Chemistry: analytical and immunological testing – Involving diffusion or migration of antigen or antibody
Reexamination Certificate
1999-06-03
2004-06-22
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving diffusion or migration of antigen or antibody
C435S007100, C435S007940, C435S287700, C435S287800, C436S518000, C436S523000, C436S538000, C436S541000, C436S810000, C436S823000, C436S824000, C422S051000, C422S067000, C422S105000
Reexamination Certificate
active
06753189
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a detection apparatus for detecting the presence of a detectable material in a sample. More specifically, the present invention relates to a detection apparatus for detecting the presence of a biological component in urine, blood, or the like. Additionally, the present invention relates to a method for the detection of a biological component in urine, blood, or the like.
Among conventional detection methods for detecting a biological component detectable material, there is an immunological detection method, which uses antibodies and antigens. This method, which provides high sensitivity and good specificity and ease of use, is used widely in the field of clinical testing. Various technologies have been recently proposed to perform testing using the immunological detection method without requiring any special techniques or training.
Of these, a group of measuring methods known as immunological chromatography has been proposed (Japanese laid-open patent publication number 9-178748) and is already being marketed to the general population. This method provides quick, easy, and accurate results.
In immunological chromatography, a marked reagent (staining indicator) using colored latex, colloidal gold, or the like, is fixed to a spreading layer. An unmarked reagent is fixed to a detection zone on the spreading layer. When the detectable material is present in the fluid sample, immunoreaction compounds are produced. The staining marker from this reaction is caught so that it can be visually observed. With the immunological chromatography method, the fluid sample can be simply applied to an application position. After a fixed period of time, the degree of coloration from the staining marker is observed at the detection zone.
With this method, the antigens or antibodies which have affinity for the detectable material must be prepared along with enzymes, fluorescent materials, luminescent materials, colored latex, colloidal gold, or the like, which serve as markers, that are chemically or physically bonded with marker antigens or marker antibodies depending on the application. However, the marking reaction involves a major problem. For example, to bond an enzyme to an antigen or antibody protein, a chemical procedure such as periodic acid oxidation can be performed (Enzyme Immunoassay, Igaku-Shoin, Tokyo, 1982). In such cases, irreversible deactivation of the antigen, antibody, or the enzyme takes place. Additionally, polymerization leading to reduction of activation or non-specific reaction can take place. As a result, the practical marker yield becomes very low.
Similarly, when physical (hydrophobic bonding) methods are used to bond colored latex to antigen or antibody proteins, the bonding to the colored latex takes place randomly so that the active sites of the antigen or antibodies are lost. This provides inadequate reactions so that excess antigens or antibodies must be used. Also, since absorption does not proceed 100%, significant antigens or antibodies are wasted.
To overcome these problems, a method that uses bivalent reagents, as a crosslinking agent, has been developed (Enzyme Immunoassay, Igaku-Shoin, Tokyo, 1982). However, the operations involved are complex and require a high degree of skill. Furthermore, since the reaction itself is highly sensitive, slight changes in reaction conditions can greatly affect the properties of the marker. Thus, the bivalent reagent method of preparing markers is considered not very reproducible.
When using the conventional high-specificity marking method, the loss of activation of the markers or the antigens/antibodies or the like is unavoidable, and 100% marking cannot be achieved since the marking operation is itself a chemical reaction.
OBJECTS AND SUMMARY OF THE INVENTION
It is an object of the present invention to provide a detection method for minimizing reduced activation and antibody loss from the marking operation while providing measurement sensitivity that is at least equal to that of the conventional technology.
It is a further object of the present invention is to provide a detection apparatus that gives highly sensitive and inexpensive immunological detection without wasting valuable antibodies.
It is another object of the present invention to provide a detection method that gives highly sensitivity and inexpensive immunological detection without wasting valuable antibodies.
It is still a further object of the present invention to provide a high-sensitivity detection apparatus and method for detecting a detectable material that does not involve restrictions of reagent amounts.
Briefly stated, the present invention provides a catcher, having an immunological epitope, fixed to a detection zone of a spreading layer. A marker, capable of easy detection, has an immunological epitope. The marker and bispecific antibodies are soluble so that they can move on the spreading layer. The bispecific antibodies include a first bispecific antibody, having specificity for the detectable material in the fluid sample and the marker, and a second bispecific antibody, having specificity for the detectable material in the fluid sample and the catcher. Pore sizes and particle diameters are set so that the reaction product in which the marking elements and the particles are bonded are caught at a catching section. The concentration of the marking elements and the particles are increased to improve detection sensitivity. The result is an inexpensive and simple detection apparatus being highly-sensitive for the immunological detection of a detectable material, without wasting valuable antibodies.
In the conventional detection apparatus, as described above, the unmarked reagent is fixed to the detection zone on the spreading layer. With this structure, the amount of reagent is restricted by its ability to bond with protein. Thus, it is difficult to use more than a fixed amount. Also, in order to detect the detectable material, the detectable material must be biologically bonded to both the reagent component and the marking component. The restriction on the amount of reagent prevents the sensitivity from going beyond a certain point.
Furthermore, the reagent is fixed to the spreading layer. Therefore, out of the time that the detectable material is chromatographically moving along the spreading layer, the reaction can only take place during the very short interval of time when the detectable material contacts the reagent. This reduces the reaction and results in low reactivity in the conventional detection device. With regard to this problem, a technology has been proposed to use magnetism (Japanese laid-open patent publication number 5-52849), but this requires the use of materials having magnetism and therefore cannot be applied to a wide range of materials.
According to an embodiment of the present invention, there is provided a detection apparatus for detecting the presence of a detectable material in a sample comprising: a spreading layer on which the sample is applied; a catcher fixed to the spreading layer at a detection zone away from a position at which the sample is applied; the catcher including an immunological epitope; bispecific antibodies supported in the spreading layer in a dry state, such that movement of the bispecific antibodies is possible when the bispecific antibodies are in a soluble state; a marker supported in the spreading layer in a dry state, such that movement of the marker is possible when the marker is in a soluble state; the marker including an immunological epitope being capable of detection; the double-specific antibody including a first double-specific antibody and a second double-specific antibody; the first double-specific antibody being specific to the detectable material in the sample as well as the marker; and the second double-specific antibody being specific to the detectable material in the sample as well as the catcher.
According to another embodiment of the present invention, there is provided a detection method for detecting the presence of a det
Narahara Kenji
Uehara Toshiyuki
Darby & Darby
Mizuho Medy Co., Ltd.
Nguyen Bao-Thuy L.
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