The feasibility of utilizing dehydrated liposomes in the development of a simple immunoassay device for point-of-care diagnostics or field assays was demonstrated. The recovery of liposomes after a cycle of dehydration and rehydration was studied using biotin-tagged, dye-loaded liposomes with antibiotin antibodies immobilized in a defined zone on nitrocellulose strips. Liposomes were vacuum-dehydrated on the strip at a location below the antibiotin zone. The strip was placed in a tube containing a carrier solution and capillary action brought the solution to the dehydrated liposomes, rehydrated them, and caused them to migrate to the antibody zone where intact liposomes were captured and measured optically. High concentrations of either trehalose or sucrose external to the liposomes and both polyvinylpyrrolidone and gelatin in the membane blocking reagent were essential for preservation of the dehydrated/rehydrated liposomes on nitrocellulose. Between 70 and 80% of the liposomes were recovered on the nitrocellulose strips after a cycle of dehydration and rehydration. The dehydrated liposomes on the strips were stable for at least 1 year when stored in vacuum-sealed plastic bags at 4°C. The technique was successfully applied to the development of a rapid one-step strip immunoassay for biotin.

The potential for development of a multi-analyte liposome immunomigration solid-phase competition (LIM-SPC) assay based on multivariate calibration of spectral reflectance measurements was investigated. A simulation of a multi-analyte LIM-SPC assay was carried out using a mixture of three analyte-tagged liposome preparations, each containing a different colored dye. The liposome preparations were combined according to a statistical experiment design and allowed to migrate up nitrocellulose test strips to a binding zone. After the liposomes were captured at this Zone, the reflectance spectrum of the zone was measured. Partial least squares regression was used to develop multivariate calibrations. For the liposome-encapsulated dyes, the coefficients of determination obtained for carboxyfluorescein, sulforhodamine B and isosulfan blue were 0.902, 0.990 and 0.977, respectively. It was concluded that the approach used was suitable for a multi-analyte LIM-SPC.

The feasibility of a simple, single-use immunomigration strip assay for alachlor was previously demonstrated. In the device, capillary action caused alachlor and alachlor-tagged, dye-containing liposomes to migrate through an anti-alachlor antibody zone, on a plastic-backed nitrocellulose strip, where competitive binding occurred. Unbound liposomes continued migration to a liposome capture zone, where they were quantified by densitometry. The amount of liposome-entrapped dye measured in this zone was directly proportional to the alachlor concentration in the sample. This report describes modifications to various components of the system, leading to improvement in the sensitivity of the assay to the point where the maximum contaminant level of alachlor, 2 ppb, can be easily detected. Measurements of liposome size and antibody cross-reactivity are also presented. The new methodology involves acid treatment of the antibody and the use of preincubation of the analyte-tagged liposomes, free analyte and anti-alachlor before initiating migration. This results in strong interactions between the anti-alachlor, liposome and strip such that liposomes that have bound to antibody do not migrate. This inhibition of migration is reversed by free analyte, which competes with the liposome for the available antibody binding sites. As in the previous assay, unbound liposomes migrate to a capture zone where they can be quantified, and the color intensity of this zone is directly proportional to the amount of analyte present. This technique produces an assay capable of detecting alachlor at levels down to 1 ppb.

Liposome-amplified competitive immunoassay systems for the herbicide alachlor, in both laboratory-flow injection and field immunomigration formats, are described. The preparation and characteristics of analyte-tagged liposomes, along with details of both types of assay are given. The laboratory assay is designed for automation and measurement of a large number of samples in the laboratory, whereas the field assay is designed for rapid field screening of large numbers of samples by non-technical personnel. The advantages of liposome immunoassay compared to more traditional formats are discussed, as are the problems posed by liposome curvature.

Single-use strip assays using immunomigration and immunoabsorption formats have been developed for the extra-laboratory determination of environmental contaminants. Dye-loaded liposomes are used as the amplification strategy in these immunoassays. Details of the assay performance and liposome characteristics are given.

A recently developed shallow-well microtiter plate, made from a specially formulated polymer that binds proteins, peptides and nucleic acids rapidly and efficiently, has been investigated for use in solid-phase immunoassays as well as for protein and phospholipid assays. The assay uses quantitation of the color intensity of a dye on the solid phase by means of a simple desk-top scanner coupled to a computer which allows the gray-scale density of the color to be easily and accurately measured. Consequently. this approach is independent of the absorption spectrum of the dye used.These studies demonstrate that the novel technology incorporated into these unique shallow-well microtiter plates has potential in both protein and immunoassays. In the latter case, there are considerable savings in both time and material costs over conventional ELISA methods. The scanning software provided for the solid-phase immunoassay is user configurable for other densitometry assays on different solid matrices and can be read directly into a spreadsheet file for subsequent data manipulation.

A novel prototype immunosensing device has been developed which incorporates the simplicity of competitive immunochromatography with the rapid signal amplification capabilities of marker-loaded liposomes. An avidin capture zone provides direct readout of the analyte concentration.

The feasibility of a simple, single-use immunomigration system has been demonstrated, using Alachlor as a model environmental contaminant. In the device, capillary action causes Alachlor and Alachlor-tagged, dye-containing liposomes to migrate through an anti-Alachlor antibody zone, on a plastic-backed nitrocellulose strip, where competitive binding occurs. Unbound liposomes continue migration to a liposome capture zone, where they are quantified either visually or by densitometry. The amount of liposome-entrapped dye that is measured in this zone is directly proportional to the Alachlor concentration in the sample.