JP2836009B2 - Fine particles for biochemistry, method for producing the same, and immunoassay using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to fine particles for biochemistry used as a solid phase in a biochemical analysis method and the like, and more particularly, to an immunoassay particularly useful for quantification of trace substances in a biological fluid. Biochemical analysis methods such as the method, purification of useful substances, used for the purification, separation, biochemical microparticles for solid phase with high reactivity and excellent suspension, its very simple production method, and its About use.

[0002]

2. Description of the Related Art In the immunological assay method at the beginning of development,
Reactions were performed in a state where all factors involved in the reaction, such as antibodies and antigens, were soluble in the liquid phase.In recent years, antibodies and antigens have been insolubilized in a solid phase for the purpose of simplifying measurement operations, etc. A way to make it happen.

[0003] An early technique for insolubilization is Sephadex, a polysaccharide polymer of Pharmacia.
An attempt was made to bind antibodies or antigens to dex), and immunoassays using the reagents were partially applied clinically as diagnostics. However, Sephadex used for the solid phase had a large particle size and high sedimentation in the liquid phase, so that it was necessary to always invert and mix during the incubation. Furthermore, the stability of the precipitated solid phase is poor in the centrifugation after the reaction, and ordinary B / F separation is not suitable.
It was complicated because it was necessary to repeat washing and re-centrifugation of the precipitate.

[0004] Thereafter, slightly fine polysaccharide polymers and fine particles mainly composed of latex are selected and used as solid phases. However, these microparticles also did not provide satisfactory results, and could not exhibit effective sensitivity in a short time especially in the measurement of a substance which is present only very slightly in a biological fluid.

[0005] As described above, the reason why the conventional solid-phase immobilization does not provide a satisfactory result is that conditions for high sensitivity or reactivity, ease of centrifugation, and precipitation of centrifugation after centrifugation. This is because the conditions for stability are contradictory.

That is, in immunological measurement, the sensitivity is one important factor together with specificity, and is particularly important when the amount of the substance to be measured is very small. And, compared with the case where the reaction between the antibody and the antigens proceeds in the same liquid phase,
When a reaction proceeds in a state in which one of them is insolubilized, that is, a reaction proceeds in a so-called heterogeneous environment, the reactivity is generally considered to be low.

As the conventional insolubilized solid phase, polystyrene tubes, glass tubes, sepharose, cellulose or polystyrene particles are generally used, and these insolubilized solid phases participate in the reaction with a liquid phase reagent. In view of its shape and rapid sedimentation, good dispersibility can not be expected in order to contribute to the water content. Also, implementations using plastic test tubes will generally have low reactivity with the liquid phase, as is the case.

[0008] In particular, in order to obtain a good result for a trace substance or the like in a limited measurement time, it is necessary to expect high reactivity of these insolubilized solid phases. It is necessary to select the conditions under which the phase assimilate with the liquid phase.

However, the conventionally provided immobilized solid phase or insolubilized solid phase which has a fast sedimentation property with respect to the measurement time is not necessarily high in reactivity, and when it takes time to dispense the reagent, During the dispensing, the insolubilized solid phase in the reagent settles out, and the measurement cannot be established at that time, or there is a risk of causing a measurement error due to the measurement.

As described above, the poor functionality and low reactivity of the insolubilized solid phase are not only disadvantageous in the measurement operation but also lead to low measurement reliability. It is necessary to immobilize an antigen or antibody using a solid phase that has better suspension properties and is rich in reaction.

As a solid phase having better suspendability and reactivity, for example, JP-A-61-97568 discloses fine latex particles having a particle size of 0.1 to 1.0 μm. Immunological measurement of a substance present at a relatively high concentration in a biological fluid, such as β2-m, is performed by using a reagent using this. However, centrifugation of latex particles by this method requires very special centrifugation conditions such as 17000 rpm, and cannot be said to be a generally applicable method.

Japanese Patent Application Laid-Open Nos. 61-97568, 62-56862 and 2-28603 also disclose methods using a fine solid phase. The preparation of the solid phase is complicated, and the binding between the solid phase and the antibody uses a simple adsorption method, which is generally considered to have a weak binding force.

