JP4024442B2 - Quantitative method and apparatus for test piece and biological material - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a test piece used for DNA analysis and immunological analysis, a method for quantifying a biological substance using the test piece, and an apparatus therefor.
[0002]
[Prior art]
It was thought that the human genome project to determine the entire base sequence of a huge human genome of about 3 billion bp and analyze them would be decided as early as 2003 from our schedule. The focus has shifted from simple nucleotide sequences to systematic functional analysis.
[0003]
The specific content of the genetic information is related to what protein is synthesized under what conditions, but the former, that is, what protein is synthesized, has been conventionally known as Western blotting, Northern blotting,・ Analysis methods such as blotting and Southern blotting are widely used. However, these methods are able to analyze all the proteins, DNA, and RNA extracted from cells at once, even though they can analyze the specific proteins, DNA, and RNA that are extracted. Was not necessarily suitable.
[0004]
On the other hand, the latter, that is, the conditions under which the protein is synthesized, was controlled by the level of transcription, and thus could not be sufficiently analyzed by conventional analysis methods. This is because the data of both the control array and the corresponding control contents were insufficient.
[0005]
However, the analysis of gene expression information has dramatically improved due to the advancement of a technology called DNA chip or DNA microarray that fixes arbitrary oligonucleotides on a carrier surface of about 1 cm square at high density. It is expected that. A DNA chip is obtained by dividing a silicon chip into a number of sections by photolithography, and directly synthesizing single-stranded DNA having a specific base sequence on each section. The DNA microarray is obtained by blotting a DNA macroarray having a spot size of about 300 μm or more blotted on a slide glass with a spot size of about 200 μm or less. The DNA chip or DNA microarray is connected to a signal reader and a computer system so that it can be known to which probe the DNA arranged on the chip or on the microarray has hybridized. It can be used for various purposes such as gene DNA mutation analysis, polymorphism analysis, nucleotide sequence analysis, and expression analysis, depending on the type of DNA and its arrangement on the DNA chip or DNA microarray.
[0006]
[Problems to be solved by the invention]
However, analysis using this DNA microarray has just begun to discuss the fabrication of microarrays and their detection devices, and still has considerable problems. For example, a microarray is called a spotter device, and is prepared by blotting cDNA. As shown in FIG. 4a, contact printing is performed in which a pin 41 is directly contacted on a slide glass 42 to place cDNA 43. As shown in FIG. 4b, there is a non-contact printing method in which cDNA 43 is blotted without contacting syringe 44 on slide glass 42 as shown in FIG. 4b. The amount of spotting varies, and even in the best case, the variation is 5 to 10% CV in the contact printing method and 3 to 5% CV in the non-contact printing method. There are even defect spots. For this reason, as shown in FIG. 5, the amount of DNA sample spotted at the points a and b of the same DNA microarray 51 and at the same point a is the same as the DNA microarray 52 produced in the same manner as the DNA microarray 51. Unlike this, there is a problem that the quantification of different DNAs prepared from one cell and the quantitative comparison of DNAs that differ in expression in the same cell depending on the timing actually contain considerable errors. ing.
[0007]
In order to solve this problem, first of all, improvement of the spotter device can be considered, but it is considered that there is a limit to the improvement for improving the reproducibility of the spotted sample amount.
[0008]
The present invention has been made in view of the above circumstances, and without paying attention to the improvement in reproducibility of each spot to be blotted by improving the spotter device, paying attention to the substance arranged on the carrier, between the spots It is intended to provide a test piece such as a DNA microarray that can be accurately quantified even if the amount of blotting varies.
[0009]
[Means for Solving the Problems]
The test strip of the present invention is a test strip used for analysis of a biological substance labeled with a labeling substance in which a plurality of different known specific binding substances are respectively arranged at predetermined positions on a carrier. The specific binding substance is labeled with a labeling substance.
