JP3076144B2 - Biological trace component inspection system - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting a biological trace component in a specimen by using a microparticle to send a reaction product by an antigen-antibody reaction or a reaction product by nucleic acid hybridization to a measurement cell. The present invention relates to a simple biological trace component inspection apparatus used in a method for detecting a trace component.

[0002]

2. Description of the Related Art Recent advances in the technology for detecting trace components of a living body have played a major role in the field of clinical examination, such as early diagnosis and prognosis of various diseases. SABerson in 1958
Et al. Have published a method for quantitatively detecting insulin by labeling with radioactive iodine, since IgE, IgG, C
Plasma proteins such as RP and microglobulin, AFP, CE
A, tumor markers such as CA19-9, TSH, T3
Hormones, blood drugs, viruses such as HBV and HIV and their antibodies, and nucleic acids such as DNA and RNA can be measured, and a large number of samples can be processed by automation. Further, an immunological method using an antigen-antibody reaction or a method of analyzing a biological trace component using nucleic acid-nucleic acid hybridization is often used.

In such an analysis method, an antibody or an antigen specifically binding to a biological trace component to be detected or a single-stranded nucleic acid as a probe is used as a solid fine particle, a bead, or a solid phase such as a reaction vessel wall. It is immobilized on the surface and subjected to an antigen-antibody reaction or nucleic acid hybridization with a substance to be detected. Further, using a labeled antibody, a labeled antigen, and a labeled single-stranded nucleic acid carrying a labeling substance having high detection sensitivity such as a fluorescent substance or a luminescent substance, an antigen-antibody complex or a double-stranded nucleic acid is used. The analyte is detected and quantified. FIG. 2 shows an example in which a fluorescent substance is used as a label to detect an antigen-antibody complex generated by an antigen-antibody reaction. The first reagent is a solid fine particle 2 in which an antibody 1 that specifically binds to an antigen in a sample is previously bound.
Consists of The second reagent consists of an antibody 5 that has been labeled with a fluorescent substance 3 and that specifically binds to an antigen 4. The first reagent
The complex 6 is obtained by reacting the antigen 4 with the second reagent. Alternatively, the first reagent may be reacted with the antigen 4 to obtain a complex, and then the second reagent may be reacted to form the complex 6.

After washing the reaction solution containing the complex 6 for the purpose of removing the unreacted second reagent as required, an optical measurement is performed. For the measurement, a method in which the reaction solution containing the complex 6 is appropriately diluted and then placed in a batch-type optical cell to measure the fluorescence intensity, or a method in which the reaction solution is sent to a flow-type optical cell to measure the fluorescence intensity Are performed, and the amount of antigen in the sample is determined from the results. A flow cytometer using the latter flow type optical cell is widely used because of its excellent measurement accuracy and sensitivity.

FIG. 3 is a configuration diagram of a measuring apparatus using a general flow cell. A reaction liquid passes through the inside of the flow part 7 a of the flow cell 7 wrapped in a sheath liquid that has become a high-speed laminar flow, and a laser light source 8 is arranged in a direction orthogonal to the flow. An imaging lens 9 is arranged on the optical axis O1 to guide the laser light L emitted from the laser light source 8 to the circulation part 7a, and further measures forward scattered light obtained from the complex in the reaction solution. For this purpose, a stopper 10, condensing lenses 11, 12 and a photodetector 13 are sequentially arranged with a flow cell 7 interposed therebetween.

On the optical axis O2 perpendicular to the optical axis O1 of the flow direction of the composite and the laser beam irradiation direction, the condenser lenses 14, 15 and the collimator lens 16, The dichroic mirrors 17 and 18 having characteristics and the reflection mirror 19 are sequentially arranged. The reflection directions of the dichroic mirrors 17 and 18 and the reflection mirror 19 indicate the barrier filters 20 and 2.
1, 22 and photodetectors 23, 24, 25 are arranged, respectively.

In FIG. 3, a laser beam L from a laser light source 8 is passed through an imaging lens 9 to a flow section 7 of a flow cell 7.
An image is formed near a. Of the scattered light of the laser light L by the complex in the reaction solution flowing through the flow part 1a, forward scattered light is condensed on the photodetector 13 by the condensing lenses 11 and 12. Here, the stopper 10 plays a role of cutting the direct light of the laser light L.