In addition, Japanese Patent Application Laid-Open No. 61-167866 discloses a technique in which a solid phase contains a magnetic powder. This method has the advantage that B / F separation is relatively easy, but requires stirring with a magnetic stirrer during incubation, and its application is also comparable to biological fluids such as IgE and ferritin. It was limited to the measurement of substances at extremely high concentrations.

The common problems of the above-mentioned conventional techniques for preparing a fine solid phase are that special techniques and complicated operations are required for the preparation of the solid phase, and that antibodies or antigens are not used for these solid phases. A new adsorption operation is required when adsorbing to the phase,
Another point is that the amount of antibody or antigen cannot be sufficiently adsorbed per unit solid phase. There is also a drawback in the production method that reduces the immunological activity of the substance to be immobilized.

As described above, since it is difficult to achieve both the reactivity and the dispersibility of the solid phase, a trace substance can be measured in a short time and with high sensitivity even by a conventional immunoassay using fine particles. Did not reach.

[0016]

In order to measure, with an effective sensitivity, a substance which is present only in a very small amount in a biological fluid, a finely divided solid phase having excellent dispersibility and stability as described above is required. Needless to say, it is also important that the solid phase can be easily prepared without affecting the immunological activity of the substance immobilized during the solid phase preparation. An object of the present invention is to provide a method for producing fine particles which satisfies all of these conditions and can be advantageously used as a solid phase in a biochemical analysis method.

[0017]

Means for Solving the Problems The present inventors have intensively studied to obtain a finely divided solid phase having excellent dispersibility and stability. By chemically bonding an aldehyde group such as glutaraldehyde under a certain optimum condition, an aggregate of an insolubilized substance was successfully generated. Also, this agglutination obtained
The lumps are immobile masses , which are aggregates of fine particles and have been found to be able to be finally dispersed as fine particles .

The antibodies and antigens are bound to the aldehyde groups remaining on the surface and inside of the aggregate by an imide bond or a chemical bond such as a bond obtained by reducing the imide bond. The inventors have found that microparticles extremely useful for immunoassay can be obtained by a simple operation such as inactivating the remaining aldehyde group with a primary amine and finely dispersing the same, thereby completing the present invention.

That is, a first object of the present invention is to form an aggregate by reacting a soluble substance having two or more amino groups with a compound having a functional group which reacts with two or more amino groups.
Thus, the present invention provides biochemical fine particles obtained by chemically binding an antibody or an antigen thereto.

Further, another object of the present invention is to provide the following steps a to
c, a. Reacting a soluble substance having two or more amino groups with a compound having a functional group that reacts with two or more amino groups to obtain an aggregate ; b. Binding antibodies or antigens to the aggregates , c. An object of the present invention is to provide a method for producing antibody- or antigen-bound fine particles, which comprises a step of inactivating the remaining functional groups of the aggregate and then dispersing the same.

[0021] Still another object of the present invention is to provide an immunological analysis method using the fine particles for biochemistry.

To produce the biochemical fine particles of the present invention,
First, a soluble substance having two or more amino groups (hereinafter, referred to as
An aggregate is obtained by reacting a compound having a functional group that reacts with two or more amino groups (hereinafter referred to as “amino-reactive compound”) with a “soluble substance”.

Examples of the soluble substance used in the production of aggregates include proteins, peptides such as polylysine, and polysaccharides such as amino sugars.

Of these, albumin and the like can be used as the protein, but gamma globulin and the like are preferable. Regardless of the species difference of gamma globulin, any one obtained from humans, pigs, horses, cows, etc. can be used, but bovine gamma globulin is suitable in terms of price.

In selecting a solubilizing substance such as a protein, care must be taken because the number and molecular weight of amino groups in the molecule affect insolubilization.

On the other hand, the amino-reactive compound is a compound having a functional group which reacts with an amino group, for example, an aldehyde group, a succinimidyl group, an isothiocyano group, a sulfonyl chloride group, etc. Among them, those having an aldehyde group as a functional group Glutaraldehyde, succinaldehyde, etc., and those whose functional groups are succinimidyl groups include ethylene glycol bis (succinimidyl succinate) (EGS), disuccinimidyl tartrate (DST), etc. When the group is an isothiocyano group, 4,4′-diisothiocyanostilbene-2,2′-disulfonate disodium salt (DIDS), paraphenylenediisothiocyanate and the like can be mentioned.

