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Oak Ridge Poster Sessions |
Stago International, 9 Rue des Frères Chausson, 92600 Asnieres, France
a author for
correspondence
Modern clinical practice requires small, easy-to-use, quantitative, automated near-the-patient test systems. Any new test system must match the clinical needs for faster and cost-effective technological approaches. The TECHNODISK concept addresses many of the key features required for rapid immunoanalysis, i.e., microfluidics, speed, safety, flexibility, and capability for multitest panels.
The system is a flat cylinder cassette named TECHNODISK composed of two plastic parts. The bottom part is a polystyrene plastic mould designed with three concentric areas separated by walls in which holes have been made that allow for the circulation of fluids from the inner area, to the middle chamber, and finally to the outer waste area. Fluid is moved between the different areas by rotation on a central drive that produces sufficient centrifugal force to allow the fluid transfers. The lid is a flexible plastic mould that closes tightly over the bottom part, forming a flat cylindrical cassette that can be turned from the bottom by the action of a peripheral drive. Two holes are punched through the plastic to allow addition of the sample and wash reagent into the middle chamber (sample hole) and the addition of fluid into the inner area of the chamber to pick up the coated microbead conjugate (inner hole). The bottom part can be moved relative to the lid to two fixed positions (clockwise and counterclockwise) that open and close valves in the cassette. In one position, holes between the inner area and middle chamber are open, and in the second position holes between the middle chamber and the waste chamber are opened. By these external actions and rotation of the system, the liquid is either held in a specific area or moved from the inner area to the middle chamber or from the middle chamber to the waste chamber. Built into the waste area is an absorbant that ensures that all the liquid is retained inside the circular cassette.
The middle chamber is coated with antibody(ies) or antigen(s) that are specific to capture the analyte in the liquid sample. The inner area receives the conjugates coated on micro beads (2-µm diameter).
The procedure is as follows:
The sample to be analyzed is introduced into the device with a pipette via the sample hole. The cassette is rotated slowly to allow the sample to circulate over the whole of the middle chamber containing the specific capture sectors. The cassette is left for an optimized incubation time during which the liquids are kept mixed by slow movement of the cassette. The relative movement of the lower portion with respect to the lid opens and closes the relevant valves, and then by faster rotation of the cassette, the sample is moved into the waste area.
Water is introduced (via the inner hole) automatically into the inner area containing the coated microbead conjugate. These are suspended before transfer to the middle chamber. A second incubation step, again using slow rotation for mixing, allows binding of the coated microbead conjugate to the captured antibodies or antigens. After incubation, the reagent is transferred to the waste by fast rotation of the cassette and the movement of the lower portion relative to the lid to open the relevant valves (for validation the suspended beads were added directly into the middle chamber).
The cassette is automatically washed several times by introducing water into the middle chamber (via the sample hole), with slow rotation to pick up the free beads and then fast rotation to transfer the wash water to the waste area.
The endpoint of the reaction is assessed by the means of a micro charge-coupled device (CCD) camera connected to software that converts the camera signal into the absolute number of events on the surface; these events are the microbeads (count particles) linked to the captured antigen or antibody. This method gives a digital signal that allows a very wide measuring range and is easily automated using a microcontroller.
The detection principle was evaluated on 96-well microplates to allow an easy comparison with fluorescence immunoassay (FIA) and ELISA methods and confirmed on the TECHNODISK device. Two different applications were used for detection and quantification of a soluble antigen and in a serological assay, respectively.
Detection and quantification of a soluble antigen.
For convenience, the antigen used in this experiment was a biotinylated monoclonal mouse antibody, CD146.
Two 96-well microplates were coated with 10 mg/L purified goat anti-mouse IgG, 100 µL per well. The middle area of a TECHNODISK was coated with 500 µL of the same solution. The coating was saturated with a solution of phosphate-buffered saline containing 30 g/L milk. The antigen solution was then incubated in both supports at various concentrations from 0.07 to 100 mg/L.
In the first 96-well microplate (for ELISA), 50 µL of a goat anti-mouse IgG peroxidase was added to each well. After washing, 100 µL of o-phenylenediamine substrate was added to each well, and the reaction was stopped after 10 min with 3 mol/L sulfuric acid. The absorbance was read at 492 nm.
In the second 96-well microplate (for FIA), streptavidin-coated fluorescent microbeads (50 µL) were added into each well. After incubation for 1 h at room temperature, the plate was washed, and the fluorescence was read on a Fluostar fluorometer (BMG).
In the TECHNODISK, streptavidin-coated fluorescent microbeads (500
µL) were added. After gentle washing, the TECHNODISK was read using
the CCD reader. The number of microbeads counted with the TECHNODISK
was correlated with the initial antigen concentration
(r2 = 0.98; Fig. 1
A).
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Detection and quantification of an antiserum (serology).
Two 96-well microplates and the TECHNODISK device were coated with 100 µL of purified keyhole limpet hemocyanin at 10 mL/L; 50 µL of mouse anti-keyhole limpet hemocyanin antiserum was then added to all three supports.
In the first microplate (for ELISA), after incubation for 1 h at room temperature, mouse anti-IgG-peroxidase was added, followed by the addition of o-phenylenediamine. The reaction was then stopped with the addition of 3 mol/L sulfuric acid. The reading was performed on an ELISA reader at 492 nm.
In the second 96-well microplate (for FIA), after incubation for 1 h, a goat biotinylated anti-mouse IgG was added. After subsequent washings, streptavidin-coated fluorescent microbeads (50 µL) were added to each well. After incubation for 1 h at room temperature, the plate was washed and the fluorescence was read on a Fluostar fluorometer.
For the TECHNODISK, streptavidin-coated fluorescent microbeads (500 µL) were added. After gentle washing, the TECHNODISK was read using the CCD reader.
The TECHNODISK offers a measuring range overranking the ELISA method by
2 Log (Fig. 1B
). The major advantage of TECHNODISK compared with the
FIA is the elimination of the classical fluorescence interferences,
thus offering a better sensitivity in low titers.
These preliminary experiments demonstrate many advantages over the conventional FIA and ELISA methods. The design of the TECHNODISK device allows the detection and quantification of up to seven different antigens simultaneously. Moreover, we have been able to apply the TECHNODISK technology to the quantification of cellular antigens, especially platelet glycoproteins. This could offer an attractive method for point-of-care monitoring of the new platelet glycoprotein IIb/IIIa antagonists.
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,
number of beads; (——–), correlation line;
r2 = 0.98. (B), 
,
particle counting by TECHNODISK;