JPH0740031B2 - Immunological measurement method - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunological measurement method utilizing a particle agglutination reaction.

[Conventional technology]

Currently, specific reagents such as hepatitis B virus antigen and its antibody, syphilis antibody, mycoplasma antibody, RF, and determination reagents such as AFP, hCG, and FDP are used as reagents utilizing the particle agglutination method. Among them, the microtiter method by indirect agglutination reaction using sensitized red blood cells is widely used in the field of immunological test because it is inexpensive and simple.

In general, this measurement method utilizes an interparticle agglutination reaction caused by a reaction between an antigen or antibody physically or chemically bound to the surface of erythrocytes or an artificial carrier and an antibody or antigen in a body fluid such as serum, and the agglutination pattern thereof. It is based on. 〔G.Takatsy et al., Acta Physiol Hun
g., Vol. 5, p. 241, 1954; JLSever, J. Immunol., Vol. 88, p.
320, 1962]. This method can be said to be an excellent method because not only can it measure with a small amount of sample, but also a large amount of sample can be processed at the same time.

Further, as the above-mentioned improved method, there has been proposed an immunological measurement method in which an antibody or an antigen is immobilized and used not only on the carrier but also on the inner wall of the measurement container. For example, JP 56-130657 A, JP 56-142459 A, JP 61-212763 A, American Journal of Clinical Pathology, 87.
No.2 267-('87) etc. The principle of any of these methods is derived from the idea called the sandwich method or the diantibody method, which is used in radioimmunoassays and enzyme-immunoassays. That is, the specimen sample is added to the measurement container in which an antibody or an antigen corresponding to the antigen or the antibody in the specimen sample is fixed to the inner wall, and the antibody or the antigen and the antigen or the antibody in the specimen sample are reacted within a certain period of time, After that, the specimen sample containing unreacted antigen or antibody is removed by washing, and then a carrier on which the antibody or antigen is immobilized is added to the reaction container, and the antigen or the antibody bound to the inner wall of the reaction container via the antibody or the antigen. Is a measuring method for observing and determining the state of aggregation generated by reacting the antibody or antigen immobilized on the carrier.

Although the above-mentioned immunological measurement method is an excellent method, it is not a technology that sufficiently satisfies the demand for increasing the measurement sensitivity in a short time.

[Problems to be solved by the invention]

In view of the current situation as described above, the present inventors can improve the sensitivity of the particle agglutination reaction, without performing the washing operation of the sample in the body fluid, prozone phenomenon is difficult to be expressed even in a high concentration range, It can be measured in a short time, has less non-specific reactivity,
The present invention has been completed as a result of an examination for the purpose of providing a method that enables measurement regardless of the type of the antigen or antibody measured in the art, regardless of its type.

[Means for solving problems]

The present invention adds a sample sample to a measurement container in which an antibody or an antigen corresponding to the antigen or antibody in the sample sample is fixed to the inner wall,
Simultaneously or subsequently with the unreacted antigen or antibody in the presence of the antibody immobilized in the measuring container or the same antibody as the antigen or insoluble carrier particles immobilized with an analogue of the specific binding to the measuring container In addition, the immunological measurement method is characterized by determining the presence or absence of an antigen or antibody in a specimen sample by the presence or absence of an agglutination reaction that occurs.

That is, according to the method of the present invention, a measurement container in which an antibody or an antigen that specifically reacts with a sample is immobilized by physical adsorption or chemical bonding is prepared in advance on the measurement container. On the other hand, in the case of insoluble carrier particles, for example, red blood cell particles, the same antibody as that immobilized on the surface of the measuring container by being chemically bound, and in the case of latex particles, being physically adsorbed. Alternatively, insoluble carrier particles to which an antigen or an analog of specific binding is immobilized are prepared. Then, a fixed amount of the diluting solution is added to the measurement container, a specimen sample is added, and then the insoluble carrier particles are added and sufficiently stirred, and then left for a fixed period of time to determine an aggregation image.

The greatest feature of the present invention is to add an analyte sample to an antigen or a measurement container having an antigen immobilized on its inner wall, and to react the antigen or antibody contained in the analyte sample with the antibody or antigen immobilized in the assay container. The point is to add insoluble carrier particles to which the antibody or the antigen or the analog of the specific binding is immobilized, while the specimen sample containing the unreacted antigen or the antibody remains in the measurement container without being washed and removed.

The above-mentioned insoluble carrier particles can be added at the same time as the addition of the sample sample to the measurement container, or after the addition, within an arbitrary time, for example, within 0 second to about 1 hour.

