JPH04204379A - Organism component analyzing method - Google Patents

Abstract

PURPOSE:To perform analysis of high sensitivity by binding an antibody to a solid phase through nucleic acid, washing a surplus labeled object, thereafter cutting the nucleic acid, freeing the labeled object captured to the solid phase through a measured object, and quantifying the freed labeled object. CONSTITUTION:Solidifying antibodies 6, 6', 6'' are antibodies relating to respective three kinds of measuring items A, B, C and left as connected to oligonucleotide having complementary base arrangement with oligonucleotide connected to a reaction plate 1. An arbitrary antibody of these antibodies 6, 6', 6'' is selected by a flow divider 5, and it is arbitrarily moved by a moving mechanism 29 in x, y, z axes. The antibody is left for a fixed time after flow division, solidified and then washed by washer 8. Next, a sample 9 is partially injected by the flow divider 5. After sample reaction, labeled antibodies 10, 10', 10'' are washed by the washer 8 and partially injected. After reaction for a fixed time, the antibody is rewashed. Next by partially injecting a detaching reagent 11, after detaching, the body is guided to a flow cell type detector 12 to optically detect measured liquid in this detector.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a biological component analysis method, and particularly to an immune biological component analysis method that utilizes antigen-antibody reactions.

[Prior Art] As a method for quantifying trace amounts of biological components, there is, for example, an immunoassay that utilizes the reaction specificity of an antibody to an antigen or a habten. Various immunoassay techniques have been developed, and one of the techniques that is theoretically the most sensitive and capable of measurement will be described in detail below.

An antibody whose antigen is the substance to be measured is bound to the surface of a test tube, microplate, glass pole, etc., and reacted with the sample. The substance to be measured in the sample is captured by the autoantibodies due to the antigen-antibody reaction. Unreacted coexisting substances are washed away, and then an antibody to which a labeled substance has been bound in advance (referred to as a labeled antibody) is reacted. As the label, a radioisotope, a fluorescent dye, an enzyme, etc., which can be detected with high sensitivity in the final step, is used. Since the labeled antibody binds to the antigen captured on the solid phase, as a result, an amount of labeled substance proportional to the amount of antigen is captured on the solid phase. After washing and removing unreacted labeled substances, the labeled substances on the solid phase are quantified. A calibration curve is created in advance using a sample with a known concentration, and the concentration of the substance to be measured is obtained by referring to the calibration curve.

In principle, highly sensitive analysis can be expected due to the specificity of the antigen-antibody reaction and the use of highly sensitively detectable labels. However, in reality, an excessive amount of labeled antibody added to the reaction system is non-specifically adsorbed to the solid phase surface of a test tube or the like without any antigen-antibody reaction, which increases the background of the measurement. As a result, the desired high-sensitivity analysis cannot be achieved.

[Problems to be Solved by the Invention] The prior art described above does not take into account the increase in background due to non-specific adsorption as described above, and has the problem of reducing measurement sensitivity.

As a countermeasure to partially reduce this, for example, in enzyme immunoassay using a glass pole as a reaction solid phase, reaction vessels may be used separately for antigen-antibody reaction and color reaction. That is, a sample and a reaction glass pole are placed in a reaction container, reacted, and washed. Next, an enzyme-labeled antibody is added and reacted, followed by washing. After this, the reaction glass pole is transferred to a new reaction container. After a chromogenic substrate is added and reacted, the absorbance or fluorescence intensity is measured.

This method eliminates the influence of non-specific adsorption of the labeled antibody onto the reaction vessel. However, even in this case, the influence of non-specific adsorption of the labeled antibody to the glass pole itself, which is the reaction carrier, cannot be avoided.

Another example is a method using a column as shown in Clinical Examination Vol. 28, pp. 909-916 (1984). In this method, an antigen-antibody reaction product is once bound to a column via a disulfide bond, and after washing, the disulfide bond is cleaved with a reducing agent to elute the immune complex.

The enzyme activity in this eluate is measured.

According to this method, it is better than the above-mentioned method of using a glass pole and transferring the reaction container, but since the reducing agent used dissociates the immune complex from the solid phase, it also denatures the antibody molecules, thereby causing Since the labeled antibody or labeled substance nonspecifically adsorbed to the solid phase surface dissociates, it is not possible to select and dissociate only the target immune complex.

In addition, when using enzymes and fluorescent dyes as labels,
Depending on the dissociation conditions, the enzyme may be denatured or inactivated.

Fading of the fluorescent dye may also occur.

An object of the present invention is to significantly reduce the influence of the non-specific adsorption and provide a highly sensitive biological component analysis method.

[Means to solve the problem] The gist of the present invention for achieving the above object is as follows.

