JPS61217764A - Method and apparatus for assaying immune protein - Google Patents

Abstract

PURPOSE:To achieve a high assaying accuracy and a shorter measuring time, by arranging a heating means and a cooling means on the downstream of a joint connection part between a third passage for supplying a reference catalyst solution to a chemically luminescent reaction section and a fourth passage connected somewhere in the course of the third passage. CONSTITUTION:A thermostating means 10 is provided to maintain solutions A and B at a constant temperature somewhere in the course of first and second passages I and II for supplying the solution A containing chemically luminescent substance and the solution B containing oxidizing substance to a flowcell Fc at a chemical reaction section R. A heating means 11 and a cooling means 12 are provided on the downstream of the joint connection part between a third passage III for supplying a reference catalyst solution C to the cell Fc and a fourth passage IV which extracts an antigen to be measured from a sample solution D to mixed into a solution C in the passage III. With the heating action of the means 11, a buret forming reaction is done quickly between the solution C and the antibody to be measured in the solution D mixed thereinto while cooling by the means 12 prevents the generation of bubbles in the passage positively thereby enabling accurate assay in a short time.

Description

[Detailed Description of the Invention] □ [Field of Industrial Application] The present invention provides a method and apparatus for quantifying immune proteins based on completely new means, and specifically, a method and apparatus for quantifying immune proteins based on completely new means. The present invention relates to a method and apparatus for quantifying the amount of a protein (the term "immune protein" here means only one of the immune proteins in an antigen-antibody relationship).

As mentioned above, the immune protein referred to here can mean either an antigen (protein) or an antibody (protein), but for convenience of explanation, the following explanation will be based on the case where the immune protein to be measured is an antigen. I will only state the following. In the explanation, by replacing the term "antigen" with the term "antibody J," it will be possible to easily infer the case where the immunological protein to be measured is an antibody.

[Conventional technology]

A method for quantifying immune proteins generally called immunoassay (
(and equipment for carrying it out) include the RIA method using a radioactive isotope as a labeling agent; the ErA method using a fluorescent substance as a dye identifying agent; Methods such as fluorescent labeling are used as identification agents, that is, labeled antibodies are labeled with 1M using radioisotopes, enzymes, or fluorescent substances to antibodies corresponding to the immunological protein to be measured (hereinafter referred to as antigens). After the labeled antibody is produced and the antigen to be measured in the sample solution is adsorbed, the unadsorbed labeled antibody and other substances are removed to leave only the I-identified antibody-antigen to be measured complex, and then,
Conventionally, it has been known to quantify the antigen to be measured in a sample solution by measuring the amount of the labeled antibody using radioisotope method, absorption method, or fluorescence method, which are known methods for quantifying proteins. .

[Problem that the invention seeks to solve]

However, in the various conventional methods described above, it is necessary to carry out labeling operations for antibodies, which are extremely complicated and time-consuming procedures, and require a long time.Also, since antibodies and labeled antibodies can only be used once, batch quantification is required. Therefore, the RIA method has the common disadvantage that it requires a long incubation time of several tens of hours for one sample, and there is a lot of waste of IIL drugs such as antibodies and labeled antibodies. The EIA method uses a dye consisting of a large molecule as a labeling agent, which causes poor immune reaction efficiency due to steric hindrance, leading to deterioration in quantitative sensitivity and accuracy. In the fluorescent labeling method, in order to use fluorescent @IJ as a labeling agent, an organic solvent must be used during the labeling operation, and therefore it is often not applicable depending on the type of antigen to be measured. As mentioned above, each method also had drawbacks regarding patency.

The present invention has been made in view of the above circumstances, and its purpose is to provide a device that can be constructed relatively simply and inexpensively.
It is an object of the present invention to provide a method and apparatus for quantifying immune proteins that can quickly, safely and economically quantify a wide variety of immune proteins with high sensitivity and precision.

[Means for solving problems]

The method and device for quantifying immune proteins according to the present invention is basically a novel method that effectively utilizes a natural phenomenon called chemiluminescence (chemiluminescence reaction), which has not previously been given any attention in the technical field. Since it was developed based on an idea, I will first explain some interesting phenomena related to the chemiluminescence reaction.

