Sui Ti A. Siebert

Liposome immunomigration field assay device for Alachlor determination

Abstract 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.

Improved liposome immunomigration strip assay for alachlor determination

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 immunosensing devices for environmental contaminant screening

Abstract 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.

Investigation of a Novel Microtiter Plate Support Material and Scanner Quantitation of Immunoassays, Proteins and Phospholipids

Abstract 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 ca...

Development of a signal measurement technique for application in multi‐analyte liposome immunomigration assays

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 car‐boxyfluorescein, 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.

Immunoassay Devices for Extra-Laboratory Measurements of Toxic Chemicals Based on Capillary Migration and Liposome Amplification

Simple, single-use biosensing devices are being developed based on the immunologic recognition of specific environmental and food commodity contaminants in combination with signal amplification strategies using marker-loaded liposomes to provide high sensitivity. Solution flow in the devices is controlled by capillary action and detection can be based on marker color or electroactivity. Several areas of investigation are being addressed: 1) immobilization of antibodies onto the solid phase of the capillary bed; 2) liposome surface density of analyte molecules conjugated to phospholipids; 3) capillarity-driven timing sequence for optimum flow and interaction of components; 4) concentration/measurement zone based on an avidin-capture mechanism; and 5) evaluation of several modes of detection/quantitation. Preliminary laboratory testing of several prototypes of this device based on optical detection has demonstrated the feasibility of this approach.