JPH04361159A - Immunoassay - Google Patents

Abstract

PURPOSE:To accurately quantify the specific component in a liquid sample, in heterogenous system immunoassay necessary for the separation of a reaction product and an unreacted substance, by allowing ultrasonic waves to act at the time of the separation of both of them. CONSTITUTION:When the specific component in a sample is analyzed using heterogenous system immunoassay, an insolubilized carrier to which a substance specifically reacting with the specific component in the sample is bonded, the sample and a labelled body wherein a label substance is bonded to the substance specifically reacting with the specific component in the sample are brought into contact with each other to be subjected to immunoreaction and, thereafter, an antigen-antibody reaction product and an unreacted substance are separated (B/F separation). In order to make the B/F separation sufficient, ultrasonic waves are allowed to act on the insolubilized carrier after immunoreaction put in a washing solution. By this method, the specific component in the liquid sample can be accurately quantified with good reproducibility by simple operation.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a heterogeneous immunoassay method that requires separation of reaction products and non-reactants.

0002

BACKGROUND OF THE INVENTION Various analytical methods have been developed for detecting trace amounts of substances contained in biological fluid samples. One of these analytical methods is based on immune reactions. Various measurement methods using this principle have been developed and are known to be highly accurate.

[0003] Immunoassays are broadly classified into homogeneous immunoassays and non-uniform immunoassays. That is, separation of antigen-antibody reaction products (Bound bodies) and non-reacting substances (Free bodies) (
They are broadly divided into non-homogeneous immunoassay methods that require B/F separation) and homogeneous immunoassay methods that do not require B/F separation. Among these, when the substance to be measured is a polymer, a non-uniform immunoassay method that requires B/F separation is used.

By the way, this non-uniform immunoassay method is based on B/F
There are problems in that the operation is complicated because it requires washing operations for separation, preparation of reagents, and addition of labeling substances, substrates, reaction stop solutions, and the like. Although various techniques have been proposed to solve these problems, the workability of B/F separation is not so good, and problems such as the time required still remain. In particular, cleaning operations must be repeated many times in order to achieve sufficient B/F separation, and improvements in workability are awaited.

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a technique that allows accurate quantification of specific components in a liquid sample with good reproducibility using simple operations. The object of the present invention is to provide a heterogeneous immunoassay method that requires separation of reaction products and non-reactants, and is characterized in that ultrasonic waves are applied during separation of reaction products and non-reactants. This is accomplished by an immunoassay method.

[0006] In order to analyze a specific component in a sample using a heterogeneous immunoassay, first, an insolubilized carrier to which a substance that specifically reacts with the specific component in the sample is bound, a sample, and a sample in the sample are prepared. A labeled substance bound to a labeled substance is brought into contact with a substance that specifically reacts with a specific component, and an immune reaction is performed. In the present invention, any form of solution, colloidal solution, etc. can be used as the sample, but fluid samples of biological origin are preferably used, such as blood, plasma, serum, cerebrospinal fluid, saliva, amniotic fluid, milk, urine, sweat, etc. Examples include meat juice.

[0007] The specific component in a fluid sample that can be measured according to the present invention is a substance (substance group) in which there may be a substance that specifically binds to the specific component. That is, examples thereof include polypeptides, proteins, complex proteins, polysaccharides, lipids, complex lipids, nucleic acids, hormones, vitamins, drugs, antibiotics, agricultural chemicals, and the like. Specifically, substances described in JP-A-62-90539 and JP-A-63-131062
(group of substances), but is not limited to these.

Labeling substances in the labeled body used in the present invention include, for example, enzymes, enzyme substrates, substances that change the activities of enzymes and enzyme precursors (enzyme inhibitors, prosthetic groups, coenzymes), enzyme precursors, Examples include apoenzyme and fluorescent substances. As a specific substance, JP-A-62-905
Examples include those described in Publication No. 39, etc., preferably enzymes or fluorescent substances, and more preferably β-D
- Enzymes such as galactosidase, alkaline phosphodase, peroxidase, glucose oxidase, glutamate dehydrogenase, amylase. When these enzymes are used as labeling substances, known enzyme reaction systems and coloring systems can be used. Specifically, Japanese Patent Application Laid-Open No. 61-292
The substances (substance group) described in JP-A No. 060, JP-A No. 62-90539, JP-A-63-131062, JP-A-63-45562, and Japanese Patent Application No. 63-219893 are listed. However, it is not limited to these. The methods for binding these labeling substances to antibodies are described in Eiji Ishikawa, Tadashi Kawai, and Kiyoshi Miyai (eds.), Enzyme Immunoassay (2nd edition), Igakushoin, 1978, and Japanese Society of Clinical Pathology (ed.), "Clinical Pathology," special issue. The method described in Special Issue No. 53, "Immunoassay for Clinical Tests - Techniques and Applications," Clinical Pathological Publishing Society, 1983, etc., can be referred to.

