JPH09196920A - Body fluid component analyzing instrument and analyzing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an analyzing instrument capable of simply and quantitatively performing immunoassay. SOLUTION: A specimen receiving port 1, a pump connection port 6, a specimen treatment region 2 having a specimen labeled with a labeling substance arranged thereto and a specimen treating and photometric region 3 having a porous material to which one of a pair of specific bonds is fixed arranged thereto and the specimen treatment region 2 and the sample treating and photometric region 3 are provided between the specimen receiving port 1 and the pump connection port 6 to be connected by a passage. Or, the sample receiving port 1, the pump connection port 6 and the specimen treating and photometric region 3 having the porous material 4 to which the specimen labeled with the labeling substance and one of a pair of specific bonds are fixed are provided and the sample treating and photometric regions 3 is provided between the specimen receiving port and the pump connection port and connected by a passage.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a body fluid component analyzer for easily measuring a target body fluid component in a body fluid component by utilizing an immune reaction.

[0002]

2. Description of the Related Art The measurement of a body fluid component using an immune reaction has started from an immunoassay using a radioactive substance as a label, and a fluorescent substance or an enzyme labeled has been developed. They have performed some or most of the complicated immunoassay procedures in parallel with the movement of freeing the operator from the hassle of operation by automating the device. The main cause of the troublesome operation in the immune reaction is the B / F separation. In many immune reactions, an operation method that requires B / F separation is used in terms of sensitivity and versatility. B / F separation is an operation to separate into those bound by the reaction (Bound) and those not bound (Free), and therefore the bottom of the microplate,
Immobilize one of the specific binding pairs such as antigen-antibody such as porous beads, glass fiber, nitrocellulose filter,
Methods have been used to react with the other and wash away unbound. STRATUS of Dade Co. and ID-1000 of Toyobo Co., Ltd. make this operation as it is, but in these devices, the reaction solution and the cleaning solution are flown vertically to the membrane and the filter, so that a large and complicated device is required. Has become.

On the other hand, a simple immunoassay instrument utilizing immunochromatography has been developed for the purpose of simplification. A typical reagent for measuring fecal occult blood is a strip of nitrocellulose filter impregnated in a glass fiber filter at one end with an excess amount of dried gold colloid-labeled antibody, anti-IgG antibody at the other end, and a center A band-shaped anti-human hemoglobin antibody is immobilized in the vicinity. At the time of measurement, a diluted solution of feces is dropped on the glass fiber filter. The excess amount of labeled antibody in the glass filter and hemoglobin in the sample solution react and flow toward the anti-IgG antibody. On the center band, hemoglobin and anti-human hemoglobin antibody react with each other to immobilize the labeled antibody. A substance that does not react with the excess amount of the labeled antibody or the anti-human hemoglobin antibody is washed away by the sample that comes later, and the excess amount of the labeled antibody binds to the end having the anti-IgG antibody. It is confirmed that a sufficient amount of the sample has flowed due to the presence of gold colloid at the end, and the presence or absence of gold colloid at the band position is visually checked to determine the presence of hemoglobin, which is a target body fluid component for measurement. As described above, this reagent can be easily measured, but since the spreading rate of the reaction solution depends on the chromatographic action (capillary action) of the porous carrier, the spreading rate (reaction time in this case) Of the antigen-antibody reaction and B due to variations in the spreading rate due to uneven physical properties of the sample and the pore diameter and density of the porous carrier, etc.
/ F cleaning time could not be controlled, and only qualitative measurement results were obtained.

An immunoassay instrument for the purpose of quantitative measurement by immunochromatography is disclosed in JP-A-7-159398. There are five areas in the labeled substance fixing area of this analytical instrument, and it is said that the number of colored areas changes depending on the concentration of the target body fluid component in the sample, so that quantification is possible. There is. However, it cannot be said to be quantitative only by distinguishing into five stages, and should be said to be semi-quantitative.

[0005]

SUMMARY OF THE INVENTION In view of the above situation, it is an object of the present invention to find an analytical instrument that enables simple and quantitative immunoassay.

[0006]

In view of the above situation, as a result of intensive investigations by the present inventors, a sample having a sample processing region and a porous material in a channel so that the liquid feeding of the sample liquid can be controlled by a pump. The present invention has been completed by finding the following analytical instrument in which a processing / photometric region is arranged.

That is, the first aspect of the present invention is that a sample receiving port, a pump connecting port, a sample processing region in which a labeling substance labeled with a labeling substance is arranged, and one of a specific binding pair are immobilized on a porous material. Has a sample processing and photometric area where
The sample processing area and the sample processing / photometric area are provided between the sample receiving port and the pump connecting port, and are connected to each other by a flow path, which is a body fluid component analyzing instrument.

A second aspect of the present invention is a sample treatment / arrangement in which a sample receiving port, a pump connecting port, and a porous material on which one of a labeling substance labeled with a labeling substance and a specific binding pair is immobilized are arranged. A body fluid component analyzing instrument having a photometric region, wherein the sample processing / photometric region is provided between the sample receiving port and the pump connecting port, and each is connected by a flow path.

Thirdly, the labeling substance is labeled with a substance that specifically binds to one recognition site of the body fluid component to be measured, and the specific substance is immobilized on a porous material. The above-mentioned analytical instrument is one in which one of the binding pairs is a substance that specifically reacts with the other recognition site of the body fluid component to be measured.

Fourthly, a labeled body in which a measurement target body fluid component and a labeling substance are bound is arranged in the sample processing area, and the porous material contains a substance which specifically reacts with the measurement target body fluid component. It is the above-described analytical instrument that is immobilized.

Fifth, in the sample processing region, a labeled body in which a labeling substance is labeled on a substance that specifically binds to one recognition site of the measurement target body fluid component, and another recognition site of the measurement target body fluid component The above-mentioned analytical instrument in which a substance that specifically binds is arranged, and a substance that can bind to a substance that specifically binds to another recognition site of the body fluid component to be measured is immobilized in the porous material. .

Sixth, in the sample treatment area, a labeled body in which a labeling substance is labeled on the first antibody that specifically binds to one recognition site of the body fluid component to be measured, and other recognition of the body fluid component to be measured. The above-mentioned analytical instrument has a complex in which biotin is bound to a second antibody that specifically binds to a site, and avidin or streptavidin is immobilized on the porous material.

In the above-described analytical instrument, the sample processing region may be provided between the sample receiving port and the sample processing / photometric region, or the sample processing region may be connected to the sample processing / photometric region by a pump. It may be provided between the mouth. Furthermore, the analytical instrument of the present invention can be provided with a waste liquid reservoir,
The waste liquid reservoir is provided adjacent to the pump connection port and upstream of the pump connection port.

Further, according to the present invention, after the sample is supplied from the sample receiving port of the above-mentioned analytical instrument, the liquid feeding of the sample is controlled by the pump and the labeling substance trapped in the porous material is measured. A method for analyzing a body fluid component, which comprises determining a target body fluid component in a sample.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION As a sample applied to the present invention,
Human, animal blood, urine, stool, etc. can be used. However, blood and stool that are dissolved in a diluent are used in order to serve also as a cleaning for B / F separation described later. This analytical instrument uses hemoglobin A _1C (hereinafter, sometimes referred to as “Hb
A _1C )), diabetes marker such as glycated albumin, and pregnancy diagnosis, fecal occult blood diagnosis, and virus infection diagnosis.

The "specific binding pair" used in the present invention means an antigen-antibody reaction, an avidin-biotin bond,
It means a substance that makes a specific bond such as a boronic acid-cisdiol bond.

The labeling substance used in the present invention is gold,
Metal colloids such as silver and selenium, pigments such as colored latex, and enzymes such as alkaline phosphatase and peroxidase are used. When using an enzyme as a labeling substance, after washing an excess sample by flowing it, a liquid containing a component that reacts with the enzyme and produces a signal such as a color is passed through the flow path to the porous material, or these are It may be contained in the sample in advance.

Preferred porous materials include nitrocellulose, cellulose acetate, nylon membranes, filter paper, glass fiber filter paper.

The present invention will be described in detail below with reference to the drawings. FIG. 1 is a typical example of the analytical instrument of the present invention. 1a is a sectional view taken along line BB ′ of FIG. 1b, and FIG. 1b is line A- of FIG. 1a.
It is A 'sectional drawing.

A plastic material is suitable as a material for forming the analytical instrument as long as it is impermeable to a light-transmissive liquid and easy to process. Typical plastic materials include polystyrene resin, acrylic resin, polyvinyl chloride resin, polycarbonate resin, polyethylene resin, polypropylene resin, polyethylene terephthalate resin and the like. Two of these plate-like members are attached to each other to form a thin-layer flow path as shown in FIG. The sample entered from the sample receiving port (1) can be moved from the sample receiving port to the pump connecting port by a pump (not shown) connected to the pump connecting port (6).