[0010]
According to the method for quantifying a biological substance of the present invention, a test strip in which a plurality of different known label-specific binding substances are arranged at predetermined positions on a carrier, respectively, from the labeling substance of the specific binding substance. Detect the amount of beacon signal emitted at each position,
A label signal that is released from a labeled substance of the bound biological substance by binding a biological substance labeled with a labeling substance different from the labeling substance that labels the specific binding substance to the specific binding substance For each position,
Based on the detection result of the amount of the labeled signal released from the labeling substance of the specific binding substance and the detection result of the amount of the label signal released from the labeling substance of the biological substance, the binding to the specific binding substance It is characterized by quantifying the obtained biological material.
[0011]
The biological substance-derived quantification device of the present invention is based on a labeling substance of the specific binding substance of a test piece in which a plurality of different known label-specific binding substances are respectively arranged at predetermined positions on a carrier. Detection means for detecting the emitted label signal;
Detects the amount of the labeled signal released from the labeling substance of the biologically-derived substance labeled with a labeling substance different from the labeling substance labeling the specific binding substance bound to the specific binding substance at each position. Detecting means for
Based on the detection result of the amount of the labeled signal released from the labeling substance of the specific binding substance and the detection result of the amount of the label signal released from the labeling substance of the biological substance, the binding to the specific binding substance And an analyzing means for quantifying the living body-derived substance.
[0012]
The “carrier” only needs to be capable of stably binding and spotting a specific binding substance, such as a membrane filter or a slide glass plate. These carriers may be pretreated in order to stably bind the specific binding substance.
[0013]
“Specific binding substances” are hormones, tumor markers, enzymes, antibodies, antigens, abzymes, other proteins, nucleic acids, cDNA, DNA, RNA, etc., which can specifically bind to biological substances Means. “Known” differs depending on the specific binding substance. For example, in the case of a nucleic acid, the base sequence and the length of the base are known, and in the case of a protein, the amino acid composition is known. Here, the specific binding substance disposed at a predetermined position of the carrier means that one type of specific binding substance is disposed at each position.
[0014]
“Biologically-derived substance” means a substance that specifically binds to a known specific binding substance arranged at a predetermined position on a carrier and is extracted, isolated, etc. from a living body, Not only those extracted directly from the living body but also those obtained by chemical treatment, chemical modification and the like are included. For example, substances such as hormones, tumor markers, enzymes, antibodies, antigens, abzymes, other proteins, nucleic acids, cDNA, DNA, and mRNA.
[0015]
A specific binding substance is “labeled with a labeling substance” (a specific binding substance labeled with a labeling substance is also simply referred to as a labeled specific binding substance) means that one side of a specific binding substance, for example, a chain molecule It may be labeled at one place like the end of, or may be labeled at several places. Normally, if one specific binding substance is labeled, the amount of the specific binding substance arranged at each position on the carrier can be detected. However, if you want to increase the detection sensitivity, When it is technically difficult or technically complicated to label only one part of a substance, a plurality of labeled portions may be provided.
[0016]
As the labeling substance for labeling the biological substance, it is preferable to select a labeling substance different from the labeling substance labeling the specific binding substance. This is because the label signal released by the labeling substance for labeling the biological substance and the label signal released by the labeling substance for labeling the specific binding substance can be detected separately at the same time. The labeling substance for labeling the biological substance may be one that labels the biological substance, or may label several places, but is preferably one. This is because in the case of biologically derived substances, the constituent substances may not be known, and it is necessary to confirm how the labeling substance is incorporated, which is technically complicated. However, as long as it is a known substance, there may be several places like the specific binding substance.
