JPH0588786B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an immunoassay device that uses a pressure difference to move a sample, and particularly to an immunoassay device that is suitable for measuring multiple items.

[Conventional technology]

Although measuring tumor markers is not a test for definitively diagnosing cancer, it is less physically taxing on the patient than histocytological or morphological tests, and since the sample is body fluid such as blood, it is easy to obtain. It is suitable as a cancer screening test.

In order for cancer screening using tumor markers to be useful, it is necessary that the test has a high rate of accurate diagnosis of disease, and also has a high rate of accurate diagnosis of no disease for benign non-cancer diseases. In other words,
The positive rate for cancer needs to be higher and the false positive rate needs to be lower. However, none of the various currently known markers independently satisfies these conditions. Therefore, the usefulness of combination assays, in which multiple markers are combined and measured, is attracting attention for the purpose of further improving diagnostic performance.

On the other hand, various immunoassays have been proposed for the purpose of measuring various tumor markers. However, any immunoassay can only measure one type of tumor marker, and if you try to perform the above combination assay, you will have to measure multiple types of tumor markers individually. It takes time and effort to use test liquids in proportion to the number of types to be tested.

The following method has been proposed as an example of a method having the characteristics of shortening measurement time and easy instrumentation (Analytical Chemistry, 57 , pp. 2754-2756 (1985), (Anal.Chem., 57 , 2754). −
2756 (1985)). In this method, antibodies are immobilized on beads and then packed into a stainless steel tube to form a microreactor, through which a sample solution to be measured and an antibody bound to a labeling substance are passed in order, allowing a reaction to occur during the passage. Finally, the amount of the target substance to be measured in the sample is calculated by measuring the amount of the labeled substance bound in the microreactor.

However, even with this method, multiple types of tumor markers cannot be measured simultaneously, and each tumor marker must be measured separately. As a result, the required amount of test liquid must be large.

[Problem that the invention seeks to solve]

With the above conventional technology, when measuring multiple types of tumor markers at the same time for combination assay, it is necessary to measure each type separately, which requires time and effort, and requires a large amount of sample liquid. I have a problem that requires .

An object of the present invention is to provide an immunoassay device that can shorten the measurement operation time and reduce the amount of sample liquid by measuring multiple types of tumor markers at once.

[Means for solving problems]

The above purpose is to use an immunoassay device that uses a liquid flow to move the sample and react with the antigen in a reaction part with immobilized antibodies provided in the flow path, which can handle multiple types of antigens to be measured. This is achieved by arranging solid phase parts in series in a flow path, each having a different antibody immobilized thereon.

[Effect]

Antibodies specifically recognize the antigens they bind to, so if solid-phase parts immobilized with antibodies corresponding to multiple antigens are connected in series and a sample is flowed through them, the sample can be absorbed without interference. The various antigens contained in the antigen will be immobilized in the corresponding carrier. In other words, with one sample injection,
It becomes possible to capture multiple types of antigens simultaneously.

〔Example〕

Examples of the present invention will be shown below.

Example 1 Commercially available glass beads (average particle size of about 10 μm) are thoroughly washed with nitric acid and then with pure water. these beads
It was immersed in a 0.1% N-β(aminoethyl)γ-aminopropyltrimethoxysilane aqueous solution for 30 minutes, then thoroughly washed with pure water, and then dried at 110°C. This was immersed in a 2.5% glutaraldehyde aqueous solution for 15 minutes, thoroughly washed with pure water, and then immediately immersed in an antibody solution (approximately 4 mg/ml solution, pH 7) whose antigen was the measurement target, and left overnight at 4°C. . After immersing in glycine solution to remove unreacted aldehyde groups, it is washed with phosphate buffer (PH7.2). This is 0.2mm in diameter,
It was packed into a stainless steel tube with a length of 4 cm to form an antibody-immobilized column.

Various antibodies were immobilized according to the procedures described above to prepare various antibody-immobilized columns.