That is, as shown in FIG. 8, luminol (CSH, N,
Ch) A solution containing a substance that exhibits chemiluminescence when oxidized, such as a solution, and a solution containing an oxidizing substance, such as a hydrogen peroxide solution (Hint solution), are potassium ferricyanide (K s When a catalyst solution C such as a [F e (CN) It is well known that reactions occur that produce fairly intense blue chemiluminescence (approximately 450 nm). In addition, the chemiluminescent substances include lucigenin and lophine in addition to the luminol, and the oxidizing substances include oxygen, ozone, hypochlorous acid, and the like in addition to the hydrogen peroxide. .

Therefore, the present inventors discovered that in such a chemiluminescence reaction system, when protein d is mixed into the catalyst solution C, the intensity of the chemiluminescence l decreases (so-called quenching), as shown by the dotted line 2 in the figure. Moreover, the amount of decrease in the intensity of chemiluminescence l is proportional (good linearity) over a wide range of amounts of the contaminant protein d (from very small amounts to considerably large amounts).
Furthermore, we discovered and experimentally found that, with a few exceptions, the rate of decrease does not depend much on the type of protein. I recognized it.

Furthermore, the present inventors have proposed that, instead of the potassium ferricyanide solution, which has the drawback of extremely high toxicity, the catalyst solution C may be a solution containing metal ions such as copper ions, Cu-, or a solution containing metal ions such as hematin, which exists in nature. It was also discovered and experimentally f! that a similar phenomenon occurs when a solution containing a metal complex is used. I recognized it. This phenomenon occurs when metal ions or metal complexes in an amount corresponding to the amount of the mixed protein out of the metal ions in the solution that serve as catalysts for the chemiluminescent reaction form a biulet with the mixed protein. It is presumed that this is because the catalytic activity of the catalyst solution as a whole decreases because the catalyst loses its catalytic activity.

Therefore, in view of the fact that both antigens and antibodies are proteins themselves, the present inventors have developed the following method and method for quantifying immune proteins (antigens or antibodies) by effectively applying the above-mentioned phenomenon. This led to the development of the device.

That is, the method for quantifying an immunological protein (for example, an antigen) according to the present invention uses an immunoaffinity chromatography system on which an immunological protein (antibody) that has an antigen-antibody relationship with respect to the immunological protein (antigen) to be measured is immobilized. A sample solution is introduced into a column (antibody-immobilized column), and the immunogenic protein (antigen) to be measured in the sample solution is exposed to the immunological protein immobilized within the column (antibody). - After capturing by antibody reaction, the inside of the column is washed to remove unnecessary substances other than the immunogenic protein (antigen) to be measured, and then an aqueous solution is introduced into the column. The immunogenic protein (antigen) to be measured is separated from the immunogenic protein (antibody) immobilized on the column and eluted from the column, and then mixed with a solution containing a substance that exhibits chemiluminescence when oxidized. The immune protein to be measured (antigen) eluted from the column is mixed into a reference catalyst solution that is supplied to a chemiluminescence reaction system with a solution containing an oxidizing substance, and in this state, the chemical A feature of this method is that it employs a procedure in which the amount of immune protein in the sample solution is measured by measuring the chemiluminescence intensity from the luminescence reaction system.

Furthermore, the apparatus for quantifying immune proteins (for example, antigens) according to the second invention is configured to be able to introduce and supply a predetermined amount of a solution containing a substance that exhibits chemiluminescence when oxidized to the chemiluminescence reaction section. a first flow path, a second flow path configured to be able to introduce and supply a predetermined amount of a solution containing an oxidizing substance to the chemiluminescence reaction section; and a predetermined amount of a reference catalyst solution to the chemiluminescence reaction section. A third flow path configured to allow the chemiluminescence to be introduced and supplied at a time, and chemiluminescence intensity measuring means for measuring the intensity of chemiluminescence emitted from the chemiluminescence reaction section,
An immunoaffinity chromatography column (antibody-immobilized column) on which an immunological protein (antibody) that has an antigen-antibody relationship with the immunological protein (antigen) to be measured is immobilized, and a state in which a sample solution is introduced into the column. and a valve mechanism that can be switched between introducing a washing solution into the column and introducing a dissociation solution into the column, and introducing a sample solution into the column. The immunogenic protein (antigen) to be measured in the sample solution is added to the immobilized immunogenic protein (antibody) as an antigen.
After capture by antibody reaction, the inside of the column is washed to remove unnecessary substances other than the immunogenic protein (antigen) to be measured, and then a dissociation solution is introduced into the column and immobilized within the column. The immunogenic protein (antigen) to be measured is separated from the immunogenic protein (antibody) obtained and eluted from the column, and the immunogenic protein (antigen) to be measured eluted from the column is transferred to the third flow path. A fourth flow path configured to be able to mix with the reference catalyst solution in the third flow path is provided in the middle of the third flow path, and the fourth flow path in the third flow path is connected to the fourth flow path. It is characterized in that a heating means and a cooling means are provided downstream of the merging connection part.