When the measurement container is a microplate, it is preferable to add the specimen sample to the measurement container and then continuously add the insoluble carrier particles. In this case, continuously means that the time until the analyte sample is added to the measurement container and then the insoluble carrier particles are added is as short as possible, which can be said to be substantially continuous. To do. Further, the insoluble carrier particles may be added at any time within about 1 hour after adding the specimen sample.

Further, if the measurement container is like a test tube, by mixing one sample portion of the reagent containing the antibody or antigen immobilized on the insoluble carrier particles and the sample sample, or simultaneously adding to the measurement container, Alternatively, the sample sample can be added to a measurement container to which a reagent containing the insoluble carrier particles for one sample has been added in advance.

The sandwich method or two-antibody method used in radioimmunoassay or enzyme-immunoassay is an antibody immobilized in a measurement container for the purpose of removing the influence of contaminants other than the detected antigen or antibody contained in the specimen sample. Alternatively, a method is employed in which the antigen and the antigen or antibody in the specimen sample are contacted for a certain period of time, and then the specimen sample containing the unreacted antigen or antibody is washed off. Even in the conventional particle agglutination reaction method using insoluble carrier particles, the technical idea of the sandwich method or the bi-antibody property has been followed.

The present inventors have been researching various immunological measuring methods utilizing particle agglutination reaction for many years. In the immunological measuring method utilizing a particle agglutination reaction, the above-mentioned radioimmunoassay method or enzyme immunoassay method is used. We have already confirmed that there is a completely different behavior from the law. As a result of conducting various studies based on such recognition, surprisingly, unreacted antigen or antibody remaining after reacting the antibody or antigen immobilized in the measurement container with the antigen or antibody contained in the specimen sample When the specimen sample containing it is subjected to the reaction with the antibody or the antigen of the insoluble carrier particles or the analog of the specific binding in the state as it is, ie, without being removed by washing, it takes 4 to 4 times in comparison with the conventional method in a very short time. It was confirmed that the agglutination reaction can be accurately determined up to an 8-fold diluted sample specimen. The expression of such an astonishing effect is apparent from the examples to be treated later, but the mechanism by which it is exerted is not clear.

The present inventors have conducted an agglutination reaction due to a reaction between an antibody or an antigen bound to the measurement container via an antigen or an antibody and an antibody or an antigen immobilized on an insoluble carrier particle or an analog of specific binding and the unreacted reaction. It is considered that the agglutination reaction between the insoluble carrier particles caused by the antigen or the antibody proceeds at the same time, which facilitates the determination of the agglutination image.

In order to exert the effect of the present invention, as described above, when an insoluble carrier particle having an antibody or an antigen or an analogue of a specific bond immobilized thereon is added to a measurement container, the unreacted antigen or antibody in the measurement container is not added. Is an important requirement.

The means for allowing the unreacted antigen or antibody to exist is not particularly limited, but the simplest means is to keep the specimen sample added to the measurement container in a state of being left without being washed and removed.

In order to further exert the effect of the present invention, particularly a surfactant and / or a diluent used for diluting a specimen sample is used.
Alternatively, a solution containing protein is used. In particular, the best effect can be expected when skim milk powder or casein is selected as the protein. The concentration of the above surfactant or protein is not particularly limited, but generally 0.001 to 10% (W /
V), preferably 0.1 to 1% (W / V).

The insoluble carrier particles used in the present invention may be particles generally used in an indirect agglutination reaction. For example, animal red blood cells, liposomes, latex particles, microcapsules,
Organic polymer carrier particles such as emulsion, glass beads,
Inorganic polymer carrier particles such as silica beads and bentonite, and other artificial carriers can be mentioned.

Their particle size can be used in the range of 0.1-50.0 μm,
The range of 0.5 to 10.0 μm is preferable.

The measuring container used in the present invention is not particularly limited in its type, material, etc., as long as it is a container used for a particle aggregation reaction. For example, a test tube made of plastic such as polystyrene, vinyl chloride, polymethacrylate, or glass, a U-type or V-type microplate, or the like can be used.

As a method for immobilizing the antigen or antibody on the measurement container or the insoluble carrier particles, a known chemical binding method or physical adsorption method can be used. For example, as the chemical binding method, a method of binding with a divalent reagent used for enzyme immunoassay such as glutaraldehyde, maleimide, etc. through a functional group such as amino group, carboxyl group, hydroxyl group, aldehyde group, etc. Can be mentioned.