[0017]
The “labeling substance” means a substance serving as a mark that is partially modified or directly added to obtain information from a specific binding substance or a biological substance. The labeling substance is not particularly limited as long as the labeling signal released from the labeling substance can be detected and the regularity taken into the specific binding substance or the biological substance is known in advance. For example, Cyber Green II ^TM , Cy5 ^TM Fluorescent dyes such as fluorescein isothiocyanate, ³² P, ³³ It is preferable to use a radioisotope such as P. The “label signal” refers to a signal that is emitted or output from the labeling substance, such as fluorescence when the labeling substance is a fluorescent dye, and radiation when the labeling substance is a radioisotope. . In this case, an isotope may be used as the specific binding substance, a fluorescent dye may be used as the biological substance, a fluorescent dye may be used as the specific binding substance, and an isotope may be used as the biological substance. A fluorescent dye may be used together with a biological substance. However, when a fluorescent dye is used for both the specific binding substance and the biological substance, it is necessary to use a fluorescent dye whose emission wavelength bands do not overlap each other, or which overlaps at least in the main detection band. is there. On the other hand, when using a radioisotope, a specific binding substance labeled with a radioisotope arranged on a carrier is used as in Japanese Patent Publication No. 5-20712 (quantitative measurement method of radioisotope in autoradiography). The photostimulable phosphor sheet is closely exposed and read with a laser. The regularity of incorporation into specific binding substances and biological substances is known in advance, for example, if the fluorescent dye CyberGreen II is weakly bound to single-stranded DNA or RNA There is a regularity corresponding to the length, and there is also a regularity that nucleotides that are also fluorescently labeled are incorporated into DNA or RNA at random or at the ends. on the other hand ³² The isotope such as P varies depending on the target to be labeled with the isotope. For example, any of the four nucleotides used as a substrate when synthesizing cDNA from mRNA has an α position ³² If something labeled with P is used, ³² P is randomly incorporated and is proportional to the base contained in the labeled nucleotide. ³² There is a regularity that P is taken in.
[0018]
“Binding a biological substance to a specific binding substance” means, for example, a case where a stable duplex is formed between complementary nucleotide sequences found in DNA or RNA (hybridization), or an antigen. It means a highly specific bond that selectively reacts only with a specific substance such as an antibody, biotin and avidin.
[0019]
“Based on the detection result of the amount of label signal released from the labeling substance of the specific binding substance and the detection result of the amount of label signal released from the labeling substance of the biological substance, the specific binding substance and “Quantifying the bound biological substance” means that the amount of the labeled signal released from the labeling substance of the specific binding substance at one position on the carrier is the amount of the specific binding substance arranged at that position. Since it is proportional, the amount of labeled signal released from the labeled substance of the biological substance bound to the specific binding substance is made to correspond to this, and the amount (concentration) of the biological substance is determined regardless of the variation of the specific binding substance. It means that it can be quantified. Taking the case where the specific binding substance is cDNA as an example, for example, the amount of fluorescence when there are s cDNAs whose one end of cDNA (specific binding substance) is fluorescently labeled at position n of the carrier ( The amount of fluorescence when the number of labeled DNA released from the labeling substance) is Ps, and one end of the molecule is hybridized with the c-number of fluorescently labeled probe DNAs (biological substances) and the nth cDNA. Let Pc. In the liquid phase system, the probe DNA that hybridizes with cDNA is said to be about 1/100, although it depends on the cDNA and conditions for hybridization. Although it is not a complete liquid phase system on the carrier, at least the concentration m of the probe DNA is proportional to the number s of cDNAs present in the n-th. Therefore, Pc is proportional to Ps and m, and the following relationship is established.
[0020]
Pc∝mPs
Therefore, if Ps and Pc are measured, the concentration m of the biological substance bound to the nth position can be obtained.
[0021]
The above is the case where only one location of the cDNA is labeled. In this case, the value of Ps is not related to the base sequence of the cDNA or the length of the base, and is arranged at the nth position. It is proportional only to the number of cDNAs. However, the labeling substance labels several places (several tens of places in some cases) to one molecule of cDNA so that the labeling substance labels a specific base or in proportion to the length of the base. In some cases, the amount of fluorescence also varies depending on the length of the base, so even if all the positions on one carrier show the same amount of fluorescence, the length of the base of the cDNA is different, so at all positions, It cannot be said that an equal amount of cDNA is blotted. Therefore, in this case, it is necessary to obtain a “characteristic value relating to cDNA”. That is, a characteristic value related to cDNA, such as the ratio of bases and lengths of cDNAs arranged on a carrier, and the amount of fluorescence when N cDNAs are contained at one location of the carrier, are calculated for each location. It is necessary to register with. What characteristic value is required depends on the labeling substance used, but the fluorescence amount Pc of the probe DNA is proportional to the concentration m of the probe DNA and the fluorescence amount Ps of the cDNA. It is inversely proportional to the number of labeling substances. Therefore, the concentration m of the probe DNA is expressed by the following formula, and a characteristic value that can determine the number of labeling substances that label one (one molecule) cDNA is required.
[0022]
m∝Pc / Ps × (number of labeling substances labeling one cDNA)
For example, when one of the four bases of a cDNA is labeled, the number of specific bases contained in one molecule of cDNA is proportional to the length of the base. And the ratio of the base in which one labeling substance is incorporated (how many bases one labeling substance is incorporated) is required.
[0023]
【The invention's effect】
According to the test piece of the present invention, since a plurality of different known specific binding substances arranged at predetermined positions on the carrier are labeled with the labeling substance, the specific binding substance is arranged on the carrier. Regardless of variations in the amount of specific binding substance that can be placed on the test strip, it can be identified.
[0024]
According to the method and apparatus for quantifying a biological substance of the present invention, a specific binding substance of a test strip in which a plurality of different known label-specific binding substances are arranged at predetermined positions on a carrier, respectively. The amount of labeling signal released from the labeling substance is detected at each position, and the biologically-derived substance labeled with a labeling substance different from the labeling substance labeling the specific binding substance is bound to the specific binding substance. The amount of the label signal released from the labeling substance of the bound biological substance is detected at each position, the detection result of the amount of the label signal released from the labeling substance of the specific binding substance, and the biological substance Based on the detection result of the amount of label signal released from the labeling substance, the biological substance bound to the specific binding substance is quantified, so regardless of variations in the amount of the specific binding substance being blotted, Biological reason It is possible to perform quantitative determination of substances. In addition, the specific binding substance and the biological substance on the test piece can be read simultaneously by making the labeling substance for labeling the biological substance different from the labeling substance that labels the specific binding substance. Is possible.
[0025]
In addition, if the test piece of the present invention, its quantification method and quantification apparatus are used, elucidation of the control content and mechanism of protein synthesis controlled at the transcription level from mRNA transcribed in cells, or synthesis in the course of disease By quantifying special proteins, it is possible to select effective drugs by quantifying various proteins expressed in accordance with the progress of cancer, for example, or to analyze EST functions widely It can be used.
[0026]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail with reference to the drawings.
[0027]
FIG. 1 is a flowchart showing an embodiment of a test piece and a manufacturing method thereof according to the present invention. Here, a DNA microarray chip will be described as an example of a test piece, cDNA as a specific binding substance, and mRNA extracted from cells as a biological substance.
[0028]
The DNA microarray 10 of the present invention is a cDNA (specific binding substance) having a plurality of different base sequences known to each other at a predetermined position of a slide glass (carrier) 3 whose surface has been pretreated with a poly-L-lysine solution. No. 1 is labeled with a fluorescent dye 5 (hereinafter abbreviated as fluorescein isothiocyanate FITC, and the fluorescent dye 5 is a labeling substance for labeling a specific binding substance).
[0029]
cDNA is prepared from known DNA, mRNA, etc. by PCR method or RT-PCR method. At this time, if dCTP labeled with FITC is used, the position of C (cytosine) among the four bases of DNA is determined as FITC. Can be prepared (hereinafter referred to as F-cDNA (labeled specific binding substance)) 2. The adjusted F-cDNA 2 is spotted at a predetermined position on the slide glass 3 by a spotter device to produce a DNA microarray 10.
[0030]
On the other hand, mRNA (biological substance) 4 to be measured is extracted from the cells, and RNA having poly A at the 3 ′ end is further extracted from the mRNA. When cDNA is synthesized from RNA having poly A at its terminal, Cy5-dUTP (Cy5, a labeling substance for labeling a biological substance) is present to produce Cy5-cDNA (probe DNA) labeled with Cy5. Moreover, if a labeled primer is used, end labeling is also possible.