In the amino-reactive compound, the number of functional groups that react with the amino group and the solubility in a solvent pose a problem. For example, when the compound is used in combination with a water-soluble protein, the compound is also water-soluble. It is preferable to select one, and glutaraldehyde or the like is most suitable in terms of supply and price. On the other hand, when the substance to be insolubilized is fat-soluble, it is preferable to use a compound such as a fat-soluble aldehyde.

In producing the aggregate of the present invention, if the amount of the solubilizing substance such as a protein or the amino-reactive compound such as an aldehyde is unilaterally excessive, an aggregate is not obtained. It is necessary to appropriately adjust the quantitative ratio of.

For example, when the solubilizing substance is gamma globulin (1 W / W%) and the amino-reactive compound is glutaraldehyde (50 W / W%), the mixed solution ratio of gamma globulin and glutaraldehyde is 2 : 1-2
Agglomerates can be obtained in the range of 00: 1.

Furthermore, aggregation for obtaining good fine particles
Setting the optimum conditions is an important factor in making a lump , including the purity and amount of the solubilizing substance and amino-reactive compound, as well as the pH, temperature, and time during the reaction. However, the optimal conditions are preferably determined experimentally because they vary depending on the type of the solubilizing substance and the amino-reactive compound used.

Next, the agglomerate thus obtained is
The antibodies or antigens are bound. The substances to be bound include
It is necessary that an amino group for reacting with and binding to a functional group such as an aldehyde group of the aggregate is present. Although an antibody always has an amino group, some antigens and the like do not have an amino group. In such a case, the amino group may be introduced by any method.

For example, for derivatives such as estradiol and aldosterone having no carboxyl group in the molecule without an amino group, a water-soluble carbodiimide or the like is used, and the carboxyl group is bonded to ethylenediamine, lysine methyl ester, gamma globulin or the like. In this way, a derivative having an amino group can be formed and bonded to the aggregate .

No special operation is required for binding the antibody or antigen to the aggregate, and the antibody or antigen is simply diffused and penetrated into the aggregate , and the amino group of the antibody or antigen is combined with the antibody or antigen.
An aldehyde group or the like in the aggregate may be chemically bonded.

Further, by inactivating a functional group which reacts with an amino group which did not participate in the reaction in the aggregate and dispersing it finely, desired biochemical fine particles can be obtained.

The inactivation of the remaining functional group is to prevent the fine particles from reaggregating after dispersion and to prevent the antigen or antibody to be measured at the time of measurement from binding to the remaining functional group.
For example, inactivation of an aldehyde group which is a functional group is performed by adding a sufficient amount of a primary amine such as glycine and reacting.

In order to finely disperse the particles, they may be suspended by stirring using a vortex mixer or by appropriately repeating suction and discharge of aggregates using pipettes.

The size of the biochemical fine particles of the present invention obtained as described above varies depending on materials and conditions.
For example, the thickness is about 0.5 to 200 μm, which can be widely used for immunological analysis using a solid phase.

For example, as long as the microparticles of the present invention have an antibody or an antigen immobilized thereon, the microparticles can be used as they are for an immunological assay in the agglutination method. If the microparticles of the present invention have a labeled antibody immobilized thereon, luteinizing hormone (LH), follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), tissue polypeptide antigen (T
PA), renin, prolactin, α-fetoprotein (AFP) and the like can be used for immunoassay of sandwiches.

Further, if the fine particles of the present invention have antigens immobilized thereon, they can be used in the immunoassay of a competitive method. For example, the fine particles of the present invention having antigens immobilized thereon can be used. If a labeled antibody is used, it is possible to carry out an immunological assay by a competition method for thyroxine (T ₄ ), triiodothyronine (T ₃ ), steroids or their free parts.

The labeling substance used for labeling the antibodies or antigens immobilized on the fine particles of the present invention is not particularly limited, and may be a known labeling substance such as a radioactive substance, an enzyme, a fluorescent substance, or a luminescent substance. It can be labeled, and this label can be an indirect label, such as with a second antibody.

The microparticles of the present invention can be applied not only to immunological measurement methods but also to various bioreactors such as immobilization of immunizing antigens, affinity chromatography and enzymes. is there.

Next, taking the thyroxine-immobilized fine particles as an example, the production method according to the present invention will be described more specifically.