The immobilized antibody column thus obtained is connected as shown in FIG. Using a liquid pump 4, a buffer solution 5 is continuously flowed through columns 1, 2, and 3 which are connected in series and have different types of antibodies immobilized thereon. The flow rate is 0.5ml/min. Introduce 50μ of the sample solution to be measured using cut valve 6. After passing through columns 1, 2, and 3, the sample is discharged from a channel 8 via a three-way tank 7 and poured into a drain reservoir 9. Next, various types of antibodies labeled with glucose oxidase were prepared and mixed to approximately the same concentration (approximately 1 mg/ml). Two minutes after the sample injection, 75 μl of this labeled antibody mixture was injected, passed through column 1, column 2, and column 3 in the same way as the sample, and then passed through the three-way tank 7 to the drain reservoir 9. pour.

After injecting the labeled antibody mixture, the column is washed for 2 minutes, and then the amount of bound labeled antibody is detected electrochemically. Detection is performed separately in columns 1, 2, and 3. First, in order to measure column 1, the controls 10, 11, 12, and 13 are operated to allow the liquid to pass through bypass 14 and bypass 15.
Avoid passing through column 3. Inject a 1% glucose solution using cut valve 6, and add it to column 1.
The H ₂ O produced by the reaction is measured by a detector 16 guided through a three-way tube 7.

Next, column 2 is measured. In this case, after operating the cocks 17, 18, 12, and 13 to pass through the bypass 19 and the bypass 15,
Perform similar measurements. Column 3 is similarly measured one after another.

Prepare samples of various concentrations using AFP (α-fetoprotein), β ₂ -microglobulin, and CEA (carcinoembryonic antigen) as antibodies to be measured, and perform measurements according to the above measurement procedure to obtain a calibration curve. Ta. The results are shown in FIG.

The results were in good agreement with those measured individually according to conventional methods. The correlation coefficients are, respectively,
The values were 1.02 for AFP, 0.95 for β ₂ -microglobulin, and 0.97 for CEA.

Therefore, it can be seen that this method allows measurement of multiple items at the same time.

Example 2 Commercially available glass wool was subjected to silica coupling treatment in the same manner as in Example 1, and then various antibodies were immobilized using glutaraldehyde. Glass tubes with a diameter of 5 mm and a length of 1 cm are each filled with glass wool immobilized with various antibodies to form a reaction column.

The three types of reaction columns obtained in this way were
connected in series as shown in the figure. A buffer solution 25 is continuously flowed through reaction columns 21, 22, and 23 in which different types of antibodies are solidified using a liquid pump 24 at a flow rate of 0.3 ml/min.

A sample to be measured is injected from the injection part 26. After passing through the columns 21 , 22 , 23 , the sample is led to a drainage reservoir 27 . Furthermore, the buffer solution is flushed to wash the column.

Next, equal amounts of various antibodies labeled with β-D-galactosidase are mixed, and 75μ of the mixture is injected from the injection port. For washing, the buffer solution is allowed to flow for a certain period of time. Subsequently, the amount of labeled antibody bound to the column is measured. O-nitrophenol-β-D- from the injection part
Inject the galactoside solution. The reaction solution that has passed through the column 21 is sent to the absorbance detector 2 via the column 28.
9 and measured the absorbance at 420 nm. Next, the column 22 is measured. The O-nitrophenol-β-D-galactoside solution is introduced into the column 22 by operating the cocks 45 and 28 and passing through the bypass 30. The reaction solution is passed through a filter 31 to an absorption detector 32
and measure the absorbance.

Next, column 23 is measured. O-nitrophenol-β by operating Kotoku 45 and Kotoku 31
-D-lactoside solution is led to column 21 via bypass 33. The reaction solution is guided to a detector 35 via a tube 34 and absorbance is measured.

AFP, β ₂ -microglobulin, and CEA were measured in the same manner as in Example 1. The results are shown in FIG. As in Example 1, the results were in good agreement with the results measured individually using the conventional method.

Example 3 Commercially available glass fiber filter paper (thickness 0.2 mm, diameter 10 mm)
is subjected to silane coupling treatment in the same manner as described in Example 1, and then the antibody is immobilized with glutaraldehyde to obtain an immobilized antibody membrane. Immobilized antibody membranes 36, 3 on which various antibodies are immobilized separately
7 and 38 are overlapped and held in a reaction container 39.