A substance that specifically binds to the antibody is captured on a solid phase, a label is bound to this, and then washed, and then quantitatively bound to the analyte captured on the solid phase. In a biological component analysis method that quantifies a substance to be measured by measuring the amount of a labeled substance, the step of binding the antibody to a solid phase via a nucleic acid, washing the excess labeled substance, and cleaving the nucleic acid. 1. A biological component analysis method comprising the steps of: releasing a labeled substance captured on a solid phase from the solid phase via a substance to be measured; and quantifying the separated labeled substance.

In the above, when an antibody or antigen is bound to a solid phase, it is bound via a nucleic acid or an oligonucleotide, and after the completion of the label binding reaction for target quantification,
In this method, only the nucleic acid or oligonucleotide is cleaved in a limited manner, and the labeled substance released from the reaction solid phase is fractionated and quantified. Nucleic acids or oligonucleotides can be easily cleaved in a limited manner by using restriction enzymes.

[Function] In the case of a nucleic acid, for example, DNA, dioxyribonucleotides as constituent units are linked by 3', 5'-phosphodiethyl ester bonds. On the other hand, proteins that form the main parts of antibodies and enzymes are polymerized amino acids formed by polypeptide bonds. Therefore, since the binding mode of nucleic acids is completely different from that of proteins, the possibility of denaturing or decomposing proteins is extremely small under reaction conditions that cleave nucleic acids. In particular, if an endonuclease called a restriction enzyme is used, it is possible to cleave only a specific base sequence in a nucleic acid, so only the nucleic acid portion can be selectively cleaved without cutting the protein portion.

Therefore, the antibody is bound to a solid phase via a nucleic acid,
The antigen and labeled antibody are captured through an antigen-antibody reaction, and after washing, they can be selectively cleaved at the nucleic acid portion. At this time, only the labeled substance involved in the target antigen-antibody reaction can be removed from the same phase without denaturing or decomposing the antibody or enzyme. Since the labeled substance is directly attached to the solid phase without intervening the nucleic acid, it will not be released by the action of restriction enzymes.This makes it possible to measure the amount of antigen by eliminating the influence of nonspecific adsorption. can be used without compromising quantitative properties.

Restriction enzymes, also called restriction endonucleases, are DN
Among the enzymes that cut A (deoxyliponucleic acid), it has the function of recognizing and cutting only a specific base sequence. This base sequence, called a recognition cleavage sequence, is unique to each restriction enzyme. Therefore, if the nucleotide sequence of the nucleic acid or oligonucleotide used for binding has at least one insertion of a recognition cleavage sequence for the restriction enzyme used, it can be cleaved by the action of a restriction enzyme as in the present invention. A base sequence that satisfies this condition can be freely set. There are currently 300 restriction enzymes.
More than one species is known, and one can be selected from these depending on the purpose.

Methods for binding nucleic acids to antibodies and solid phases include, for example, the method of Bosch et al. [Ghosh: Bioconjuga
teChem, 1, pp. 71-76 (1990)]
[Corey: 5cienc] method of Corey et al.
e, 238.

1401-1403 (1987)], Method 1 of Bhan et al.: Bhan: Bioconjugata Ch.
Em, 1, pp. 82-88 (1990)]
, the method of Doan et al.
Chem, 1, l O8-113 (1990
)], and many others have been introduced, and these methods can be applied.

[Example code] The present invention will be specifically explained with reference to Examples.

[Example 1] An immobilized antibody microplate was prepared as shown in the schematic diagram of FIG.

limit! Base sequence A that can be cleaved with l element Hae T
Oligonucleotide 17 with GGOCT was synthesized, and anti-CEA (carcinoembryonic antigen) was added to this.

Carcino Embryonic Antigen
) Antibody 16 was coupled by maleimide-thiol coupling according to the method of Bosch et al., supra. A, G, T
, C are constituent units of nucleotides, and represent an adenine base, a guanine base, a thymine base, and a cytosine base, respectively.

A complementary strand 18 to the synthetic oligonucleotide was synthesized and bound to each well (well) 19 of a microplate having an amino group as a surface active group using the same method as described above.

An immobilized antibody microplate was obtained by complementary binding the anti-CEA antibody-oligonucleotide complex to a complementary oligonucleotide immobilized on a microplate.

Next, a CEA standard sample was measured using the microplate. The reaction procedure is schematically shown in FIG. 2. CEA Sakura quasi-samples 20 prepared at various concentrations were placed in each well 19, reacted for 15 minutes, and washed with a buffer solution. Next, anti-CEA antibody 1 labeled with horseradish peroxidase 21
6 (labeled antibody) was reacted. After washing, restriction enzyme Hae
The antigen-antibody reaction complex was separated from the solid phase using I22 and fractionated into another microplate. The amount of peroxidase contained in the fractionated liquid was reacted with 3-(4-hydroxyphenyl)propionic acid as a substrate, and the fluorescence produced by the reaction was measured using a fluorometer.

For comparison, we investigated cases in which antibodies were immobilized on microplates by direct adsorption and N-succinimidyl-
Measurements were also carried out when the antibody was immobilized on a microplate via a disulfide bond using 3-(2-pyridyldithio)propionate).

The conditions for the sample, antibody, and antigen-antibody reaction were the same.

However, when using a microphone plate on which antibodies were directly immobilized, the color reaction was performed while the microphone plate was used during the reaction. When using a microplate with immobilized antibodies immobilized via disulfide bonds, after reacting the sample and the labeled antibody, the disulfide bonds are reduced with a reducing agent (disthreitol) and the immune complexes are dissociated. After transferring to another microplate, a color reaction was performed.

The results are shown in FIG.

The present example is shown by curve a, the case where a disulfide bond is used is shown by curve b, and the case where a direct bond is used is shown by curve C.

As is clear from the figure, when the CEA concentration is zero (blank), that is, looking at the background value, a, b,
It increases in the order of c. Also, b and c are each 1
At concentrations below 0"M, IO"M, the fluorescence intensity is no longer proportional and becomes flat. In other words, it is shown that quantification cannot be performed below this value.

[Example 2] When measuring a low-molecular compound hapten such as a drug, highly sensitive measurement can be performed using a mirror table method.

As in Example 1, the anti-digoxin antibody was immobilized, that is, the anti-digoxin antibody was bound to an oligonucleotide having the same base sequence. This and the oligonucleotide used in Example 1 were dispensed into a microplate on which was immobilized, and complementary binding was performed.

Each fixed concentration of fluorescently labeled digoxin was added to a sample containing digoxin, dispensed into a microplate, and allowed to react for a fixed period of time. After washing each well to which the antibody had been bound, it was cut with restriction enzyme Hae I, and the fluorescent label dissociated into the solution was measured using a fluorometer. This hapten could also be measured with high sensitivity.

In addition, as in this example, by using antibodies bound to oligonucleotides with the same base sequence, it is possible to easily immobilize any antibody to be measured in the same reaction vessel. In addition, when configuring the device, measurement items can be measured in any arrangement depending on the purpose.

In other words, it has the advantage of random access.

[Example 3] A schematic diagram of the configuration of an apparatus based on the above example is shown in FIG. 4.

A reaction microplate 1 on which oligonucleotides have been immobilized is placed on a reaction container holder 2. The reaction microplate 1 has the same oligonucleotide immobilized thereon so that it can be commonly used for various measurement items. The reaction vessel holding stand 2 is connected to a moving mechanism 3 and a constant temperature controller 4 so as to maintain a predetermined reaction temperature. The reaction container holding stand 2 is provided with a test tube chamber so that it can accommodate, for example, test tubes in addition to microplates.

Antibodies 6.6' and 6' for immobilization are antibodies for three types of measurement items A, B, and C, respectively, and are oligonucleotides having base sequences complementary to the oligonucleotide bound to the reaction microplate. is combined.

Therefore, antibodies for any measurement item can be bound to the same reaction microplate 1.

The controller 7 selects an antibody for an arbitrary measurement item to be measured from among the immobilized antibodies 6, 6' and 6'' using the dispenser 5, and injects it into the container 1 for each well. 28, and is moved to a selected position by a moving mechanism 29 that can move arbitrarily in the XYZ axes.

The containers 30 are moved well by well in synchronization with the injection. For example, the measurement items are A, A, B, and C.

If you want to measure in the order of B, antibodies 6, 6.6', 6",
Inject into each well in the order of 6' and immobilize.

After dispensing, the antibody is left to stand for a certain period of time, and after the antibody is immobilized, it is washed with a washing liquid 27 using a washer 8. Next, the sample 9 is dispensed using the dispenser 5. The controller 7 is controlled so that the samples are also measured in an arbitrary order.

Note that the sample should be diluted if necessary.

After the sample reaction, the sample is washed with a washer 8, and then 10, 10' and 10'' antibodies are dispensed. Measurement items A, A, B, C, which were specified at the beginning.
Also labeled antibodies according to B. 10.10.10', 10''.

10' are automatically selected by the controller 7.

After the reaction for a certain period of time, cleaning is performed again using the washer 8. Next, an elimination reagent 11 containing a restriction enzyme is dispensed. After desorption, it is guided to a flow cell type detector 12. The detector 12 is a so-called flow injection analyzer, and the liquid to be measured is finally introduced into a flow cell and optically detected therein.