A fluorescent substance is labeled on the complex, and condensing lenses 14 and 15, a collimator lens 16, dichroic mirrors 17 and 18, which are arranged on the optical axis O 2.
Barrier filters 20, 21, 22; photodetectors 23, 2
Using 4 and 25, fluorescence measurement is performed using a plurality of channels based on side scattered light.

From the fluorescence data thus obtained,
The amount of label is calculated by an arithmetic unit (not shown), and the amount of antigen in the sample is obtained.

[0010]

The measurement by the flow cytometer is excellent in both accuracy and sensitivity, but it is a pump for sending the reaction solution to the apparatus and a method for sequentially sending the complex in the reaction solution to the flosser. Since a large amount of sheath liquid is required, there are problems such as an increase in the size of the apparatus and a large amount of test waste liquid derived from the sheath liquid.

An object of the present invention is to solve the above problems and to provide a small-sized, simple, and small amount of test waste solution inspection apparatus for a living body.

[0012]

The gist of the biological trace component inspection apparatus according to the present invention for achieving the above object is to provide a first substance which is active on a biological trace component in a specimen on the surface of solid fine particles. Reacting a first reagent, which is bound to the above, the sample, and a second reagent which is preliminarily labeled with a second substance which is active on the biological trace component and is different from the first substance. In a test apparatus for generating a complex and qualitatively or quantitatively detecting the presence of a biological trace component in the sample by measuring the labeling amount of the complex, a reaction tank for performing a reaction for producing the complex, A flow cell for measuring the complex, and a waste liquid tank provided with a liquid absorbing member at a connection portion with the flow cell.

[0013]

The biological trace component inspection apparatus having the above-described configuration is
The reaction tank, the flow cell, and the waste liquid tank are integrated, and the reaction liquid flows from the reaction tank to the waste liquid tank by the absorbing power of the liquid absorbing member, so that the complex in the reaction liquid does not use the sheath liquid, and the flow cell is used. Are sent sequentially.

[0014]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a block diagram showing an embodiment of the present invention; FIG. 1 is a configuration diagram of an embodiment. Reaction tank 3
0. The capillary flow cell 31 and the waste liquid tank 32 are integrated, and the flow cell 31 is formed of an optically transparent material such as glass or plastic. Flow cell 31
Absorbing member 33 is disposed in the connection portion between waste water tank 32 and waste liquid tank 32. Absorbing member 33 is made of paper such as cotton or filter paper, or highly absorbent polymer such as polyacrylamide or cellulose. Any material can be used as long as the material absorbs the reaction solution. The reaction tank 30 is provided with an injection port 34,
A stirrer 35 is provided inside.

A laser beam L is provided beside the flow cell 31.
A laser light source 36 and an imaging lens 37 are provided, and a stopper 38 is provided on the opposite side of the flow cell 31.
A first condenser lens 39 and a first photodetector 40 are arranged. Further, a second condenser lens 41 and a second photodetector 42 are arranged at right angles to the longitudinal direction of the flow cell 31 and the direction of the laser beam L.

Reagents, specimens, and the like are injected into the reaction tank 30 from the injection port 34, the first reagent and the second reagent are mixed in the reaction tank 30, a complex formation reaction is performed, and the temperature is controlled if necessary. . In addition, stirring of the reaction solution is necessary for promptly and uniformly proceeding the complex formation reaction, and a stirring tool 35 is inserted from the injection port 34 and stirred. In addition, as a stirring method, a rotor may be inserted and rotationally stirred by magnetic force, or an ultrasonic stirrer or the like may be used. When the reaction solution is diluted with a diluent containing water as a main component after the completion of the reaction, when the reaction solution passes through the measuring section of the floesser 31, the dilution ratio is set so that the complexes are sequentially sent one by one. Then, the reaction solution is permeated into the capillary flow cell 31 by capillary action.
Then, the reaction liquid is absorbed by the absorbing member 33 disposed at the outlet of the flow cell 31, so that the reaction liquid continuously flows out of the reaction tank 30 into the waste liquid tank 32.

The reaction liquid flowing into the flow cell 31 is applied to the light source 3
Irradiated by the laser light L from 6. The forward scattered light of the laser beam L by the complex 6 in the reaction solution is measured by the first photodetector 40 via the first condenser lens 39. Further, the fluorescent component is measured by the second condenser lens 41 and the second photodetector 42 provided in a direction perpendicular to the direction of the laser light L, and the label amount of the reaction solution is measured.

In the above-mentioned example, a fluorescent substance is used as a labeling substance. However, when a luminescent substance such as a chemiluminescent substance or a bioluminescent substance is used as a label, the laser light source 3
6 without irradiating the laser light,
In, the second light emission of the complex 6 can be measured by the photodetector.