[0031]
Cy5-cDNA is adjusted to a predetermined solution and gently placed on the DNA microarray 10 to perform normal hybridization.
[0032]
Next, an apparatus for quantifying Cy5-cDNA hybridized on the DNA microarray 10 shown in FIG. 1 will be described with reference to FIG. FIG. 2 is a diagram showing the configuration of an embodiment of the quantitative device of the present invention. The quantification apparatus 100 shown in the figure has a transparent sample stage 20 on which a DNA microarray 10 in which FITC-labeled F-cDNA is distributed is placed and placed at a predetermined position, and an emission wavelength suitable for exciting the FITC. An argon laser (or SHG laser (excitation wavelength 473 nm)) 21 that emits laser light L1 and a He—Ne laser that has excitation wavelength 633 nm that emits laser light L2 having an emission wavelength suitable for exciting Cy5. (Or a semiconductor laser (excitation wavelength: 635 nm)) 22, a first dichroic mirror 23 that transmits the laser light L1 and reflects the laser light L2, and fluorescence emitted from the fluorescent dye of the DNA microarray 10 is excited photoelectrically. A photomultiplier (hereinafter referred to as PMT) 90 to be detected, and laser beams emitted from the lasers 21 and 22. The light is irradiated to the DNA microarray 10 placed on the sample stage 20 and the fluorescence emitted from the DNA microarray 10 is guided to the PMT 90 by this irradiation, and the optical path from the optical head 50 to the PMT 90 A filter set 80 provided to be switchable between two types of band-pass filters 81, 82, a main scanning means 60 for moving the optical head 50 at a constant speed in the arrow X direction, and the lasers 21, 22, A sub-scanning means 70 that integrally moves the optical head 50, the filter set 80, and the PMT 90 in the arrow Y direction (direction orthogonal to the arrow X direction); an amplifier 91 that amplifies the detection signal detected by the PMT 90 logarithmically; An A / D converter 92 that performs A / D conversion on the amplified detection signal, and A / D converted data are input in advance. The analysis device 93 that analyzes the data on the DNA microarray 10 that is being analyzed and the control to emit the laser beams L1 and L2, and one of the bandpass filters 81 and 82 of the filter set 80 are selected, In this configuration, a control unit 95 is provided for controlling the selected bandpass filter to be disposed on the optical path.
[0033]
Next, operation | movement of the fixed_quantity | quantitative_assay apparatus 100 of this embodiment is demonstrated.
[0034]
A DNA microarray 10 in which a cDNA labeled with FITC and a Cy5-cDNA labeled with Cy5 are hybridized with the cDNA labeled with FITC is placed on the sample stage 20, and the control unit 95 selects the laser beam L1 and the laser beam L2. Thus, the lasers 21 and 22 are controlled so as to be emitted, whereby the laser light L 1 is emitted from the laser 21 and the laser light L 2 is emitted from the laser 22. On the other hand, the control unit 95 controls the filter set 80 so that the first filter 81 is disposed on the optical path between the optical head 50 and the PMT 90, and the filter set 80 controls the first filter 81. It arranges on the optical path.
[0035]
The laser beam L1 emitted from the laser 21 passes through the dichroic mirror 23 and proceeds in the direction of the arrow X. The laser beam L1 incident on the flat mirror 51 of the optical head 50 is reflected upward in the figure, passes through the small hole 52a of the perforated mirror 52, enters the lens 53, passes through the lens 53, and is placed on the sample stage 20. A minute region of the prepared DNA microarray 10 is irradiated. At this time, the optical head 50 is moved in the arrow X direction at high speed and at the same speed by the main scanning means 60, and the laser light L1 scans the DNA microarray 10 in the arrow X direction. In addition, for F-cDNA present in a minute region irradiated with the laser beam L1, FITC is excited by the irradiated laser beam L1 to emit fluorescence K1.