As a diluting solution, 0.001-10% (W / V), preferably 0.1
A buffer containing ~ 1% (W / V) skim milk powder, casein, BSA or various animal sera such as normal rabbit sera, or 0.
The above-mentioned buffer solution containing 001 to 10% (W / V), preferably 0.1 to 1% (W / V) nonionic, amphoteric or anionic surfactant can be used. For the buffer,
The pH may be 4.0 to 9.0, and those generally used in this field can be used. As the surfactant, sorbitan fatty acid ester, glycerin fatty acid ester,
Decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene Nonionic surfactants such as alkyl phenyl ether, polyoxyethylene castor oil, hydrogenated castor oil, polyoxyethylene lanolin and amphoteric surfactants such as betaine acetate, polyoxyethylene alkyl ether sulfate, polyoxyethylene alkyl Anionic surfactants such as ether acetate can be used.

Further, the same antigen or antibody to be immobilized on the insoluble carrier and the measurement container may be used, but the antibody may be the same in that it recognizes the same antigen. In this case, the origin may be different, and in the case of a monoclonal antibody, two or more kinds of antibodies having the same or different epitopes on the antigen may be used.

Further, an analog of specific binding refers to a substance that exhibits substantially the same behavior regarding the binding affinity as a counterpart of specific binding of an antigen or antibody, and this analog is also insoluble like an antigen or an antibody. It can be used by fixing it to a carrier and a measuring container.

Alternatively, the antigen or antibody may be immobilized in the measurement container, then removed by suction, and if necessary, a known blocking agent such as bovine serum albumin may be blocked. The measurement container on which the antigen or antibody is immobilized in this manner is a known method generally used in enzyme immunoassay, for example,
It can be stored for a long period of time by adding a 1% BSA solution containing 0.1% NaN ₃ or by freeze-drying.

The measurement target of the present invention is not particularly limited as long as it is an antigen or antibody measured in the art, and known substances can be measured. Typical examples include hepatitis B virus antigen and its antibody, syphilis antibody, mycoplasma antibody, C-reactive protein, α-fetoprotein, carcinoembryonic antigen, various immunoglobulins,
Examples include complement, streptolysin O, rheumatoid factor and the like.

[Work]

INDUSTRIAL APPLICABILITY As described above, the present invention fixes an antibody or an antigen against an antigen or an antibody contained in a specimen sample to both carrier particles and a measurement container, and further includes the antibody or antigen fixed to the measurement container and the specimen sample. The specimen sample containing unreacted antigen or antibody remaining after reacting with the antigen or antibody is left as it is, that is, without being removed by washing, and the sample of the antibody or antigen of the insoluble carrier particles or an analog of specific binding is obtained. By subjecting the sample to the reaction described above, the agglutination reaction can be accurately determined in an extremely short time and up to a diluted specimen sample that is 4 to 8 times larger than the conventional method, and the sensitivity of the agglutination reaction can be significantly increased and the measurement sensitivity can be improved. A clear aggregation pattern is obtained in a short time because it is not easily affected by the shape of the microplate and the addition of surfactants, etc., and the prozone phenomenon is generally recognized. UNA is what has become can also be measured in a high density area.

Hereinafter, the present invention will be described with reference to examples. The present invention is not limited to these examples.

Example 1 Measurement of HBs antigen 1. Preparation of antibody-immobilized carrier particles To one volume of high specific gravity composite particles (manufactured by Tokuyama Soda) suspended in PBS (pH 7.6) at 2% (W / V), a mouse was placed. Using 1 volume (1 mg / ml) of anion-exchange column chromatography-purified fraction of the anti-HBs monoclonal antibody obtained by the conventional method, 37
The reaction was carried out at 0 ° C for 3 hours. Then, it was washed with PBS and treated with 1% (W / V) casein (Merck) at 4 ° C. overnight to suppress nonspecific adsorption. This was washed with 100 mM Mc Ilvaine buffer containing 0.1% (W / V) Tween 20, 1% (W / V) casein, and suspended in the same buffer to 0.3% (W / V), The anti-HBs antibody was used as solid particles.

2. Preparation of antibody-immobilized assay container The anti-HBs monoclonal antibody (10 μg / m) diluted in PBS was added to each well of a 96-well microplate (Sumitomo Bakelite Co., Ltd.).
l) was dispensed in 50 μm aliquots. After leaving this at 37 ℃ for 2 hours, P
An antibody-immobilized measurement container was prepared by washing with BS, adding 200 μ of 1% BSA-PBS solution, leaving at 37 ° C. for 1 hour, and washing with purified water. However, if it is not used immediately, fill each hole with the BSA-PBS solution containing a preservative such as sodium azide, store at 2 to 4 ° C, or add an excipient such as glucose and freeze-dry. Using.