In the analytical instrument of the present invention, the washing for B / F separation is performed by the sample itself, so that it is not necessary to separately prepare a washing solution for the measurement, and the sample after washing also has a waste liquid pool in the analytical instrument. The analytical instrument can be discarded as it is after use because it does not stain the analytical instrument because it accumulates in the.

In the present invention, the sample and the reaction solution are not moved in the porous material as in the immunochromatography, but are transferred under the control of the pump in the flow path, so that the physical characteristics such as the viscosity of the sample solution are used. Not affected. Further, it can be applied to both the sandwich method and the competitive method used in the immunoassay, depending on the type of the reagent arranged in the channel and the combination of the reagents bound to the porous material.

In the case of the sandwich method, a labeled substance in which an excessive amount of a substance that specifically binds to a target body fluid component in a sample is labeled with a labeling substance (hereinafter referred to as "first substance")
Are placed in the sample processing area. Sample is sample processing area (2)
When the sample is transferred to the sample body fluid component in the sample, the first substance in the sample processing region reacts with each other to form a complex. The first substance in the sample processing region is excessively arranged with respect to the measurement target body fluid component contained in the sample.
There are both substances to which the body fluid component to be measured is bound and substances that are not bound to the substance. After waiting the time required for the reaction in the sample processing area, the sample is sucked by the pump and transferred to the sample processing / photometric area. In the sample processing / photometric region (3), a complex of the first substance and the body fluid component to be measured can be bound, and a substance not bound to the first substance to which the body fluid component to be measured is not bound In the book, "second substance") is immobilized on the porous material (4). The second substance immobilized on the porous material is a substance capable of recognizing a recognition site different from the recognition site of the measurement target body fluid component for the first substance. The sample is sent to the waste reservoir (5) after waiting for the time required for the sample processing and reaction in the photometric region. At this time, the first substance bound to the measurement target body fluid component remains in the porous material because it is trapped in the porous material, but the unbound first substance moves to the waste liquid reservoir as the liquid is fed.
Therefore, the measurement target bodily fluid component can be measured by optically measuring the amount of the labeling substance captured in the porous material. In this case, since the first substance and the second substance do not react with each other, they may be in the same place.

In the above-mentioned sandwich method, avidin or streptavidin can be immobilized on the porous material. At this time, in the sample processing area, an excessive amount of the first substance with respect to the measurement target body fluid component in the sample and a second substance that binds to the measurement target body fluid component in the sample at a recognition site different from the first substance Place the one with biotin bound to it. In this case, the body fluid component to be measured in the sample is sandwiched between the first substance and the second substance in the sample treatment region, and further immobilized in the porous substance and biotin bound to the second substance in the sample treatment / photometry region. It is captured by binding to avidin or streptavidin. Since the surplus first substance is washed away by the sample flowing later, it is possible to measure the target body fluid component by optically measuring the amount of the labeling substance captured in the porous material.

The sample processing area may be arranged not only between the sample receiving port and the sample processing / photometric area but also on the pump connection port side of the sample processing / photometric area. in this case,
The optical background of the sample may be measured by impregnating the sample with a porous material, reacting it with the first substance and the second substance, and pumping the sample back. In the present invention, since the labeling substance in the porous material is optically measured, the area having the porous material is used as the sample processing / photometric area. The photometry may be either transmitted light or reflected light.

The use of the avidin-biotin bond is commercially advantageous because immobilization on a porous material can be made common regardless of measurement items.

In the case of the competitive method, a target substance fluid component labeled with a labeling substance is placed in the sample processing area.
The measurement target bodily fluid component may be the measurement target bodily fluid component itself, or may be a modification of the measurement target bodily fluid component so that the labeling substance is easily bound. A substance that specifically binds to the body fluid component to be measured is immobilized on the porous material. When the sample is supplied from the sample receiving port, the sample is mixed with the measurement target body fluid component labeled with the labeling substance in the sample processing region, and then transferred to the porous material. The substance that specifically binds to the measurement target body fluid component immobilized on the porous material is the ratio of the concentration of the measurement target body fluid component in the sample to the concentration of the labeled measurement target body fluid component that was placed in the sample treatment area. Join. Therefore, by measuring the concentration of the labeling substance that has been washed away and remains, the concentration of the measurement target body fluid component in the sample can be known from the amount of the labeled measurement target body fluid component that is arranged in advance in the sample processing region.