A sample of 500 μm to be measured is poured into the reactor and introduced into the reaction membrane by suction by the peristaltic pump 40 connected to the bottom. Then add 10 ^-2 M phosphate buffer to 10
ml to wash the reaction membrane.

Three kinds of peroxidase-labeled antibodies are sequentially introduced into the reaction membrane in an amount of 50μ each. again
Wash the reaction membrane with 10 ml of 10 ^-2 M phosphate buffer.

The reaction membranes are taken out and measured one by one. A reaction membrane 36 is held in a reaction vessel 41 for detection, a homovanillic acid solution is introduced from the upper part of the reaction vessel, reacted with the reaction membrane, and then measured with a fluorescence detector 42. AFP,
The results of measuring β ₂ -microglobulin and CEF are shown in FIG.

As in Examples 1 and 2, the measurements can be made in good correlation with the results measured individually using the conventional method.

Although Examples 1, 2, and 3 show examples in which glass is used as a carrier, it goes without saying that materials used as general immobilization carriers are useful in common.

〔Effect of the invention〕

According to the present invention, multi-component measurement items can be measured simultaneously, reducing operation time and
This has the effect of reducing the amount of sample. That is, since the amount of sample used does not depend on the number of measurement items, the amount required per measurement item is inversely proportional to the number of measurement items measured simultaneously, and the effect becomes greater as more items are measured. For example, when measuring five components,
This means that a sample amount that is about one-fifth of the conventional amount is sufficient.

[Brief explanation of the drawing]

Figure 1 is a schematic diagram of an apparatus for carrying out Example 1 of the present invention, Figure 2 is a diagram showing the measurement results of Example 1, and Figure 3 is a diagram showing the measurement results of Example 1.
The figure is a schematic diagram of the apparatus for carrying out Example 2, Figure 4 is a diagram showing the measurement results of Example 2, Figure 5 is a schematic diagram of the apparatus for carrying out Example 3, and Figure 6 is the measurement results of Example 3. FIG. Explanation of symbols, 1... Reaction column, 2... Reaction column, 3... Reaction column, 4... Liquid pump, 5... Buffer solution, 6... Cut valve, 7... Three-way pot, 8... Channel, 9... Drainage reservoir, 10...Kotsuku, 11...Kotsuku,
12...Kotuku, 13...Kotuku, 14...Bypass,
15...Bypass, 16...Detector, 17...Kottoku,
18... Kotoku, 19... Bypass, 20... Drainage reservoir,
21... Reaction column, 22... Reaction column, 23... Reaction column, 24... Liquid pump, 25... Buffer solution, 2
6... Syringe, 27... Drainage reservoir, 28... Kotoku, 29
...Detector, 30...Bypass, 31...Kotsuku, 32
...Detector, 33...Bypass, 34...Kotuku, 35
...detector, 36...immobilized antibody membrane, 37...immobilized antibody membrane, 38...immobilized antibody membrane, 39...reaction container, 4
0...Pump, 41...Reaction container for detection, 42...Detector, 43...Drainage reservoir, 44...Pump, 45...Kotuku.

Claims (1)

[Scope of Claims] 1. A plurality of antibody-immobilized columns having the same shape, in which antibodies corresponding to multiple types of antigens to be measured are immobilized on separate solid phases, and each of these solid phases is packed separately. are arranged in series through a flow path selection means for selecting a flow path in a series of flow paths, and the sample liquid to be measured is sequentially moved through the plurality of types of antibody-immobilized columns by the flow path selection means. , a solution containing at least a plurality of types of enzyme-labeled antibodies is sequentially moved through the plurality of antibody-immobilized columns by the flow path selection means, and the antigen and the enzyme are transferred to the single antibody-immobilized column by the flow path selection means. A solution containing at least a substrate that reacts with the enzyme portion of the conjugate with the labeled antibody is introduced, and the solution containing the reaction product produced by the reaction is caused to pass through the other antibody-immobilized column by the flow path selection means and is The reaction product is transferred to a detector for detecting the reaction product to perform detection on the corresponding antigen, and the detection on each of the antigens is measured separately for each of the corresponding antibody-immobilized columns. Item immunoassay device. 2. The multi-item immunoassay device according to claim 1, wherein the solid phase is composed of solid particles.