A reaction condition adjusting reagent 25 and a reaction reagent 13 such as a substrate are added to the liquid to be measured led from the tube to the detector 12.
The mixture passes through the reaction coil 26 and is mixed and reacted. Optical measurement can measure fluorescence or absorbance, and when the label is an enzyme, a fluorescent or chromogenic substrate suitable for this is selected as the reaction reagent.

If the label is a fluorescent dye, no particular reaction reagent is required.

The reaction condition adjustment reagent 25 is used to adjust reaction conditions such as pH and ionic strength suitable for optical measurements and enzymatic reactions.

The measured values are processed by the data processor 14 and displayed by the display 15.

As the labeled antibody, an antibody specific to the measurement target and a labeled secondary antibody therefor may be used.

As the detector 12, in addition to the above-mentioned flow type, a normal spectrophotometer can also be applied. In that case, the liquid to be measured after desorption is transferred to a container different from the reaction container, and then the reaction reagent 13,
Add adjustment reagent 25.

[Effects of the Invention] According to the present invention, non-specific adsorption of labeled antibodies can be avoided and measurement can be performed with low background, so biological components can be measured with high sensitivity.

In addition, if complementary oligonucleotides are bound to the solid phase and the antibody, and the complementary binding is made during use, the solid phase force can be shared regardless of the type of antibody, so any item can be randomly The present invention can be applied to a so-called random access mode device that can perform measurements in a straight line.

[Brief explanation of the drawing]

Fig. 1 is a schematic diagram showing an example of antibody immobilization in an example of the present invention, Fig. 2 is a schematic diagram showing the operation flow of the same example, and Fig. 3 is a graph showing the relationship between CEAQ degree and fluorescence intensity. FIG. 4 is a schematic diagram showing the configuration of an apparatus based on an embodiment of the present invention. l Reaction plate i・, 2 Reaction vessel holder, 3 Movement mechanism, 4 Constant temperature controller, 5 Dispenser, 6゜6', 6''...
Antibody for immobilization, 7 Controller, 8... Cleaner, 9...
・Sample, 10.10', 10''・Labeled antibody, 11 ・
Desorption reagent, 12. Detector, 13. Substrate, 14. Data processor, 15. Display device, 16.. Antibody, 17. Oligonucleotide, 18. Complementary strand, 19.. Well, 20..
CEA standard sample, 21... Horseradish peroxidase, 22... Restriction enzyme (Hae I), 25-=- Reaction condition adjustment reagent, 26... Reaction coil, 27... Washing solution, 28... Nozzle, 29... - Moving mechanism, 30... Container. Agent Patent Attorney Akio Takahashi -1 Figure 1 16 Antibody 17 Oligonucleotide 18 Complementary strand 19 Well 20 Standard sample 21-1
111122... im+i Fermentation chart Figure 2 Figure 3 CEA concentration (M) 30... Container Figure 4: -

Claims (1)

[Claims] 1. Using an antibody, a substance that specifically binds to the antibody is captured on a solid phase, a label is bound to this, and the substance is washed and then captured on the solid phase. In a biological component analysis method that quantifies a target substance by measuring the amount of a label quantitatively bound to the target substance, the step of binding the antibody to a solid phase via a nucleic acid, and removing the excess label After washing, the method is characterized by comprising the steps of: releasing the labeled substance captured on the solid phase from the solid phase via the substance to be measured by cleaving the nucleic acid; and quantifying the separated labeled substance. Biocomponent analysis method. 2. The biological component analysis method according to claim 1, wherein a restriction enzyme is used to cleave the nucleic acid. 3. The biological component analysis method according to claim 1, wherein one terminal of the nucleic acid is bound to a solid phase and the other terminal is bound to an antibody. 4. The biological component analysis method according to claim 1, wherein the antibody is bound to a solid phase by a connector, a part of the connector is composed of a nucleic acid, and the nucleic acid is cleaved. 5. The biological component analysis method according to claim 1, wherein the connector is composed of a molecule having affinity selected from antibodies, lectins, biotin, avidin, and protein A and a nucleic acid. 6. The biological organism according to claim 1, wherein one end of the nucleic acid is bound to a solid phase, and one end of the nucleic acid having a complementary base sequence to the solid phase is bound to an antibody, and the nucleic acid is used in a complementary manner. Component analysis method. 7. The biological component analysis method according to any one of claims 1 to 6, wherein the nucleic acid is an oligonucleotide. 8. The base sequence of the oligonucleotide is made common regardless of the analyte, and the same oligonucleotide is bound to each receptor for the analyte, forming a complementary strand pair immediately before the reaction to form an immobilized receptor. The biological component analysis method according to claim 1, characterized in that: