Liposomes in Immunological Trials

Introduction

The completion of immunological experiments relies on various immunoassay techniques. While carrier microspheres such as polystyrene latex particles, dextran gels, and red blood cells are often used in various agglutination tests and immunoassay techniques involving solid phase separation, when liposomes are used as carriers in immunoassays, this immunoassay technique is referred to as the liposome immunoassay (LIA).

Concept, Composition and Mechanism

LIA is a class of immunoassay techniques characterized by the release of a content label indicator by the lysis of liposomes to indicate antibody-antigen reactions, which was first created by the Japanese scholar Kataoka in 1968. After continuous refinement and development, LIA has been widely used for immunological experiments for a variety of clinical indicators such as drugs, bacterial as well as viral antigens or antibodies, tumor markers, etc. Liposomes used for immunoassays are generally immune-encapsulated liposomes with a complex phospholipid composition.

Fig. 1 Schematic diagram for the liposome immunoassay. (Darwish IA, 2006)

The basic principle of LIA is very similar to the traditional lysis dish test. Firstly, immunoliposomes with some kind of marker molecules wrapped inside are prepared, and these immunoliposomes specifically bind to the corresponding antigen or antibody in the sample to be tested with the help of ligand molecules bound on their surface. The liposomes are then ruptured by adding liposome cracking agents (e.g. complement, cytolysin, or reagents that cause special changes in the structure of the liposomes, etc.), releasing the internal markers, which are then detected by the corresponding assays. The liposomes can contain high concentrations of marker molecules, which makes the assay highly sensitive.

Markers and Assays

Markers that have been applied include organic molecules (e.g. drugs and fluorophores), enzymes, enzyme substrates, (small) proteins, RNA, DNA, ions and radioisotopes. Fluorescein and hydroxyfluorescein are the most commonly used as they are easy to measure. Larger molecules such as enzymes and enzyme cofactors can also be used, which have the advantage of more significant amplification in the assay and less leakage of the larger molecules through the liposome membrane, although they also limit the amount of liposome encapsulation. Electroactive molecular markers can also be used. For example, small inorganic salts that can be detected by detection currents, or larger ions can be encapsulated as chelates and measured by time-resolved fluorescence. Electron spin labels, such as TEMPO-choline, can also be used and detected by electron paramagnetic or spin resonance (ESR). Radioactive labels (e.g. 67Ga-deferoxamine complex) can also be used in clinical studies. In addition to hydrophilic markers, lipid-soluble substances (e.g. xylene derivatives, etc.) can also be used as markers, which can solve the problem of liposomes leaking markers.

Classification

The two main applications of liposomes in immunological experiments are liposome immunoassays and as separate carriers to assist other immunological assays. In turn, LIAs can be divided into homogeneous LIAs and non-homogeneous LIAs.

LIA can also be classified according to the type of immunoassay, such as competitive and non-competitive, direct, and indirect assays. Non-competitive assays are where the antigen is placed between two antibodies and the liposome solidifies one antibody while the other (detection) antibody is labeled. In a competitive assay, competition occurs between the antigen (sample) and the labeled antigen to obtain a limited number of antibodies. If the antibody is labeled, the competitive assay is called an immunoassay, and if the antigen is labeled, it is called an immunoassay. Direct assays use only primary antibodies against the antigen, while indirect assays rely on labeled secondary antibodies against primary antibodies, or the interaction between avermectin or streptavidin and biotin.

Individual Carriers

Liposomes can be used as individual carriers for immunological assays just like red blood cells and latex microspheres. For example, immunoliposomes can produce agglutination on the corresponding antigen or antibody, which is known as the liposome agglutination test (LAT). Liposomes can increase the sensitivity of the latex agglutination test, known as the liposome-enhanced agglutination assay (LEA). They can also be used as solid phase carriers in FIA and RIA as well as simplifying the steps of HEIA and improving the reproducibility of the procedure.

Homogeneous Liposome Immunoassays

In homogeneous assays, all components are mixed and reacted in the same vessel, and the activity of the marker changes upon antibody binding, so the antigen is selectively detected against the label (antigen).

Homogeneous liposome immunoassays can be divided into three main categories according to the liposome lysis agent: complement-mediated, cytolysin-mediated, and hexagonal phase change assays.

Complement is the most commonly used reagent for cleaving liposomes associated with antibodies, but some liposomes will be cleaved even if they are not bound to antibodies. Complement-mediated methods are sensitive and simple to perform, but complement composition is relatively complex and difficult to standardize.

The cytolysin-mediated method uses cell membrane lysins (e.g. bee toxin, streptococcal hemolysin O, etc.) to directly lyse liposomal membranes, which remain unchanged when covalently coupled to semi-antigens and their analogs, but lose their liposomal lytic activity when semi-antigens bind to antibodies, probably due to spatial blockage that prevents the two from binding. This method is generally a competitive test.

Some phospholipids can form two structures under certain conditions, a liposome, and an unstable hexagonal form, respectively, and the two can be interchanged. The use of some inducers such as divalent cations or the use of some target-sensitive immunoliposomes can lead to a phase change in the liposome after antibody-antigen binding and leakage of the marker for detection.

Heterogeneous Liposome Immunoassays

Heterogeneous detection involves one or more separation steps to remove unreacted excess reagents before non-selective detection can be performed. This separation is achieved by adsorbing or binding one of the immunogenic components to the solid phase (liposome).

Heterogeneous liposome immunoassays can be performed by adding a detergent (e.g. Triton X-100, sodium deoxycholate, etc.) or by hexagonal phase change to cause the release of markers from the liposomes. Heterogeneous liposome assays are less common than homogeneous liposome immunoassays and are mostly non-competitive. Advantages include improved sensitivity and better stability of the liposome label.

Conclusion

Liposomes have been widely used as solid phase carriers for immunological assays in therapeutic drug monitoring (TDM), detection of microbial antigens or antibodies, detection of other serological indicators, radioimmunoassays, flow cytometry assay techniques, Liposome immunosensor, etc. Liposome immunoassay has the advantages of high sensitivity, easy operation, good objectivity, easy automation and short detection time, although there are still problems such as easy oxidative degradation of phospholipids and limited preservation time of immunoliposomes, with the emergence of various new immunoliposomes, it still has good prospects in the field of immunological assays.

About BOC Sciences

We can design a variety of liposome products containing the most complex compounds. Our scientists can customize liposomes (any size and concentration) to meet your different needs. We have the ability to perform the entire program from liposome design to PK modeling.

Reference

1. Darwish IA. Immunoassay Methods and their Applications in Pharmaceutical Analysis: Basic Methodology and Recent Advances. Int J Biomed Sci. 2006 Sep; 2(3): 217-35.