First, the fine particles used for immobilization are produced by insolubilizing 5 ml of a 1% bovine gamma globulin solution with 200 μl of a 50% glutaraldehyde solution. The resulting aggregates are then washed with distilled water to remove unreacted glutaraldehyde, and immediately diffuse and permeate 10 μg of thyroxine into the aggregates .

After the thyroxine binding, the aldehyde group remaining in the aggregate is inactivated by glycine, followed by washing, and finely dispersed by stirring. Finally, the dispersed thyroxine-immobilized fine particles are appropriately diluted to obtain a thyroxine-bound fine particle suspension.

The measurement of free thyroxine using the thyroxine-immobilized fine particles obtained as described above may be carried out as follows.

First, 50 μl of a test sample or standard serum of each concentration was taken, and this was used as the thyroxine-bound fine particle suspension 1.
00 μl and 100 μl of ¹²⁵ I-labeled anti-thyroxine antibody
And a reaction at 37 ° C. for 1 hour to allow free thyroxine in the test sample or standard serum of each concentration to compete with thyroxine-bound fine particles on a ¹²⁵ I-labeled anti-thyroxine monoclonal antibody.

Next, in order to increase the separation efficiency, 0.4%
-2000 g after adding 1 ml of polyvinyl acetate resin
Centrifuge at for 30 minutes and remove the supernatant. Finally, the radioactivity of the precipitate is measured, and the concentration of free thyroxine in the test sample is determined from a standard curve obtained from a standard serum.

The thyroxine-bound fine particles of the present invention used in the above-described measurement method maintain a good dispersion state during the reaction (incubation), and the ¹²⁵ I-labeled anti-thyroxine monoclonal antibody can be maintained in a static incubation time of only about 1 hour. Reacted well.

In the actual measurement method, it is necessary to separate thyroxine-bound fine particles or thyroxine-bound fine particles bound to ¹²⁵ I-labeled anti-thyroxine monoclonal antibody from the reaction solution, and the most widely used means is centrifugation. There is separation, but in some cases, the stability of sedimentation after centrifugation is poor due to the fineness of extremely small fine particles, which may cause inconvenience when the supernatant is subsequently separated.

In such a case, the technique devised by the present inventors (see JP-A-3-154868) can stabilize the sedimentation, and is particularly advantageous in using the fine particles of the present invention. It is.

That is, the fine particles produced according to the present invention and the technique of stabilizing the sedimentation are effective in any heterogeneous reaction, and are a simple method for stabilizing the sedimentation with high reactivity of the solid-phased substance. And very useful. Therefore, in the immunoassay using the competitive method and the sandwich assay using two antibodies and antigens which are widely used, it is possible to provide a solid phase in which the production of the solid phase is low cost and which maintains high activity. This is an effective means for objective measurement.

[0052]

EFFECTS OF THE INVENTION The biochemical fine particles of the present invention have an advantage in production that they can be easily and inexpensively produced, a high ability to immobilize antibodies and antigens, a good dispersibility and It has both physical advantages of having reactivity. Hereinafter, these advantages will be sequentially described.

(Easy Production Method) In most of the conventional methods for producing solid-phased fine particles, previously finely divided fine particles are used, and antibodies or antigens are simply adsorbed or chemically bonded thereto.
However, when an antibody or the like is chemically bonded to previously miniaturized particles, the fine particles sometimes bond to many of the functional groups of the antibody or the like, and as a result, the fine particles are bonded so as to surround the antibody or the like. May impair important immune activity.

On the other hand, in the present invention, the aggregate (insolubilized substance) prepared before binding the antibodies is an immobile gel, and after binding the antibody or the like, the functional group such as an aldehyde group is inactivated. Because they are dispersed finely, they do not secondarily surround the antibodies. As described above, the present invention has a feature that antibody / antigen-bound fine particles can be produced while maintaining immunological activity.

Further, the production method can be completed by a series of operations from the production of aggregates to the chemical bonding of antibodies or antigens, and the production time is as short as several hours to at most two days. Furthermore, no special reagents, techniques and equipment are required for the production, and when gamma globulin is used for preparing microparticles, the only required reagents are gamma globulin, glutaraldehyde, and a routine buffer. The instruments considered are beakers and pipettes and ordinary centrifuges. For this reason, it is possible to prepare solid-phased microparticles anytime and anywhere in a simple laboratory.