[Effect]

The effects exhibited by the method and apparatus of the present invention are as follows.

That is, according to the first method of the invention, an immune protein (antigen) that has an antigen-antibody relationship with the immune protein (antigen) to be measured is obtained.
By using an immunoaffinity chromatography column (antibody-immobilized column) with immobilized antibodies (antibodies), the immune protein (antigen) to be measured in the sample solution can be efficiently separated and eluted. There is no need to perform troublesome procedures and time-consuming sign operations, and
Since the immunological protein (antibody) immobilized on the column can be used repeatedly, continuous quantification is possible, and therefore,
It has become possible to rapidly quantify one sample in a very short time of about 15 minutes compared to the conventional several tens of hours, and it has also become possible to significantly reduce wastage of reagents.

In addition, the amount of the immunological protein (antigen) to be measured is determined in the sample solution mixed in the reference catalyst solution that is supplied to the chemiluminescent reaction system of the chemiluminescent substance-containing solution and the oxidizing substance-containing solution. There are a small number of compounds that can be used proportionally with good linearity over a wide range of amounts, from small amounts to very large amounts, and that have some degree of catalytic activity for chemiluminescent reactions. The intensity of chemiluminescence from the chemiluminescence reaction system decreases at an almost constant rate regardless of the type of immune protein (antigen) to be measured. )
can be quantified with high sensitivity and precision over a wide measurement range.

By the way, according to experiments conducted using the method of the present invention,
In the case of human serum albumin, a measurement value of 5°ng (absolute amount) was obtained, which was two orders of magnitude better than the conventional method.

Furthermore, the method of the present invention does not use a radioactive isotope labeling agent unlike the conventional RIA method, and can be used instead of the normally used highly toxic potassium ferricyanide solution as the catalyst solution for the chemiluminescent reaction system. In addition, since a solution containing non-toxic metal ions such as copper ions or metal complexes such as hematin is used, quantitative operations can be performed safely.

Furthermore, the method of the present invention can be carried out using a relatively simple and inexpensively configured apparatus and inexpensively available reagents, and as mentioned above, there is no need for pretreatment of the sample such as labeling operation, and there is little wastage of reagents. Therefore, the initial cost and running cost are low, making it extremely economical.

According to the immunological protein quantification apparatus according to the second invention, which is constructed by applying the above-mentioned first invention method, the excellent basic effects of the above-mentioned first invention method can be exerted as is. In particular, a third flow path configured to be able to introduce and supply a reference catalyst solution containing metal ions such as copper ions or metal complexes such as hematin into the chemiluminescence reaction section in predetermined amounts, and a reference catalyst within the third flow path. A heating means is provided on the downstream side of the confluence connection part with the fourth flow path which is connected in the middle of the third flow path so that the sample solution containing the immunological protein to be measured can be mixed into the solution. Since the device is equipped with a cooling means, the heating effect of the heating means causes the biulet-forming reaction between the reference catalyst solution and the immunological protein to be measured in the sample solution to occur quickly and without shortage. At the same time, the cooling effect of the cooling means reliably prevents the generation of bubbles in the flow path, which would be a major hindrance to stable measurement of luminescence intensity, allowing more accurate quantification to be performed in a shorter time. be able to.

〔Example〕

Embodiments of the present invention will be described below with reference to the drawings (FIGS. 1 to 7).

Figure 1 (a), (b), (c), (d) and Figure 2 (
A) and (B) are for explaining the basic procedure of the method for quantifying an antigen as an immune protein according to the present invention.

First, as shown in FIG. 1 (a), an immunoaffinity chromatography plate is used to immobilize an antibody (anti-human serum albumin) Ab that has an antigen-antibody relationship against the antigen to be measured (for example, human serum albumin) H3A. In column CU (hereinafter referred to as antibody-immobilized column, the specific structure of which will be described later), as shown by the dotted line in the figure, the antigen to be measured H3A was added.
Introduce a sample solution containing...etc.

Then, as shown in FIG. 1 (b), only the specific antigen, that is, the antigen to be measured H3A... in the sample solution, reacts with the antibody Ab... immobilized in the column CU. The other unnecessary substances fXX, which are bound and captured by the antigen-antibody reaction, do not bind to the antibody Ab and remain free. In this state, the cleaning solution E is caused to flow through the column CU as shown by the dotted line in the figure.