3. Measurement of antigen Normal human serum (1000 ng / ml) to which purified HBs antigen was added was diluted 2.5 times with purified water. This diluted serum 25μ, 0.1%
(W / V) Tween 20, 100 mM containing 1% (W / V) casein
25 μl of Mc Ilvaine buffer of 3 μl was diluted in each well of the antibody-immobilized microplate. Then, in each hole
A suspension of HBs antibody-immobilized particles was added in an amount of 25 μm each, and the plate was shaken with a micromixer for 1 minute and then allowed to stand at room temperature for 20 minutes to determine the aggregation pattern. As a negative sample,
Normal human serum to which HBs antigen was not added was used. or,
As a control experiment, the diluted serum to which the purified HBs antigen was added was similarly double-diluted in each well of the antibody-immobilized microplate, and then allowed to stand at room temperature for 1 hour, followed by 0.1% (W / V) Tween 20, 100 mM Mc Ilvai containing 1% (W / V) casein
Washed thoroughly with ne buffer. Add 25 μl of the above buffer or normal human serum double-diluted with this buffer to each well.
Each was added. Further, 25 μm of each suspension of the antibody-HBs antibody-immobilized particles was added to each well, the plate was shaken with a micromixer for 1 minute, and allowed to stand at room temperature for 20 minutes to determine the aggregation pattern. The results are shown in Table 1.

As is clear from the results of the blood aggregation pattern determination shown in Table 1, no non-specific reaction was observed in this method, and
A marked increase in sensitivity was recognized as compared with the conventional method in which the washing operation was performed.

Example 2 Measurement of HBs antibody 1. Preparation of HBs antigen-immobilized carrier particles Purified against 1 volume of high specific gravity complex particles (manufactured by Tokuyama Soda) suspended in 2% (W / V) in PBS (pH 7.6) HBs antigen (20 μg / m
l) 1 volume was added and reacted at 4 ° C. for 1 hour. Then PBS
Wash with 1% (W / V) BSA- to suppress non-specific adsorption.
Treated with PBS at 4 ° C overnight. This was washed with 100 mM Mc Ilvaine buffer and suspended in the same buffer to give 0.3% (W / V) to obtain anti-HBs antibody-immobilized particles.

2. Preparation of antigen-fixed measurement container Purified HBs antigen diluted to 1 μg / ml with PBS was dispensed into each well of a 96-well microplate (Sumitomo Bakelite Co., Ltd.) in an amount of 50 μm. This was left overnight at 4 ° C, washed with PBS, and washed with 1% BSA.
-A PBS solution (200 µm) was added, the mixture was left at 4 ° C for 2 hours, and washed with purified water to prepare an antibody-immobilized measurement container.

3. Antibody measurement Normal human serum to which anti-HBs monoclonal antibody was added at a concentration of 500 ng / ml was diluted 2.5 times with purified water. This diluted serum 25
μ contains 0.5% (W / V) BT-9, 2% (W / V) BSA 10
25 μl of 0 mM Tris-HCl buffer was used and diluted in each well of the antigen-immobilized microplate by doubling. Then for each hole
A suspension of HBs antigen-immobilized particles was added at 25 μm each, and the plate was shaken with a micromixer for 1 minute and then allowed to stand at room temperature for 20 minutes for determination. In addition, as a control experiment, a comparison was made with a microplate that was washed as in Example 1. The results are shown in Table 2.

Example 3 Measurement of HBs antigen 1. Preparation of antibody-immobilized sheep red blood cells 2.5% (W / V) glutar against 4 volumes of sheep red blood cells suspended in 5% (V / V) in PBS (pH 7.6) 1 volume of a solution of aldehyde in PBS was added and reacted at room temperature for 2 hours. Then PBS
Immobilized sheep red blood cells were prepared by washing with. 1 volume of 0.005% (W / V) tannic acid PBS solution was added to 1 volume of 5% suspension of this immobilized sheep red blood cell, and the mixture was reacted at room temperature for 30 minutes and washed with PBS to obtain tannic acid-treated immobilized sheep red blood cells. . An anti-HBs monoclonal antibody (2.
5 mg / ml) 1 volume was added, and the mixture was reacted at room temperature for 3 hours. Then, wash with PBS and adjust to 1% (V / V) to 0.5% (W / V).
V) Suspended in PBS containing normal rabbit serum to obtain antibody-immobilized sheep red blood cells.