On the other hand, this aggregate can be preserved without immediately binding to an antibody or the like as long as the deactivation of the aldehyde group is prevented, and the aggregate can be bound without deactivating the substance as described above. Due to its characteristics, it can be applied to various bioreactors such as immobilization of antigens for immunization, affinity chromatography and immobilization of enzymes.

(Inexpensive Production Method) When bovine gamma globulin is used as a water-soluble substance required for production and glutaraldehyde is used as an amino-reactive compound, the raw material cost is low and the production method is simple. It can be kept very low and appears to be one of the cheapest of all the microparticle production methods.

One of the features is that the dispersion state of the finally obtained fine particles can be easily controlled by changing the amount of bovine gamma globulin or glutaraldehyde within the optimum range for preparing the aggregate. It is. For example, if a large amount of bovine gamma globulin is used, the particles become slightly larger, while if a small amount is used, the particles become small.

[0059] (high solid phase capability) On the other hand, fine particles of the present invention is to use a large amount of glutaraldehyde in the course of making this, coagulation resulting in reaction
Many aldehyde groups are bound to the aggregate while maintaining the activity.

For this reason, a large amount of antibodies and antigens having an amino group can be immediately bound to aggregates . For example, when 10 μg of thyroxine or triiodothyronine is bound to about 50 mg of fine particles, the unreacted fraction Since almost no specific light absorption or immunological activity of thyroxine or triiodothyronine was observed in J. , it was judged that most of them had bound to the aggregate . This bond is not a simple adsorption having a weak bonding force but a covalent bond, and the bond is considered to be stable.

(Good Dispersibility) In order to maintain a good dispersion state, the size of the particles needs to be small. At the same time, it is important that the specific gravity of the particles is relatively close to the reaction solution. In recent years, a method has been used in which magnetic fine particles are used as fine particles and B / F separation can be performed by a magnet at the end of an immune reaction. However, even if such magnetic fine particles are extremely fine, a magnetic substance is contained in the particles. Therefore, it has a disadvantage that it is heavier than the reaction solution and sediments in a short time.

In some cases, it is necessary to incubate with stirring to compensate for such disadvantages. On the other hand, the microparticles of the present invention made of proteins and the like have extremely good suspending properties, and are characterized in that they are fine in size but also excellent in floating properties.

(Good Reactivity) The effect of high reactivity by using fine and dispersible fine particles is as follows.
This becomes clear when compared with the case where a plastic test tube or the like is simply used for the solid phase. The present inventors have obtained a similar result in a so-called sandwich immunological assay method by changing the antibody solid-phase plastic test tube to the antibody-immobilized fine particles in approximately one third of the reaction time.

In addition, when a plastic test tube was used for the solid phase, incubation by shaking was necessary, while for the antibody-immobilized fine particles, static incubation was sufficient. This can be understood that the fine particles showed a good dispersion state in the reaction solution, and as a result, high reactivity was obtained.

As is clear from the above, the biochemical fine particles of the present invention have both advantages in production and properties, and are advantageous in a widely used competitive method and sandwich measurement method. It is a very good thing that can be used for.

[0066]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

EXAMPLE 1 Production of Thyroxine-Binding Fine Particles and Determination of Free Thyroxine Concentration: (1) Preparation of Insolubilized Substance (Agglomerated Agglomerate)
Dissolve it in a 1% concentration in an mM-phosphate buffer (pH = 6.5), take 5 ml of this solution in a glass beaker, add 200 μl of a 50% -glutaraldehyde solution, and gently stir for about 10 seconds. I do. Under these conditions, BγG
Is crosslinked with glutaraldehyde to form an aggregated gel mass (insolubilized substance).

(2) Production of thyroxine-bound fine particle suspension The aggregate obtained in (1) was washed several times with the above-mentioned phosphate buffer to remove unreacted reagent, and then a thyroxine solution adjusted to 2 μg / ml was prepared. Add 5 ml and let stand overnight. (Thyroxine is dissolved in 50% -DMSO.) In this step, the amino group of thyroxine is chemically bonded to the free aldehyde group remaining after aggregation with BγG. Next, after gently removing the supernatant,
0.5 M-glycine buffer (p
H = 8.5) Add 50 ml to block unreacted aldehyde groups. (This operation is repeated twice.)