Here, the reagent to be used will be described in detail. The first reagent is, for example, polystyrene fine particles having a particle diameter of about 1 μm, to which a first substance active to a trace component generated in a sample is bound. This bonding is generally made by physical bonding or chemical bonding.

When an antigen-antibody reaction is used for the reaction to form a complex, the first substance may be, for example, a natural or synthetic peptide, protein, enzyme, saccharide, lectin, virus, bacterium, nucleic acid, DNA, RNA. , Antibodies and the like. Among them, the following are listed as clinically particularly useful substances.

(A) immunoglobulins such as IgG and IgE, complement, plasma proteins such as CRP, ferritin, α1 or β2 microglobulin, and antibodies thereof;

(B) Tumor markers such as α-fetoprotein, carcinoembryonic antigen (CEA), CA19-9 and CA-125 and their antibodies: luteinizing hormone (LH), follicle stimulating hormone (FSH), human cilia Gonadotropin (hCG)

(C) Hormones such as estrogen and insulin and their antibodies

(D) Virus infection-related substances such as HBV, HCV, HIV and ATL and their antibodies

(E) Bacteria such as diphtheria, botulinum, mycoplasma, treponema pallidum and their antibodies

(F) Protozoa such as Toxoplasma, Trichomonas, Leishmania, Trypanosomes, Malaria parasites and their antibodies

(G) Antiepileptic drugs such as phenytoin and phenobarbital; cardiovascular drugs such as quinidine and digoxin; antiasthmatic drugs such as theophylline; drugs such as chloramphenicol and gentamicin;

(H) Other enzymes, bacterial exotoxins (such as Strelizin O) and their antibodies, etc., and substances which cause an antigen-antibody reaction with the substance to be measured in the sample are appropriately determined according to the type of the substance to be measured. To choose.

When nucleic acid hybridization is used in the reaction, for example, a base sequence complementary to a base sequence of a nucleic acid to be tested is used as an active first substance with respect to a target substance in a sample. Is used.

The second reagent is a second substance that is active for the substance to be detected and is different from the active first substance used in the first reagent, and a labeling substance such as a fluorescent substance or a luminescent substance. Is used. Usually, the second substance and the labeling substance are chemically bonded using a cross-linking agent such as a polyvalent amine and carbodiimides.

Both the first reagent and the second reagent are dispersed in a dispersion medium containing water as a main component. In addition, a pH buffer,
Proteins, surfactants, water-soluble polymer compounds and the like are added as appropriate.

[0032]

As described above, the biological trace component inspection apparatus according to the present invention does not require a pump for sending out a reaction solution, so that the apparatus is simple. Further, since no sheath liquid is used, the inspection waste liquid can be greatly reduced. It is possible to reduce it.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of a first embodiment.

FIG. 2 is a model diagram of a complex formation reaction reaction between a reagent and a biological trace component (antigen).

FIG. 3 is a configuration diagram of a measurement device using a conventional floresail.

[Explanation of symbols]

 Reference Signs List 30 reaction tank 31 flow cell 32 waste liquid tank 33 absorption member 34 injection port 35 stirrer 36 laser light source 40, 42 light detector

Continuation of the front page (72) Inventor Matsuomi Nishimura 3-30-2 Shimomaruko, Ota-ku, Tokyo Inside Canon Inc. (72) Inventor Ken Miyazaki 3-30-2, Shimomaruko 3-chome, Ota-ku, Tokyo Inside Canon Inc. (56) References JP-A-61-132869 (JP, A) JP-A-4-36636 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/543 G01N 21 / 01 G01N 35/08

Claims (2)

(57) [Claims] 1. A first reagent comprising a surface of solid fine particles and a first substance which is active with respect to a biological trace component in a sample, and a first reagent which is active with respect to the sample and the biological trace component. The second substance different from the substance is reacted with a second reagent pre-labeled to form a complex of these substances, and the amount of the complex is measured to measure the amount of the biological trace component in the sample. In an inspection device for qualitatively or quantitatively detecting the presence, a reaction tank for performing a reaction for producing the complex, a flow cell for measuring the complex, and a waste liquid in which a liquid absorbing member is disposed at a connection portion with the flow cell A biological trace component inspection device comprising a tank. 2. An optical measurement is performed by the flow cell.
2. The biological trace component inspection apparatus according to claim 1, further comprising: