Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
1993-06-28
2001-07-10
Housel, James (Department: 1648)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C436S520000, C436S524000, C436S525000, C436S531000, C436S533000, C436S534000, C436S527000, C436S806000, C210S222000, C422S068100
Reexamination Certificate
active
06258607
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Filed of the Invention
The present invention relates to an indirect agglutination immunoassay and an apparatus therefor, and more particularly to an indirect agglutination immunoassay using an antigen-antibody reaction and magnetic particles or magnetic-material-containing particles, and an apparatus for conducting the indirect agglutination immunoassay.
2. Discussion of Background
Indirect agglutination used in an immunoassay, in which the combining reaction by the antigen-antibody reaction is intensified by use of antigen- or antibody-bonded particles, is called “passive agglutination” or “reverse passive agglutination” and is widely used in practice in a simple immunoassay for a large number of test samples.
The immunoassay utilizing indirect agglutination has the advantages over the conventional EIA (enzyme immunoassay) and RIA (radioimmunoassay) in that the assay is simple in operation and does not require any particular device for detecting the occurrence of the antigen-antibody reaction. However the immunoassay utilizing indirect agglutination has the drawbacks that it is extremely difficult to conduct the assay automatically, and it has not as yet been developed beyond the semi-automation stage.
There are two methods for forming detection patterns during indirect agglutination.
In one method, a particle-containing reagent is added to a diluted solution of a test sample containing a desired analyte placed in a “U” well or “V” well microplate, the mixture is stirred and then allowed to stand, and the occurrence of an antigen-antibody reaction is detected from a sedimentation pattern of the particles of the reagent formed at the bottom of the well. Hereinafter this method is referred to as “the standing method”.
In another method, a particle-containing reagent is added to a diluted solution of the test sample placed in a “U” well or “V” well microplate, and the mixture is stirred and then centrifuged to precipitate the particles onto the bottom of the well. The microplate is then inclined, so that the presence or absence of an antigen-antibody reaction is detected from the absence or presence of slippage observed of a coating of the precipitated particles at the bottom of the well of the microplate. Hereinafter this method is referred to as “the centrifugation method”.
In the case where the occurrence of an antigen-antibody reaction is detected from the sedimentation pattern by the standing method, it is extremely difficult to detect the pattern automatically because the pattern is easily distorted by slight vibrations during the standing thereof. As a result, for instance, the pattern is deformed, the area of the pattern is decreased, and slippage of the pattern along the bottom of the well takes place. In addition, the standing method has the drawback that about 0.5 to 3 hours are required before the pattern is formed in a suitable fashion for the assay, although the necessary time period for this of course depends upon the type of particles employed.
In contrast, in the case of the centrifugation method, the sedimentation can be finished within a few minutes by use of a centrifuge, and the pattern can be read after the microplate is slanted for several minutes. Furthermore, the concern about the distortion of the pattern caused by vibrations applied thereto during the formation of the pattern is entirely unnecessary. However, it is difficult to perform the immunoassay automatically by use of a centrifuge in practice.
As test samples that can be used for the above-mentioned conventional standing method and centrifugation method, for instance, blood serum, urine, and other body fluids can be given. In conventional methods, the test samples are usually diluted and used. However, when whole blood is used without separating out of the blood corpuscles and blood serum, the sedimentation pattern tends to be centered at one point of the bottom of the well of the microplate, so that the blood components adversely affect the shape of the agglutination or sedimentation pattern. It is known that this will distort the results of the assay.
In other conventional immunoassays, such as the EIA (enzyme immunoassay) and RIA (radioimmunoassay), the separation of blood corpuscles and blood serum is conducted as a pre-processing step in order to avoid non-specific reactions.
SUMMARY OF THE INVENTION
It is therefore a first object of the present invention to provide a simplified, automatic indirect agglutination immunoassay with an increased magnetic sedimentation rate.
A second object of the present invention is to provide an apparatus for performing the above agglutination immunoassay automatically.
According to the present invention, the first object of the present invention can be achieved by an indirect agglutination immunoassay comprising the steps of:
providing, in a container, an immunoassay system comprising a test sample containing a desired analyte, and a reagent comprising magnetic particles or magnetic-material containing particles containing iron, wherein the magnetic particles or magnetic-material containing particles have been sensitized to allow specific binding to the desired analyte, and have a particle size in the range of 1 to 5 &mgr;m, with the content of the iron being in the range of 8 to 20 wt. %, precipitating the magnetic particles or magnetic-material containing particles by the application of magnetic force,
allowing the container to stand at an inclination, and
detecting the presence or absence of an immune reaction from the absence or presence of slippage observed of the precipitated magnetic particles or magnetic-material containing particles on the bottom of the container.
In the above magnetic agglutination immunoassay, the magnetic particles can be precipitated by use of either an electromagnet or a permanent magnet.
The second object of the present invention can be achieved by an apparatus for conducting an indirect agglutination immunoassay comprising (a) a container which contains a test sample for immunoassay, and sensitized magnetic particles or sensitized magnetic-material containing particles, having a particle size in the range of 1 to 5 &mgr;m, which contain iron in an amount in the range of 8 to 20 wt. %, (b) a magnetic sedimentation means for magnetically precipitating the components containing the magnetic particles at the bottom of the container, and (c) an inclination means for allowing the container to stand at an inclination after removal of the magnetic sedimentation means.
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Production and Use of Magnetiziable Particles in Immunoassay, M. Pourfarzaneh, Ph.D., et al., The Ligand Quarterly, vol. 5, No. 1, 1982, pp. 41-47.
Davis et al., “Microbiology”, 2nd Edition, Harper & Row Publishers, Inc., Hagerstown, MD, p. 393, (1973).
Ikeda Mikio
Saito Tomo
Fujirebio Inc.
Housel James
Nelson Brett
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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