key words: Liposome immune lysis assay; lytic antimycobacterial antibodies; tuberculosis, human; test automation.

Homogeneous immunoassay for α2 plasmin inhibitor ( α2PI) and α2PI-plasmin complex : Application of a sandwich liposome immune lysis assay (LILA)_technique

The measurement of the α 2 plasmin inhibitor ( α 2PI and α 2PI-plasmin complex is important for a complete understanding of fibrinolytic conditions. Using monoclonal antibodies against α 2PI, a sandwich liposome immune lysis assay (LILA) has been used for the quantitation of α 2PI and the α 2PI-plasmin complex. In the assay system for the measurement of α 2PI, anti- α 2PI monoclonal antibodies were covalently coupled to liposomes and specific lysis of liposomes was observed when the liposomes were incubated with the α 2PI antigen, TNP haptenized second monoclonal antibody against α 2PI and complement activating rabbit anti-TNP antibody. The same liposomes and rabbit anti-plasminogen antibody could be used for the homogeneous determination of the α 2PI-plasmin complex. The former assay suggests that monoclonal antibodies lacking complement-activating ability can be used in the sandwich LILA technique. The second application suggests that the LILA technique is capable of measuring heterocomplexes. These assays, which involve the same analytical system, are simple, fast and highly sensitive. They are potentially useful in determining the fibrinolytic status of patients.

Application of liposomes to generation of monoclonal antibody to glycosphingolipid: production of monoclonal antibody to GgOse4Cer.

Liposomes were applied to the immunization with GgOse4Cer and screening for production of monoclonal antibody to GgOse4Cer. Four-week-old and 22-week-old Balb/c mice were immunized with GgOse4Cer and Salmonella minnesota R595 lipopolysaccharides incorporated liposomes which were composed of dipalmitoyl-phosphatidylcholine and cholesterol. Since antibody response to GgOse4Cer was higher in 22-week-old than 4-week-old Balb/c mice after immunization, 22-week-old Balb/c mice were used for the immunization prior to generation of the monoclonal antibodies to GgOse4Cer. The screening of monoclonal antibodies was performed by complement-dependent liposome immune lysis assay using GgOse4Cer-containing liposomes. Six kinds of monoclonal antibodies, AG-1, -2, -3, -4, -5, and -6, of the IgM class were established. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay using various glycosphingolipids incorporated in liposomes and by thin-layer chromatography (TLC) with immunostaining. All of the monoclonal antibodies reacted only with GgOse4Cer in the liposome immune lysis assay. In addition, the monoclonal antibodies reacted only with GgOse4Cer in the TLC immunostaining. However, none of the monoclonal antibodies obtained was capable of removing natural killer activity from C3H/He mice spleen cell suspensions in vitro. Liposomes may be useful in the procedures of immunization and screening for generation of antiserum and monoclonal antibody to GSLs.

Liposome immune lysis assay (LILA): Application of sandwich method to determine a serum protein component with antibody-bearing liposomes

A complement-dependent liposome immune lysis assay (LILA) using carboxyfluorescein (CF)-entrapped liposomes bearing antibody was developed to measure C-reactive protein (CRP) antigen as a model of protein antigens in human sera. Goat anti-CRP antibody was covalently coupled to liposomes, and a specific lysis of the liposomes could be observed when the liposomes were incubated with both rabbit anti-CRP antibody (secondary antibody) and CRP antigen in sera in the presence of guinea pig complement. In this assay system, so-called sandwich assay, CRP (a multivalent antigen) bound to the liposomes bearing anti-CRP antibody and subsequently secondary antibody, which activated complement efficiently. The amount of CF released by a complement-dependent liposome immune lysis was proportional to CRP concentrations. This sandwich assay was simple, fast, highly sensitive, and covered the ranges 10–300 ng of CRP/ml in a homogeneous mode, that is, one where no separation step was employed. The results correlated well with those obtained by single radial immunodiffusion and enzyme immunoassay. This assay system would be applicable to the measurement of other protein antigens.

Liposome immune lysis assay (LILA): a simple method to measure anti-protein antibody using protein antigen-bearing liposomes

A new simple immunoassay technique using immune lysis of liposomes was developed to measure antibody against protein antigens. Multilamellar liposomes were composed of dipalmitoylphosphatidylcholine, cholesterol and phosphatidylethanolamine substituted with the hetero-bifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP). The protein antigen (human IgG) was coupled to these liposomes after treatment with SPDP and mild reduction. As a release marker, carboxyfluorescein (CF) was entrapped in the liposomes. The CF release was specific to anti-human IgG antibody and depended on the presence of complement. This technique could detect 10 −15 mol of anti-human IgG antibody or human IgG. The liposomes were stable over 8 months at 4°C under nitrogen gas.