[0028]

EXAMPLE Next, an example is shown in which hemoglobin A _1C was measured using a combination of blue microparticles and an anti-human HbA _1C mouse monoclonal antibody as the first substance and an anti-human hemoglobin antibody as the second substance.

A) Manufacturing method a-1) Preparation of anti-hemoglobin antibody-immobilized filter (second)
Immobilization of substance on porous material) 300 μg / anti-hemoglobin antibody in neutral phosphate buffer
Mix to make up to ml. In addition, a nitrocellulose filter with a pore size of 8 μm (manufactured by Millipore) (hereinafter,
"NCF") was impregnated and solid-phased at room temperature for 2 hours with gentle shaking. Immobilization amount is 45 μg / c
m ² . Next, in order to prevent non-specific adsorption of this NCF, it was washed with a neutral phosphate buffer solution, then immersed in a neutral phosphate buffer solution containing 1% milk casein, and gently shaken at room temperature for 2 hours. After blocking, it was washed with a neutral phosphate buffer. This was dried at 37 ° C. for 1 hour and punched into 5 mmφ with a punch to obtain an anti-hemoglobin antibody-immobilized filter.

A-2) Preparation of anti-hemoglobin A _1C monoclonal antibody-immobilized blue microparticles (hereinafter referred to as “bmP”) (preparation of the first substance) Anti-hemoglobin A _1C monoclonal antibody and blue microparticles were added to HEPES buffer. 1.5 mg each
/ Ml, 1.25% was mixed. As the blue microparticles, blue colored polystyrene beads having a diameter of 200 nm (Bang Laboratories) were used. This was gently shaken at room temperature for 2 hours to be solid-phased. 1 mg per 1 ml of 1% blue microparticles
Met. This solution was centrifuged at 30,000 × G for 1 hour, and the supernatant was removed to separate blue microparticles. The supernatant was removed and dispersed in a PIPES buffer solution containing the same volume of 1% milk casein and gently shaken at room temperature for 2 hours for blocking. 30,0 after blocking
It was washed by centrifuging at 00 × G for 1 hour, removing the supernatant, and dispersing in HEPES buffer. This washing operation was repeated 3 times for sufficient washing, and then dispersed in HEPES buffer solution to a concentration of 2% to obtain bmP.

A-3) Preparation of analytical instrument b in the sample processing area formed between two polystyrene plates
The mP was dried, and then laminated with an anti-hemoglobin antibody-immobilized filter sandwiched in the sample processing / photometric region to produce the analytical instrument of FIG. The thickness of the flow path was 0.5 mm at the portion corresponding to the waste liquid reservoir and 0.2 mm at the other portions.

B) Measurement b-1) Preparation of HbA _1C sample (preparation of sample) Erythrocytes were separated from human blood containing various HbA _1C % by centrifugation, dispersed in physiological saline and washed. The supernatant after centrifugation was removed, and it was again dispersed in physiological saline. This operation was repeated 3 times to thoroughly wash the red blood cells. Finally, it was dispersed in HEPES buffered saline (containing a hemoglobin denaturing agent) so as to have an appropriate concentration. These were hemolyzed by repeating freeze-thawing to obtain samples. A HbA ₀ purified product (manufactured by EXOCELL) was used as a HbA _1C 0% sample.

B-2) Reaction 100 μl of HbA _1C sample was dropped into the sample receiving port, and the liquid was sucked with a suction pump until the liquid tip reached the dried bmP tip.
The mP was dispersed. At this position, stop the liquid for 3 minutes, bm
P was reacted with hemoglobin in the sample. Suck again,
The liquid tip was advanced to the tip of the anti-hemoglobin antibody-immobilized filter. After stopping at this position for 5 minutes, the HbA _1C -bmP complex and hemoglobin were allowed to bind to the anti-hemoglobin antibody. At this time, since only a fixed amount of hemoglobin binds to the anti-hemoglobin antibody, HbA _1C % in hemoglobin
Corresponding bmP is coupled to the filter. Then 70μ
aspirating 1 minute, and excess bmP and HbA _1C −
The bmP complex was rinsed into the waste sump.

B-3) Using a photometric color difference meter (manufactured by Nippon Denshoku Co., Ltd.), the reflectance (R) of the anti-hemoglobin antibody-immobilized filter at 640 nm was measured.