[0036]
The fluorescence K1 emitted by the laser light L1 spreads and exits from the lower surface of the DNA microarray 10, and the emitted fluorescence K1 is converted into a lower beam by the lens 53 of the optical head 50. Similarly, the perforated mirror 52 of the optical head 50 is used. Is incident on. The fluorescence K1 is reflected by the reflecting surface of the perforated mirror 52 and travels in the direction along the arrow X direction. The fluorescence K1 traveling in the direction of the arrow X is blocked by the first bandpass filter 81 from light other than the fluorescence K1, and only the fluorescence K1 passes through and enters the PMT 90. The fluorescence K1 incident on the PMT 90 is amplified and photoelectrically detected by the PMT 90, read as a corresponding electric signal, amplified by the logarithmic amplifier 91, converted into a digital signal by the A / D converter 92, and sent to the analyzer 93. Is output.
[0037]
When reading by one main scanning is completed in this way, the optical head 50 is returned to the original position by the main scanning means 60, while the sub-scanning means 70 includes the lasers 21 and 22, the dichroic mirror 23, and the optical head. 50, the filter set 80 and the PMT 90 are integrally sub-scanned in the arrow Y direction. Then, by repeating the above main scanning and sub-scanning, the entire surface of the DNA microarray 10 is irradiated with the laser light L1, and the fluorescence K1 corresponding to each position of the DNA microarray 10 is converted into a digital signal and acquired.
[0038]
When the data of the fluorescence K1 is acquired up to the last position of the DNA microarray 10 after the main scanning and the sub-scanning, the optical head 50 is returned to the first position, and then the laser beam L2 emitted from the laser 22 is similarly used. The emitted fluorescence K2 is made incident on the PMT using a second bandpass filter 82 that blocks transmission of light other than the fluorescence K2. Thereafter, the fluorescence K2 is converted into a digital signal and acquired by repeating the main scanning and the sub-scanning similarly to the fluorescence K1. In addition, although the fluorescence K1 about all the positions of the DNA microarray 10 was read and this was preserve | saved as data and the fluorescence K2 was read next was demonstrated here, of course for every position of the DNA microarray 10, The operation of irradiating the laser L1 and reading the fluorescence K1 and then irradiating the laser L2 to the same position and reading the fluorescence K2 may be repeated for all positions.
[0039]
As shown in FIG. 3, the analysis device 93 that has acquired the digital signal corresponding to each position of the DNA microarray 10 includes bases (A, A, F) that are arranged at each position of the DNA microarray 10. G, C, T) ratio data is registered. Therefore, the concentration of Cy5-cDNA at that position is calculated from the ratio of the base per registered F-cDNA and the fluorescence amount of F-cDNA, and the measured fluorescence amount of F-cDNA and fluorescence amount of Cy5 cDNA. Can be sought. For example, the fluorescence amount of F-cDNA at position 1-1 is expressed as P ₁ , The amount of fluorescence of Cy5-cDNA is P ₂ Then, the concentration m of Cy5-cDNA is m∝P. ₂ / P ₁ × 20. If all positions of the DNA microarray 10 are similarly analyzed, the concentration of each position of Cy5-cDNA can be obtained.
[0040]
Here, FITC and Cy5 are used as fluorescent dyes, but other fluorescent dyes or isotopes can also be used. In that case, the length of the base of the known cDNA at each position, the ratio of the constituent base, the fluorescence amount or the radiation dose of the known amount of cDNA, etc. are registered in advance in the analyzer 93 according to the labeling substance. In this case, the concentration of the probe DNA at each position can be determined by measuring the fluorescence amount or radiation amount of the cDNA of the newly prepared DNA microarray and the fluorescence amount or radiation amount of the probe DNA.
[0041]
In the embodiment of the present invention, a DNA microarray is used as a test piece, cDNA is used as a specific binding substance, and mRNA extracted from a cell is used as an organism-derived substance. However, the present invention is limited to this. It is not a thing.