2. Measurement of antigen Normal human serum (1000 ng / ml) to which purified HBs antigen was added at a concentration of 1000 ng / ml was diluted 2.5 times with purified water. 25 μl of this diluted serum was added to 1% (V / V) normal rabbit serum and 1% (W / V).
V) PBS containing Triton X-100 was used and diluted in each well of the antibody-immobilized microplate prepared by the method described in Example 1 by doubling. Next, 25 μm of a suspension of the sheep red blood cells immobilized with the anti-HBs antibody was added, and the plate was shaken with a micromixer for 1 minute and then allowed to stand at room temperature for 1 hour to determine the aggregation pattern. Normal human serum was used as a negative sample, and a microplate to which no antibody was bound was used as a control experiment. The results are shown in Table 3.

Example 4. Measurement of C-reactive protein (CRP) 1. Preparation of antibody-immobilized carrier particles High specific gravity complex particles (made by Tokuyama Soda) suspended in 2% (W / V) in PBS (pH 7.6) 1 On the other hand, immunoglobulin fraction 1 volume (1n (1n) obtained by salting out anti-CRP goat serum (manufactured by Biotest) and DEAE-cellulose column chromatography)
(g / ml) was added, and the mixture was reacted at 37 ° C for 3 hours. Then PBS
And washed with 1% (W / V) BSA at 4 ° C. overnight to suppress nonspecific adsorption. 0.5% (W / V) Triton X-
The cells were washed with 250 mM Mc Ilvaine buffer containing 100% 1% (W / V) BSA and suspended in the same buffer to give 0.3% (W / V), to give anti-CRP antibody-immobilized particles.

2. Preparation of antibody-immobilized assay container Each well of a 96-well microplate (Sumitomo Bakelite) was diluted with PBS to the anti-CRP goat antibody concentration of 10 μg / ml.
Dispensed in 50 μm increments. This was left at 37 ° C. for 2 hours, washed with PBS, added with 200 μl of 1% BSA-PBS solution, and blocked at 37 ° C. for 1 hour to prepare an anti-CRP antibody-immobilized microplate.

3. Antigen measurement Purified CRP antigen (manufactured by Experimental Biomedical Research Institute) was prepared to a concentration of 300 μg / ml with 100 mM Tris-HCl buffer (pH 8.0) containing 1 mM calcium chloride and 1% (W / V) BSA. did. 25 µC of this CRP standard solution was diluted twice in each well of an antibody-immobilized microplate using 250 µM of 250 mM Mc Ilvaine buffer containing 1% (W / V) Triton X-100 and 1% BSA. Next, 25 μm of each suspension of the anti-CRP antibody-immobilized particles was added to each well, the plate was shaken with a micromixer for 1 minute and then allowed to stand at room temperature for 20 minutes to determine the aggregation pattern. As a control experiment, a microplate to which no antibody was bound was used.
Incidentally, 1 mM calcium chloride used to dilute the CRP antigen,
100 mM Tris-HCl buffer (pH8.0) containing 1% (W / V) BSA
Was used as the CRP negative solution. The results are shown in Table 4.

As shown in Table 4, in the conventional method, agglutination reaction is not observed in the high concentration range of CRP, whereas in the present method, the prozone phenomenon is strong even in the high concentration range. It was found to exhibit cohesiveness.

〔The invention's effect〕

According to the immunological measurement method of the present invention, the sensitivity of the agglutination reaction is remarkably increased as compared with the conventional method, a clear agglutination pattern is obtained in a short time, and the prozone phenomenon is generally recognized in a high concentration range. It has become possible to measure at. Moreover, there is also an advantage that the operation itself is simpler because the washing operation performed in the conventional method is unnecessary.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Naofumi Takahashi 5-5-8 Sagamihara, Sagamihara City, Kanagawa Prefecture Garden House 501 (56) References JP-A-56-142459 (JP, A)

Claims (1)

[Claims] 1. A sample container is added to a measurement container having an inner wall fixed with an antibody or an antigen corresponding to the antigen or antibody in the sample sample, and the unreacted antigen or antibody is simultaneously or subsequently present in the measurement container. The presence or absence of the antigen or antibody in the specimen sample depending on the presence or absence of the agglutination reaction that is caused by adding insoluble carrier particles to which the same antibody or antigen as the antibody or antigen immobilized on An immunological measurement method characterized by determining.