Then, as shown in FIG. 1(C), the inside of the column CU is washed, the other substances XX... are removed, and the substances captured by the immobilized antibodies Ab are left in the column CU. Only the measurement antigen H3A... will remain.

In this state, the dissociation solution F is caused to flow through the column CU as shown by the dotted line in the figure.

Then, as shown in FIG. 1(d), the antigen to be measured H3A.
... is separated and eluted into the Mtfj solution F. The separated and eluted antigen H3A to be measured, as shown by the dotted line in the same figure, is fed into the reference catalyst solution supply line 4 to the chemiluminescence reaction system Q, which will be described next, together with the dissociation solution F. It will be mixed into the reference catalyst solution C.

On the other hand, as shown in FIG. 2 (a), luminol is used as solution A containing a substance that exhibits chemiluminescence when oxidized.Hydrogen peroxide (HxO8) is used as solution B that contains a substance that exhibits oxidizing properties. A reference catalyst solution C is introduced into a chemiluminescence reaction system Q configured to mix and supply a predetermined amount of water to each of the flow cells Fc constituting the chemiluminescence reaction section R.
A predetermined amount of only a solution containing metal ions such as copper ions Cu1+ is supplied via the reference catalyst solution supply line 5, and in this state, the intensity of chemiluminescence emitted from the flow cell Fc of the chemiluminescence reaction section R is (This is taken as the reference chemiluminescence intensity v0), photosensor 1, amplifier 2. Measurement is performed by chemiluminescence intensity measuring means S consisting of a recorder 3 and the like. At this time, only the dissociation solution F may be mixed into the reference catalyst solution supply line 5, as shown by the imaginary line in the figure.

Next, as shown in FIG. 2(b) [this corresponds to the steps in FIG. 1(c) and FIG. 1(d) above], the aforementioned reference catalyst solution supply line 4 is Figure 1 (c)
The state of It! Antibody-immobilized column C in (antigen capture state)
By introducing the dissociation solution F into the reference catalyst solution supply line 5 through U, as shown in FIG. . measure.

Then, the antigen to be measured H3A... is added to the reference catalyst solution C.
By comparing the chemiluminescence intensity V measured when the light is mixed into the interior (at the time of dimming) and the reference chemiluminescence intensity V, specifically, the portion S shown by hunting in FIG. Based on the reduction in the catalytic activity of the reference catalyst solution C due to the antigen H3A to be measured in the sample solution... The amount of antigen H3A to be measured is quantified.

In addition, in the said FIG. 2 (a) and (b), Pl.

P and Ps respectively indicate constant flow pressure pumps for transferring each solution, and M1. M *, M x
indicate mixed connectors, respectively.

FIG. 4 shows a schematic configuration of an apparatus for quantifying an antigen as an immune protein according to the second invention, which is constructed by applying the method of the first invention.

In the figure, ■ is a first channel configured to be able to introduce and supply a predetermined amount of a luminol solution as the chemiluminescent substance-containing solution A to the flow cell Fc in the chemiluminescent reaction section R, and ■ is the oxidizing substance-containing solution. A second channel configured to be able to introduce and supply a predetermined amount of hydrogen peroxide solution as the containing solution B to the flow cell Fc; The third flow path serves as a reference catalyst solution supply line configured to be able to introduce and supply a predetermined amount of catalyst solution to the flow cell Fc, and each of the flow paths 1. II and Ill is provided with a closed solution tank T.
+, Tt, Tx and flow meters U+, Ut, Us with needle valves for flow rate adjustment are provided, respectively. Each of the channels 1. As the solution transfer energy source for U and Ill, instead of the normally used pressure pump, a pressure regulator 6 common to each of these channels +, n, and m is used to prevent pulsation in the fluid flow. A pressurized inert gas (eg nitrogen gas) cylinder 7 is used.

Further, for the flow cell Fc of the chemiluminescence reaction section R, a photosensor l such as a photomultiplier tube, an amplifier 2.
A chemiluminescence intensity measuring means S consisting of a recorder, 31-inch grater 4, etc. is provided.

In the middle of the third flow path (2), there is a fourth flow path (2) configured so that the antigen to be measured can be extracted from the sample solution and mixed into the reference catalyst solution C in the third flow path (2). are connected together.

