Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2000-03-30
2001-08-21
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C422S068100, C435S006120, C435S007100, C435S007900, C435S007920, C435S287200, C435S808000, C435S810000, C436S164000
Reexamination Certificate
active
06277654
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method and an apparatus that detect organism-originated substances by using an array chip that is employed in deoxyribonucleic acid (DNA) analysis and immunological analysis.
2. Description of the Related Art
In recent years, techniques in the field of generic engineering have rapidly developed, and the human genome project in which one of the objects is to interpret as many as 100,000 base sequences of the human genome has been developed.
On the other hand, an enzyme immunoassay method, a fluorescent antibody method and the like taking advantage of an antigen-antibody reaction have been utilized in diagnoses and research, and techniques of searching for DNA having influence on various genetic diseases have also advanced. As one of the methods, attention has been paid to an array technique.
In this array technique, an array chip (also called a DNA chip), such as the one shown in
FIG. 1
, is employed as an array chip
1
. In the array chip
1
, a large number of known complementary DNAs (an example of a specific binding substance) differing from one another, which have already been interpreted, are disposed with high density in matrix form on a carrier
2
such as a membrane filter, a slide glass and the like. For instance, DNA (an example of an organism-originated substance) originated from the cell of healthy subject A is labeled with a labeling substance consisting of a fluorescent dye or a radioactive isotope, and similarly, DNA originated from the cell of subject B having a genetic disease is labeled with the labeling substance. The labeled DNAs of the subjects A, B are dropped on separate array chips and are hybridized with the complementary cDNAs on the separate array chips. Each hybridized cDNA on each array chip is scanned by laser light that excites each labeling substance, and the fluorescent light or radiation emitted from each cDNA is detected by a photo or radiation detector. Next, a labeled signal representing this result of detection, corresponding to the light-emitting position (spot position) on the array chip, is obtained, and based on this labeled signal, it is judged which cDNA has been hybridized by the cDNA of each subject. The ratio or difference of the labeled signals obtained between both subjects is calculated, and between both subjects, the hybridized cDNAs are compared. In this way, the gene manifested or lost due to the above-mentioned disease (hereinafter referred to as change in manifestation) is specified. Note that if two images represented by the labeled signals are printed or output to a monitor, the change in manifestation can be visually recognized and specified. On the other hand, the ratio between the labeled signals that are obtained from both subjects becomes greater (or smaller) at a position where the change in manifestation exists. Therefore, in the order that the ratio of the labeled signals between both subjects is greater (or smaller), the signal value ratios at
50
spot positions, for example, are caused to correspond to the positions at which the labeled signals were obtained, and these corresponding relations are output as a measurement-result table representing measurement results. Based on this measurement-result table and images displayed on a monitor, the positions at which the labeled signals were obtained are calculated, whereby the change in manifestation can be specified.
The signal value corresponding to each spot position is obtained by setting a template, on which circular ROIs (regions of interest) are disposed at positions corresponding to the spot positions, onto images represented by the labeled signals and detecting a signal value at a position corresponding to the position of the ROI on this template.
However, the above-mentioned method of disposing spot positions on the array chip varies depending on manufacturers of array chips and test purposes. For this reason, if other array chips are employed, in the case where a template is used only for array chips made by a specific manufacturer or for a specific test purpose, the detection of signal values cannot be performed because the spot positions on the array chip do not correspond to the ROI positions on the template. In such a case, if a new template, on which ROIs are disposed at positions corresponding to the spot positions on an array chip used, is generated, signal values can be detected. However, an array chip has as few as 500 spot positions or as many as a few tens of thousand spot positions, and the method of disposing spot positions varies from array chip to array chip. Thus, the operation of generating templates is fairly difficult and time consuming.
SUMMARY OF THE INVENTION
The present invention has been made in view of the aforementioned circumstances. Accordingly, the object of the present invention is to provide a method and an apparatus which are capable of detecting signal values at spot positions even when array chips differing in disposition of spot positions are employed.
To achieve the above object and in accordance with one aspect of the present invention, there is provided a method comprising the steps of:
respectively binding organism-originated substances of a subject labeled with a labeling substance to a plurality of known specific binding substances differing from one another, the specific binding substances being disposed at a plurality of predetermined positions on a carrier on an array chip;
obtaining an image represented by labeled signals emitted from the labeling substances of the organism-originated substances bound to the specific binding substances;
setting a template of a type corresponding to the array chip onto the image, the template having ROIs disposed at positions corresponding to the plurality of predetermined positions on the array chip; and
detecting values of the labeling signals at positions on the image which correspond to the ROIs;
wherein the template of a type corresponding to the array chip is read out from template storage means in which various types of templates are stored, and based on the template read out, the signal values are detected.
Here, the ROIs are disposed at positions corresponding to the positions of the specific binding substances disposed on an array chip to be used. The expression “the template of a type corresponding to the array chip is read out” means to read out a template on which ROIs are disposed at positions corresponding to the positions of specific binding substances disposed on an array chip to be used.
Also, the expression “based on the template, the signal values are detected” means to set the template onto an image represented by labeled signals and to detect values of the labeling signals at positions on the image which correspond to the ROIs.
The word “carrier” may be any type if a specific binding substance can be stably bound and spotted on it. For instance, the carrier may be a membrane filter, a slide glass plate, etc. These carriers may be preprocessed to stably bind a specific binding substance.
The aforementioned “specific binding substance” may be hormones, a tumor marker, enzyme, an antibody, an antigen, abzyme, other proteins, a nucleic acid, cDNA, DNA, RNA, etc., and means a substance bindable specifically with an organism-originated substance. The word “known” varies depending on a specific binding substance. For example, in the case of a nucleic acid, the word “known” means that the base sequence, the base length, etc., are known, and in the case of protein, the word “known” means that the composition and the like of the amino acid are known. Here, the expression “specific binding substances disposed at a plurality of predetermined positions on the carrier” means that one kind of specific binding substance has been disposed for each position.
The aforementioned “organism-originated substance” is a substance that binds specifically with a known specific binding substance disposed at a predetermined position on the carrier, and means substances extracted, isolated and the like
Fuji Photo Film Co. , Ltd.
Le Long V.
Padmanabhan Kartic
Sughrue Mion Zinn Macpeak & Seas, PLLC
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