Next, the glycine buffer solution was removed, 15 ml of a new glycine buffer solution was added, and the thyroxine-bound aggregates were micronized by stirring with a pipette operation. Centrifuge. The supernatant after centrifugation was removed, and the precipitate was resuspended in a glycine-phosphate buffer (pH = 6.4) to reduce the total volume to 2%.
Make up to 5 ml. The obtained thyroxine-bound fine particle suspension has a thyroxine concentration of 400 ng / ml.

(3) Preparation and Assay of Suspension of Thyroxine-Binding Fine Particles The suspension obtained in (2) was appropriately diluted with the glycine-phosphate buffer, and then 100 μl each and ¹²⁵ I-labeled anti-thyroxine antibody. 100 μl of the mixed solution was reacted at 37 ° C. for 1 hour, and after adding 0.4% -polyvinyl acetate resin 1 ml,
After centrifugation, the supernatant was removed. The amount of radioactivity of the precipitate was measured to determine the thyroxine concentration of the thyroxine-bound fine particle suspension showing about 50% of the total radioactivity, and was found to be about 10 ng /
ml (1 ng / 100 μl). Therefore, 10 ng / ml was selected as the final concentration of the thyroxine-bound fine particle suspension used for immunological measurement.

On the other hand, 400 ng / ml of the fine particle suspension was
^As determined by a competition method using ¹²⁵ I thyroxine, the active concentration was about 1/4, that is, 100 ng / ml. Therefore, the thyroxine activity concentration of the final prepared thyroxine-bound fine particle suspension is about 2.5 ng / ml (0.25 ng / 10
0 μl).

The method (1% -BγG5 ml-50%)
The thyroxine binding capacity in glutaraldehyde (200 μl) is 5 mg or more, and the total amount of 10 μg / 5 ml of thyroxine used here is chemically bound. This binding capacity was determined by performing a competitive reaction using ¹²⁵ I thyroxine on the supernatant obtained after chemically binding thyroxine solutions of each concentration of 10 mg / ml or less under the same conditions.
It was determined from the remaining amount of thyroxine.

(4) Measurement of Free Thyroxine Amount and Preparation of Standard Curve Using Thyroxine-Bound Fine Particle Suspension 50 μl of test sample (or standard serum containing free thyroxine at a predetermined concentration), thyroxine-bound fine particle suspension 100
mix 100 μl of ¹²⁵ I-labeled anti-thyroxine
The reaction is allowed to proceed at 7 ° C. for 1 hour, and free thyroxine in the test sample (or standard serum at each concentration) and thyroxine-bound fine particles are competed with ¹²⁵ I-labeled anti-thyroxine.

Next, in order to increase the separation efficiency, 0.4%
-Add 1 ml of polyvinyl acetate resin, and 2,000 g
After centrifugation for 0 minutes, the supernatant is removed, and the radioactivity of the precipitate is measured. From the free thyroxine concentration in the standard serum and the amount of radioactivity in the precipitate, a standard curve for the free thyroxine is obtained, and the free thyroxine concentration in the specimen is obtained therefrom.
The relationship between the free thyroxine concentration of the standard serum and the radiation dose for obtaining the standard curve is shown below.

[0075]

Example 2 Production of Triiodothyronine-Binding Fine Particles and Measurement of Free Triiodothyroxine: An insolubilized substance (agglomerated mass) obtained from BγG and glutaraldehyde was obtained in the same manner as in Example 1, (1). 20 mM phosphate buffer (pH = 6.5)
And washed several times to remove unreacted reagent. To this insolubilized substance, 5 ml of a triiodothyronine solution adjusted to 1 μg / ml was added, and the mixture was allowed to stand overnight. (Triiodothyronine is dissolved in 50% -DMSO.) In this step, BγG
The amino group of triiodothyronine is chemically bonded to the free aldehyde group remaining after the aggregation.

Next, after gently removing the supernatant, the triiodothyronine-bound aggregate was added to a 0.5M-glycine buffer solution (pH =
8.0) Add 50 ml to block unreacted aldehyde groups. (This operation is repeated twice.) Next, the glycine buffer is removed, 15 ml of a new glycine buffer is added, and triiodothyronine-bonded aggregates are micronized by stirring with a pipette operation. Transfer to a centrifuge tube and centrifuge at 1000 g for 30 minutes. The supernatant after centrifugation was removed, and the precipitate was resuspended in a glycine-phosphate buffer (pH = 6.4) to reduce the total volume to 25 m.
l. This triiodothyronine-bound fine particle suspension has a triiodothyronine concentration of 200 ng / ml.