That is, the fourth flow path (2) includes an open tank T that accommodates the cleaning solution E, a first constant flow pump Pa for transferring the cleaning solution E in predetermined amounts, and the first pump Pa. A constant volume (50 μm in this example) is located downstream of the
) A first valve mechanism 8 consisting of a seven-way valve equipped with a sampling loop and an aspiration/discharge syringe W;
An open tank T4 containing a sample solution containing the antigen to be measured is connected to the valve mechanism 8, and a confluence of the third flow path (■) and the fourth flow path (■) from the outflow portion of the first valve mechanism 8. A second section consisting of a six-way socket is inserted in the middle of the flow path leading to the section.
a valve mechanism 9; an immunoaffinity chromatography column (antibody immobilization column) CU connected to the second valve mechanism 9;
It consists of an open type tank Th that accommodates the dissociation solution F, and a constant flow rate pressure-feeding second pump pb for transferring the solution F by a predetermined amount to the second valve mechanism 9, and each of the valve mechanisms 8, 9 to the appropriate state (see section 6 for details).
(explained later using the figure), a predetermined amount (50 μm) of the sample solution is sucked into the sampling loop, and the sample solution in the sampling loop is immobilized with the antibody by washing solution E. Column C
A state in which the dissociation solution F is introduced into the antibody-immobilized column CU by flowing into the antibody-immobilized column CU, and the inside of the antibody-immobilized column CU is washed with the washing solution.
It is configured so that it can be switched to a state where it is introduced into the confluence of the flow path -■ and the fourth flow path ■.

Furthermore, there are channels in the middle of the first flow path (■) and the second flow path (■)! , a common constant temperature means 10 for maintaining each solution in II at a constant temperature to stabilize the chemiluminescence reaction.
(for example, a constant temperature water bath of approximately 25°C), and a heating water bath such as a high-temperature water bath is installed downstream of the merging connection portion of the fourth flow path (■) in the third flow path (■). Means 1
1 (for example, about 95°C) followed by a cooling means 12 (for example, about 0°C) such as a low-temperature water bath,
Due to the heating action of the heating means 11, the biulet-forming reaction between the reference catalyst solution C and the antibody to be measured in the sample solution mixed therein is carried out quickly and sufficiently in the third flow path (2). In addition, the cooling action of the cooling means 12 is configured to reliably prevent the generation of bubbles in the wave path, which would be a major hindrance to stable measurement of luminescence intensity.

In addition, the chemiluminescence reaction part R1 first flow path summer, second flow path ■
, the piping parts in the third flow path ■, the fourth flow path ■, etc.
All Teflon (polytetrafluoroethylene: DuPont trademark)
In addition, in order to prevent the sample from dispersing inside the piping, the inner diameter of all the piping must be relatively small (for example,
．． 5- below) is desirable.

Furthermore, in FIG. 4, M L+ M z and M s each indicate a mixed connector.

FIG. 5 shows a specific example of the structure of the immunoaffinity chromatography column (antibody immobilization column) CU.
and filter member 13C.

A large number of glass beads 14 each having an antibody immobilized thereon in an antigen-antibody relationship with respect to the antigen to be measured are filled inside the cylindrical body 13 having 130 at both ends. The antibody-immobilized glass beads 14 are prepared as follows. That is, the porous glass beads are H(1!, HN
After washing with O, solution, etc., 1% concentration T-glycidoxypropyltrimethoxysilane is applied to form glycidoxy type glass beads, and then a pH 3 HCIt solution is applied to form diol type glass beads. Furthermore, aldehyde-type glass beads are made by reacting with sodium periodate, and a predetermined antibody is bonded to the aldehyde-type glass beads to obtain imine-type bonded antibody-immobilized glass beads, and sodium borohydride is reacted with the glass beads. This process produces antibody-immobilized glass beads.

FIGS. 6(a), (b), (c), (d), and (e) are for explaining the quantitative operation procedure using the antigen quantitative determination apparatus configured as described above.

That is, first, the first valve mechanism 8 in the fourth flow path (2) is put into the state shown in FIG. Inhale a predetermined amount (in this case, 50μ) of the sample solution.

Next, the second pump pb in the fourth channel (2) is operated, and the second valve mechanism 9 is set to the state shown in FIG. 6 (B), and only the dissociation solution F is supplied to the third channel (2). In this state, that is, in a state in which substantially only the reference catalyst solution C is supplied to the flow cell Fc of the chemiluminescence reaction section R, the intensity of chemiluminescence emitted from the flow cell Fc (reference chemiluminescence intensity V, ), that is, the zero point, is measured by the chemiluminescence intensity measuring means S.