B-4) Calibration curve The obtained reflectance was converted into a K / S value, and the K / S value was plotted against HbA _1C % separately measured by the HPLC method.
The following calibration curve was obtained.

As described above in detail, according to the present invention, quantitative immunoassay can be easily performed.

[Brief description of drawings]

FIG. 1 shows a typical example of an analytical instrument of the present invention. 1a is a cross-sectional view taken along the line BB ′ of FIG. 1b. 1b is a cross-sectional view taken along the line AA ′ of FIG. 1a.

FIG. 2 is a diagram of a calibration curve obtained in the example.

[Explanation of symbols]

 1; Sample receiving port 2; Sample processing region 3; Sample processing and photometric region 4; Porous material 5; Waste liquid reservoir 6; Pump connection port

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Momoko Okuyama One day at 9 Techno Park, Sanda City, Hyogo Prefecture Inside the Hyogo Plant of Meiji Physics Co., Ltd. (72) Inventor Shigeru Fujioka 1-1-13 Kudankita, Chiyoda-ku, Tokyo No. 5 Inside Tokyo Headquarters of Nihon Medi-Physics Co., Ltd.

Claims (14)

[Claims] 1. A sample treatment in which a sample receiving port, a pump connection port, a sample treatment region in which a labeling substance labeled with a labeling substance is disposed, and a porous material in which one of specific binding pairs is immobilized are disposed. A body fluid component having a combined photometric region, wherein the sample processing region and the sample processing / photometric region are provided between the sample receiving port and the pump connection port, and each is connected by a flow path. Analytical instrument. 2. A sample processing / photometric region in which a sample receiving port, a pump connecting port, and a porous material having one of a labeling substance labeled with a labeling substance and a specific binding pair immobilized thereon are arranged. The body fluid component analysis instrument, wherein the sample processing / photometric region is provided between the sample receiving port and the pump connecting port, and each is connected by a flow path. 3. A specific binding pair in which the labeling substance is labeled with a substance that specifically binds to one recognition site of a body fluid component to be measured, and which is immobilized on a porous material. The analytical instrument according to claim 1 or 2, wherein one of the substances is a substance that specifically reacts with another recognition site of the body fluid component to be measured. 4. A labeled body in which a measurement target body fluid component and a labeling substance are bound to each other is arranged in a sample processing area, and a substance which specifically reacts with the measurement target body fluid component is immobilized on the porous material. The analytical instrument according to claim 1, which is provided. 5. A sample treatment area is provided with a labeled substance in which a labeling substance is labeled on a substance that specifically binds to one recognition site of a body fluid component to be measured, and to another recognition site of a body fluid component to be measured. 3. The analysis according to claim 1 or 2, wherein a substance that binds to is disposed, and a substance that can bind to a substance that specifically binds to another recognition site of the target body fluid component is immobilized on the porous material. Equipment. 6. A sample processing region, a labeled body in which a labeling substance is labeled on a first antibody that specifically binds to one recognition site of a body fluid component to be measured, and another recognition site of a body fluid component to be measured. The analytical instrument according to claim 5, wherein a complex in which biotin is bound to a second antibody that specifically binds is arranged, and avidin or streptavidin is immobilized on the porous material. 7. A sample processing area is provided between the sample receiving port and the sample processing / photometric area.
6. The analytical instrument according to any one of 6. 8. The analytical instrument according to claim 7, wherein a waste liquid reservoir is provided between the sample processing / photometric region and the pump connection port. 9. The analytical instrument according to claim 1, wherein the sample processing region is provided between the sample processing / photometric region and the pump connection port. 10. The analytical instrument according to claim 9, wherein a waste liquid reservoir is provided between the sample processing region and the pump connection port. 11. The analytical instrument according to claim 1, wherein the labeling substance is a metal colloid or colored latex particles. 12. The labeling substance according to claim 1, which is an enzyme.
0. The analytical instrument according to 0. 13. The analytical instrument according to claim 1, wherein the sample processing area, the sample processing / photometric area, the waste liquid reservoir, and the flow path connecting them are formed in a thin layer. 14. After supplying the sample from the sample receiving port of the analytical instrument according to any one of claims 1 to 13, the liquid feeding of the sample is controlled by a pump to remove the labeling substance trapped in the porous material. A method for analyzing a body fluid component, which comprises obtaining a measurement target body fluid component in a sample by measuring.