[0009] Substances that specifically bind to a specific component or label in a fluid sample that can be applied to the present invention include antibodies, antigens, lectins, protein A, etc., depending on the object to be measured. It is particularly preferred that the binding reaction of the binding substance is an antigen-antibody reaction. The origin of the antibodies used in the present invention is not particularly limited. Antiserum obtained by administering and immunizing mammals with antigens, ascites fluid as is, or sulfuric acid using a conventionally known method. Sodium precipitation method, ammonium sulfate precipitation method, gel filtration method using Sephadex gel, ion exchange cellulose chromatography method, electrophoresis method, etc. (Shunsuke Migita, "Immunochemistry")
Nakayama Shoten, pp. 74-88). Alternatively, hybrid cells (hybridoma) are obtained from spleen cells or myeloma cells (myeloma) of a mammal (e.g. mouse) infected with an antigen, and a monoclonal antibody is created, which is then used as a substance that can specifically bind to a specific component. It is preferable to use it as the specificity improves. In addition, these antibodies can be used as IgG, IgM, IgA, IgD, and IgE fractions, or these antibodies can be treated with enzymes to produce active antibody fragments such as Fab, Fab', or F(ab')2. May be used. Furthermore, these antibodies may be used alone or in combination.

[0010] Reaction types for the immunoassay of the present invention include a competitive method, a two-antibody method, and a sandwich method. Furthermore, immunoassay methods using other biologically active substances (eg, biotin, avidin) can also be applied. In the present invention, an immune reaction is mentioned as a reaction type to measure a specific component in a fluid sample, but a specific reaction of a substance that exhibits biological activity similar to an immune reaction (in this specification, this specific reaction is also referred to as an immune reaction) It is also possible to use the reaction (inclusion in the reaction).

The antigen used in the present invention reacts with specific antibodies and contains haptens and derivatives thereof. The insolubilized carrier to which the antibody is bound is not particularly limited, but
It is preferable to use granules having a size of 2 mm or less. Materials for the insolubilizing carrier include agarose, cellulose, cross-linked dextran, polyacrylamide, cellulose, microcrystalline cellulose, cross-linked agarose, cross-linked polyacrylamide, glass, silica gel, diatomaceous earth, titanium dioxide, barium sulfate, zinc oxide, lead oxide, In addition to various synthetic resins such as silica sand and polystyrene, materials for porous layers,
Furthermore, magnetic fine particles can be used. Preferred are agarose, cross-linked agarose, cross-linked dextran, polyacrylamide, cross-linked polyacrylamide, glass, silica gel, polystyrene, cellulose, microcrystalline cellulose, etc., and more preferred are polyacrylamide, cross-linked polyacrylamide, polystyrene, microcrystalline cellulose, etc. . Several kinds of the above-mentioned insolubilizing carriers may be used in combination.

[0012] Antibodies or antigens can be chemically and/or physically bound to these insolubilized carriers directly or indirectly by methods known to those skilled in the art. Regarding binding methods, published by Kodansha in 1976, edited by Ichiro Chibata and two others, "Experimental and Applied Affinity Chromatography" (1st printing)
You can refer to "Affinity Chromatography" (1st edition), published by Kodansha, 1975, edited by Makoto Yamazaki and two others.

[0013] In order to eliminate non-specific reactions of labeled antibodies or antigens, it is possible to carry proteins that are not involved in the specific reaction to be measured. Typical examples thereof include mammalian normal serum proteins, albumin, skim milk, lactic acid fermentation products, collagen, gelatin, and decomposition products thereof. An insolubilized carrier bound to a substance that specifically reacts with a specific component in the sample, a sample, and a labeled body bound to a labeling substance to a substance that specifically reacts with a specific component in the sample are brought into contact and immunoreacted. Afterwards, B/F separation is performed. In this B/F separation, conventionally, the insolubilized carrier after the immune reaction was simply placed in a washing solution and left to stand, but in the present invention, the insolubilized carrier after the immune reaction was placed in the washing solution. Ultrasonic waves are applied to the subsequent insolubilized carrier.

[0014] The washing liquid used for this B/F separation includes, for example, physiological saline, phosphate buffer, and a surfactant such as Twin 20 (Wako Pure Chemical Industries, Ltd.) added at about 0.02%. A solution, etc. is used, and the frequency of the ultrasonic wave is 16 to 100 KHz, and the output of the oscillator is 20 W to 20 KHz.
It is sufficient to use a material of about KW and allow it to act for about 1 to 30 seconds each time.

The method for measuring the signal caused by the labeled substance after B/F separation differs depending on the type of label, but for example, if the labeled substance is a fluorescent substance, it is sufficient to measure the fluorescence intensity; If it is an enzyme, add a suitable substrate, or if necessary, a solution containing the enzyme or coloring system, and after incubating for a certain period of time, measure the absorbance or reflection density of light at a wavelength compatible with the coloring system (depending on the type of substrate, fluorescence). The signal strength can be measured by measuring the luminescence intensity). The substrate and coloring system used for this purpose can be appropriately selected according to the type of labeled enzyme. The signal caused by the labeled enzyme can be detected by absorbance method (colorimetric method), fluorescence method, or luminescence method.Measurement methods include rate measurement method that measures the change in signal over time or signal after a certain period of time. can be measured using endpoint assays that measure The absorbance method is preferable, and the absorbance method (colorimetric method) can utilize ultraviolet light, visible light, and near-infrared light. For example, when serum and plasma are used as fluid samples, the absorbance by the serum and plasma is It is preferable to use green light, red light or near-infrared light to reduce the influence.

[0016]

EXAMPLES The present invention will be explained in more detail below with reference to Examples, but the present invention is not limited to these Examples. [Example 1] Mouse monoclonal antibody (Ig
M) was dissolved in 0.1M carbonate buffer (pH 9.5) containing 0.5M sodium chloride to a concentration of 10 μg/ml, and then polystyrene beads (Sekisui Chemical Co., Ltd. #80) were added thereto. was soaked and fixed overnight at 4°C to obtain an immobilized antibody.

This immobilized antibody was dissolved in 0.05 ml of 0 U/ml GAT standard solution and 1 M sodium chloride.
It was placed in a mixture of 0.2 ml of 0 mM phosphate buffer (pH 6.5) and incubated at 37° C. for 2 hours. Next, after washing with phosphate buffered saline (PBS), 0.25 ml of a 1% BSA-PBS solution (0.5 μg/ml) of peroxidase-labeled anti-GAT monoclonal antibody, which recognizes a site different from that of the immobilized antibody, was added. Add 1 at room temperature
Incubated for hours.

After this, 1 ml of PB was added to the immobilized antibody.
S was added and ultrasonic cleaning was performed for 5 seconds. In addition, this ultrasonic cleaning uses a container volume of 3 liters and a frequency of 45 KH.
z, conducted using an ultrasonic oscillator with an output of 60 W. After this ultrasonic cleaning, the cleaning liquid was suction filtered and discarded, and the above operation was repeated a predetermined number of times. [Comparative Example 1] The same procedure as in Example 1 was carried out except that instead of the ultrasonic cleaning, shaking and standing were performed.

[Characteristics] After the above ultrasonic cleaning operation and shaking,
After static cleaning work, 0.02% H2 O2 and 3mg
Add 0.3 ml of o-phenylenediamine solution at room temperature for 30 minutes, then add 1 ml of 1N sulfuric acid.
1 was added to stop the coloring reaction, and the absorbance of the coloring reaction solution was measured at 492 nm (n=25). The results are shown in Table 1.

[0020]According to this, it can be seen that B/F separation is effectively performed by applying ultrasonic waves during B/F separation. For example, it can be seen that the background value becomes approximately zero after two or three washing operations.

[Example 2] 0 U/ml in Example 1
The same procedure was performed using the serum of an ovarian cancer patient instead of the GAT standard solution. [Comparative Example 2] In Example 2, the same procedure as in Comparative Example 1 was carried out.

[Characteristics] After the above ultrasonic cleaning operation and shaking,
After static cleaning work, 0.02% H2 O2 and 3mg
Add 0.3 ml of o-phenylenediamine solution at room temperature for 30 minutes, then add 1 ml of 1N sulfuric acid.
1 was added to stop the coloring reaction, and the absorbance of the coloring reaction solution was measured at 492 nm (n=25). The results are shown in Table 2.

Table 2 Number of cleanings
Example 2 Comparative example 2
Absorbance coefficient of variation Absorbance coefficient of variation 1 time 0.
625 10.2% 0.768 11.6%
2 times 0.501
2.3% 0.630 8.8%
3 times 0.502
2.2% 0.515 7.6%
4 times 0.501 2
．． 4% 0.503 3.1%
5 times 0.501 2.3
% 0.501 2.2% According to this, B/
By applying ultrasound during F separation, B/F
It can be seen that separation is effectively performed and accurate measurements can be made quickly.

Claims (1)

[Claims] [Claim 1] A heterogeneous immunoassay method that requires separation of reaction products and non-reactants, characterized in that ultrasonic waves are applied during separation of reaction products and non-reactants. Immunoassay.