Note that the operating procedures shown in FIGS. 6(a) and 6(b) may be performed before or after the above.

Subsequently, the second pump pb is stopped while the first pump is stopped.
Activating the pump Pa and switching the first valve mechanism 8 and the second valve mechanism to the state shown in FIG. 6(c) to flow the cleaning solution E through the sampling loop. The sample solution in the sampling loop is supplied to the antibody-immobilized column C port, and the specific antigen in the sample solution, that is, the antigen to be measured, is transferred to the antibody immobilized in the column C port. Binding by antigen-antibody reaction, followed by washing solution E
By causing the sample to flow through the column C port, substances other than the antigen to be measured remaining in the column C port are removed and discharged.

Next, the first pump Pa is stopped while the second pump pb is started again, and the second valve mechanism 9 is switched to the state shown in FIG. The antigen to be measured is separated and eluted from the immobilized antibody in the opening of the column C into the dissociation solution F by flowing through the opening of the column C, and the separated and eluted antigen to be measured is separated and eluted together with the dissociation solution F. A fourth line serving as a reference catalyst solution supply line to the chemiluminescence reaction section R
It is mixed into the reference catalyst solution C in the flow path (2). In that state, that is, in a state in which a mixed solution of the reference catalyst solution C and the dissociation solution F containing the antigen to be measured is supplied to the flow cell F of the chemiluminescence reaction section R (at the time of dimming). In this step, the intensity of chemiluminescence emitted from the flow cell F is measured by the measuring means S, and the predetermined amount of the sample is calculated from the reduced light amount calculated by the inch grater 4 based on the reference chemiluminescence intensity V. The amount of antigen in the solution is quantified.

Finally, the second pump pb is stopped and the first valve mechanism 8 is switched to the state shown in FIG. The sample solution in the syringe W is discharged to prepare for the next quantitative determination.

Next, the results of tests conducted using the antigen quantification device configured as described above will be described.

The outline of the test conditions is as follows.

〔reagent〕

All reagents were commercially available special grade products.

■Chemiluminescent substance-containing solution A 0.1 mol/da' of boric acid and 0.1 mol/da3
A buffer solution of pH 10.2 containing potassium hydroxide (B
uff-1) to give 1.0 Xl0-'mol/
A luminol solution of dm' was prepared.

■ Oxidizing substance-containing solution B Dilute 0.3% by weight hydrogen peroxide with pure water and
10-'mol/da' of hydrogen peroxide solution was prepared.

■Standard catalyst solution C A stock solution of copper ion Cu0 of 2,OX 10-'mol/da' is diluted with the buffer solution (Buff-1), and Cu'° of 2,OX 10-'mol/dfll' is diluted with the buffer solution (Buff-1).
A solution was prepared.

■Washing solution E This also serves as a buffer solution for the sample solution.
In order to suppress ionic sample adsorption by the antibody-immobilized column CU during sample elution in the antibody-immobilized column CU, a mixed solution of potassium phosphate (K H! P 04 ) and sodium phosphate (Na, HPO, ) is added. A small amount of potassium chloride was added to prepare a washing phosphate buffer solution (Buff-II) with a pH of 7.2.

(2) Sample solution The sample antigen was diluted with the phosphate buffer solution (Buff-II).

■ Solution 111 Molten solution F (Glycine-HCjl> solution and (tartaric acid-sodium tartrate) solution were also tried, but (H
(1!-K(1) solution was optimal, so this was used.

[Device adjustment]

■The inner diameter of all piping has been unified to 0.5m++s.

■All solutions A, B, C, D, E are 1 cm'/+wi
A pressurized inert gas cylinder 7 and a pressure regulator 6. Flowmeter U with needle valve for flow rate adjustment
, Ut, us, constant flow pressure pumps Pa, Pb, etc. were adjusted.

■The constant temperature means 10 is set to 25°C, and the heating means 11 is set to 95°C (
0.64 m), and the cooling means 12 was maintained at 0°C (0.40 m).

(2) The residence time of the solution in flow cell F was adjusted to 3.4 seconds.

〔Test results〕

(2) In the case of human serum albumin, an excellent measurement sensitivity of 50 ng (absolute amount) was obtained.

■It is possible to quantify one sample in a short time of about 15 minutes.

■ Constant temperature means 10. By using the heating means 11 and the cooling means 12, stable reference chemiluminescence intensity can be achieved. It has been confirmed that this is a very effective means of obtaining

FIG. 6 shows another embodiment of the antigen quantification device according to the second invention.

This system includes a controller 13, a plurality of sample solutions, etc., so that almost all operations required for quantitative determination in the apparatus shown in FIGS. 4 to 6 can be automatically performed.
...... An autosampler 14 is provided for automatically switching and supplying D7 sequentially, and cleaning solution E is provided.
The dissociation solution F is also transported using a pressurized inert gas cylinder 7.

That is, the autosampler 14 is connected to a second valve mechanism 9 consisting of a six-way valve having a sampling loop and a suction/discharge passage 16 equipped with a check valve 15 and a suction pump P8, via a multistage switching valve 17. A plurality of open tanks T41 each containing a plurality of sample solutions D1......D, respectively.
4. It is configured by connecting.

In this case, the tank T containing the cleaning solution E and the tank T containing the dissociation solution F are constructed in a sealed type, and the outflow passage is equipped with a flow meter U4. US is interposed respectively. And a flow meter UI, Uz, Us with 5 needle valves for flow rate adjustment.

Ua, Us, the multistage switching valve 17, and the suction pump P are configured to be automatically controlled by the controller 13 according to a predetermined sequence. Other configurations are similar to those of the previous embodiment.

In each of the above Examples, a luminol solution was used as the chemiluminescent substance-containing solution, hydrogen peroxide was used as the oxidizing substance base solution B, and metal ions such as copper ions were used as the reference catalyst solution C. The chemiluminescent substance-containing solution A uses lucigenin, lophine, etc. instead of the luminol, and the oxidizing substance-containing solution B uses hydrogen peroxide instead of the luminol. Furthermore, as the reference catalyst solution C, a solution containing a metal complex such as hematin may be used instead of the metal ion-containing solution.

In addition, when quantifying an unknown amount of immune protein using the method and device of the present invention, it is recommended to quantitate a known amount of the same type of immune protein as a prior calibration and compare and refer to the results. Needless to say, it is possible to perform quantitative determination with even higher accuracy.

〔Effect of the invention〕

As is clear from the detailed description above, according to the method for quantifying an immunological protein according to the method of the present invention, the immunological protein to be measured in a sample solution can be efficiently separated and eluted using an immunoaffinity chromatography column. As a result, there is no need to perform extremely complicated and laborious procedures and labeling operations that require a long time as in conventional methods, and the immunological protein immobilized on the column can be used repeatedly, so it can be used continuously. Therefore, the quantification time for one sample can be quickly performed in a short time of about 15 minutes, and waste of reagents can be extremely reduced. Regardless of the type of immunological protein being measured, it can be quantified with high sensitivity and precision over a wide measurement range.Furthermore, unlike the conventional RrA method, there is no need to use a radioactive isotope labeling agent, and the above-mentioned chemical Since a solution containing non-toxic metal ions such as copper ions or metal complexes such as hematin is used as the catalyst solution for the luminescent reaction system, various excellent effects such as safe quantitative operations are demonstrated. Ru. Furthermore, the method of the present invention can be carried out using relatively simple and inexpensive equipment and inexpensively available reagents;
Moreover, as mentioned above, there is no need for pretreatment of the sample such as labeling operation, and there is little wastage of reagents, so the initial cost and running cost are low, resulting in an extremely economical effect.

In addition, according to the immunological protein quantification device according to the second invention, which is constructed by applying the above-mentioned first invention method, the excellent basic effects of the above-mentioned first invention method can of course be exhibited as is. In particular, downstream of the confluence connection portion between a third flow path configured to introduce and supply a reference catalyst solution in a predetermined amount to the chemiluminescent reaction section and a fourth flow path connected midway through the third flow path. Since the heating means and subsequent cooling means are provided on the side, the biulet-forming reaction between the reference catalyst solution and the immunological protein to be measured in the sample solution is carried out quickly and without any shortage. In addition, since the generation of air bubbles in the flow path is reliably prevented, it is also possible to perform quantitative determination with higher accuracy in a shorter time.

[Brief explanation of drawings]

1 to 7 show embodiments of the present invention, and FIG. 1 (
A), (B), (C), (D) and Figure 2 (A), (
B) is an explanatory diagram of the procedure of the immunological protein quantification method according to the second invention, FIG. 3 is a graph of a measurement example, and FIG. 4 is a general schematic configuration of the immunological protein quantification apparatus according to the second invention. Figure 5 is an enlarged vertical sectional side view of the main part, Figure 6 (a), (b),
(c). (D) and (E) are explanatory diagrams of the procedure for quantitative determination using the apparatus, and FIG. 7 is an overall schematic diagram of the immunological protein quantitative determination apparatus according to another embodiment. Further, FIG. 8 shows an explanatory diagram of the foreground technique of the present invention. A: chemiluminescent substance-containing solution,
B......Oxidizing substance-containing solution, C...
・・・・・・・・・Reference catalyst solution, D・・・・・・・・・
A sample solution containing the immunological protein to be measured, E...
・・・・・・Washing solution, F・・・・・・・・・Dissociation solution, ■,・・・・・・Reference chemiluminescence intensity, ■
・・・・・・・・・Chemiluminescence intensity (at dimming), Q
・・・・・・・・・Chemiluminescence reaction system,■・・・・
...First channel, ■...Second channel,
■・・・・・・・・・Third flow path, ■・・・・・・・・・
4th flow path, CU...Immune affinity chromatography column (antibody immobilization column), S...
...Chemiluminescence intensity measuring means, 7...
・・Pressurized inert gas cylinder, 8.9・・・・・・・・・
...Valve mechanism, 10... Constant temperature means, 1
1...Heating means, 12...
... Cooling means. Figure 3 Figure 5 Figure 6 (a)

Claims (5)

[Claims] (1) A method for measuring the amount of immune protein in a sample solution, in which the sample solution is placed in an immunoaffinity chromatography column on which an immune protein that has an antigen-antibody relationship with the immune protein to be measured is immobilized. the immune protein immobilized in the column to capture the immunogenic protein to be measured in the sample solution through an antigen-antibody reaction, and then wash the inside of the column to capture the immunogenic protein to be measured in the sample solution. After removing unnecessary substances other than the immunogenic protein, a dissociation solution is introduced into the column to separate the immunological protein to be measured from the immunological protein immobilized within the column and elute it from the column. After that, elution from the column is performed with respect to a reference catalyst solution to be supplied to a chemiluminescent reaction system of a solution containing a substance that exhibits chemiluminescence when oxidized and a solution containing a substance that has oxidizing properties. the immunological protein to be measured,
A method for quantifying an immune protein, comprising: measuring the chemiluminescence intensity from the chemiluminescence reaction system in this state, thereby measuring the amount of the immune protein in the sample solution. (2) A device for measuring the amount of immune protein in a sample solution, which is configured to be able to introduce and supply a predetermined amount of a solution containing a substance that exhibits chemiluminescence when oxidized to a chemiluminescence reaction section. a first flow path, a second flow path configured to be able to introduce and supply a predetermined amount of a solution containing an oxidizing substance to the chemiluminescence reaction section; and a predetermined amount of a reference catalyst solution to the chemiluminescence reaction section. A third flow path configured to be able to introduce and supply the chemiluminescence to the immunological protein to be measured, and a chemiluminescence intensity measuring means for measuring the intensity of chemiluminescence emitted from the chemiluminescence reaction section are provided. an immunoaffinity chromatography column immobilized with related immune proteins, and
A valve mechanism is provided that can switch between a state in which a sample solution is introduced into the column, a state in which a washing solution is introduced into the column, and a state in which a dissociation solution is introduced into the column. A sample solution is introduced and the immune protein to be measured in the sample solution is captured by an antigen-antibody reaction against the immunological protein immobilized within the column, and then the inside of the column is washed and the immune protein to be measured is captured by the immunological protein immobilized within the column. After removing unnecessary substances other than the immunological protein to be measured, a dissociation solution is introduced into the column to separate the immunological protein to be measured from the immunological protein immobilized within the column. A fourth flow path configured such that the immune protein to be measured eluted from the column can be mixed into the reference catalyst solution in the third flow path is provided in the middle of the third flow path. and a heating means and a subsequent cooling means are interposed downstream of the merging connection portion of the fourth flow path in the third flow path. Protein quantification device. (3) The immunological protein quantification device according to claim (2), wherein a constant temperature means is interposed between the first flow path and the second flow path. (4) A pressurized inert gas cylinder is used as the solution transfer energy source in the first flow path, the second flow path, and the third flow path, as set forth in claim (2) or (3). Immune protein quantification device. (5) The chemiluminescent reaction section, the first flow path, the second flow path, the third
Quantification of an immune protein according to any one of claims (2) to (4), wherein all piping parts in the flow path, the fourth flow path, etc. are made of Teflon (polytetrafluoroethylene). Device.