[Brief description of the drawings]
FIG. 1 is a flowchart showing an embodiment of a test piece of the present invention and a manufacturing method thereof.
FIG. 2 is a diagram showing a configuration of an embodiment of a quantitative device of the present invention.
FIG. 3 is a diagram showing an embodiment of data held by the quantitative device of the present invention.
FIG. 4 is a view showing an embodiment of a spotter and blot of a DNA microarray.
FIG. 5 is a perspective view of a conventional DNA microarray.
[Explanation of symbols]
1 cDNA (specific binding substance)
2 Label specific binding substance
3 slide glass (carrier)
4 mRNA (biological material)
5 Fluorescent dye (labeling substance)
6 Fluorescent dye (labeling substance)
10 DNA microarray
93 Analysis device (analysis means)
100 quantitative device

Claims (14)

  A plurality of different known specific binding substances that are different from each other are arranged at a plurality of predetermined positions on the carrier, and a biological substance labeled with a labeling substance is bound to the specific binding substance, and the living body is separated at each position. A test piece used for analysis for detecting a labeling signal released from a labeling substance of a derived substance to quantify the biologically derived substance, wherein the specific binding substance is labeled with a labeling substance, and the biologically derived substance is labeled A test piece, wherein the labeling substance to be used is a labeling substance different from the labeling substance labeling the specific binding substance.  The test piece according to claim 1, wherein the specific binding substance is cDNA.  The test piece according to claim 1 or 2, wherein the labeling substance that labels the specific binding substance is a fluorescent dye.  The test piece according to claim 1 or 2, wherein the labeling substance that labels the specific binding substance is a radioisotope.  The amount of the label signal emitted from the labeling substance of the specific binding substance of each test piece in which a plurality of different known label-specific binding substances are arranged at a plurality of predetermined positions on the carrier, for each position. A biological substance labeled with a labeling substance different from the labeling substance that is detected and labeled with the specific binding substance is bound to the specific binding substance and released from the labeled substance of the bound biological substance. The amount of labeled signal detected is detected at each position, the detection result of the amount of labeled signal released from the labeling substance of the specific binding substance, and the amount of labeling signal released from the labeling substance of the biological substance A method for quantifying a biological substance, characterized in that a biological substance bound to the specific binding substance is quantified based on the detection result.  The method for quantifying a biological substance according to claim 5, wherein the specific binding substance is cDNA. 7. The method for quantifying a biological substance according to claim 5, wherein the quantification is further quantified based on a characteristic value relating to cDNA. The method for quantifying a biological substance according to claim 5, 6 or 7 , wherein the labeling substance that labels the specific binding substance is a fluorescent dye. The method for quantifying a biological substance according to claim 5, 6 or 7 , wherein the labeling substance that labels the specific binding substance is a radioisotope.  Detection means for detecting a label signal emitted from the labeling substance of the specific binding substance, in a test strip in which a plurality of different known label-specific binding substances are respectively arranged at predetermined positions on the carrier; The amount of the label signal released from the labeling substance of the biological substance labeled with a labeling substance different from the labeling substance that is bound to the specific binding substance and labeled with the specific binding substance for each position Based on the detection means for detecting, the detection result of the amount of the label signal released from the labeling substance of the specific binding substance, and the detection result of the amount of the label signal released from the labeling substance of the biological substance, An apparatus for quantifying a biological substance, comprising: an analyzing means for quantifying a biological substance bound to a specific binding substance.  The apparatus for quantifying a biological substance according to claim 10, wherein the specific binding substance is cDNA. 12. The apparatus for quantifying a biological substance according to claim 10 or 11, wherein the analyzing means further quantifies based on a characteristic value relating to cDNA. The biological substance-derived quantitative apparatus according to claim 10, 11 or 12 , wherein the labeling substance that labels the specific binding substance is a fluorescent dye. 13. The apparatus for quantifying a biological substance according to claim 10, 11 or 12 , wherein the labeling substance labeling the specific binding substance is a radioisotope.

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