After appropriately diluting this suspension with the glycine-phosphate buffer, a mixture of 100 μl of each and 100 μl of ¹²⁵ I-labeled anti-triiodothyronine antibody was reacted at 37 ° C. for 1 hour to give 0.4% After adding 1 ml of polyvinyl acetate resin, the mixture was centrifuged and the supernatant was removed. The radioactivity of the precipitate was measured to determine the triiodothyronine concentration of the triiodothyronine-bound fine particle suspension showing about 50% of the total radioactivity, which was about 2 ng / ml (0.2 ng / 100 μl).
Met. Therefore, the final concentration of the triiodothyronine-bound fine particle suspension used for the immunological measurement was 2 ng / m 2.
l was selected.

On the other hand, 200 ng / ml of the fine particle suspension was
^As determined by a competition method using ¹²⁵ I triiodothyronine, the active concentration was about 1/5, that is, 40 ng / ml.
Therefore, the triiodothyronine activity concentration of the finally prepared triiodothyronine-bound fine particle suspension was about 0.4 ng /
ml (0.04 ng / 100 μl).

The measurement of free triiodothyronine using the suspension of triiodothyronine-bound fine particles obtained above is performed as follows. That is, first test subject (or standard serum at each concentration) 50 [mu] l, triiodothyronine binding particle suspension 100 [mu] l, ¹²⁵ mixed I-labeled anti-triiodothyronine antibody 100 [mu] l, reacted at 37 ° C., ¹²⁵ Free triiodothyronine and triiodothyronine-bound microparticles in the test sample (or standard serum at each concentration) are subjected to a competitive reaction on an I-labeled anti-triiodothyronine antibody.

Next, in order to increase the separation efficiency, 0.4%
-Add 1 ml of polyvinyl acetate resin, and 2,000 g
After centrifugation for 0 minutes, the supernatant is removed, and the radioactivity of the precipitate is measured. Finally, a standard curve was prepared from the radiation dose of the standard serum, and the concentration of free triiodothyronine in the test sample was determined therefrom. The relationship between the free thyroxine concentration of the standard serum and the radiation dose for obtaining the standard curve is as follows.

[0082]

Example 3 Preparation of Anti-FSH Monoclonal Antibody-Bound Fine Particles and FS
Measurement method of H concentration: B was measured in the same manner as in Example 1 (1).
The insolubilized substance (aggregate) obtained from γG and glutaraldehyde was washed several times with a 20 mM phosphate buffer (pH = 6.5) to remove unreacted reagent. Next, 5 ml of an anti-FSH monoclonal antibody solution adjusted to 1 mg / ml is added to the insolubilized substance, and the mixture is left overnight. In this step, the amino group of the anti-FSH monoclonal antibody chemically bonds to the free aldehyde group remaining after aggregation with BγG.

Next, after gently removing the supernatant, 50 ml of 0.5M-glycine buffer (pH = 8.0) is added to the aggregates bound with the anti-FSH monoclonal antibody to block unreacted aldehyde groups. (This operation is repeated twice.) Then, the glycine buffer was removed, 15 ml of a new glycine buffer was added, and the anti-FS was stirred by pipetting.
After the H monoclonal antibody-bound aggregates are micronized,
Transfer the microparticle suspension to a centrifuge tube and centrifuge at 1000 g for 30 minutes.

The supernatant after centrifugation was removed, and the precipitate was washed with 1% -B
Resuspend in phosphate buffer (pH = 6.8) containing SA to bring the total volume to 25 ml. The antibody concentration of this anti-FSH monoclonal antibody-bound fine particle suspension was 200 μg / m
l.

The suspension was appropriately diluted with the above-mentioned phosphate buffer containing 1% -BSA, 100 μl each, 100 μl of each concentration of FSH antigen, and the antibody used for the microparticles were FS.
A mixture of 100 μl of ¹²⁵ I-labeled anti-FSH monoclonal antibody having a different recognition site for H was reacted at room temperature for 1 hour,
After adding 1 ml of 0.4% -polyvinyl acetate resin, the mixture was centrifuged and the supernatant was removed to measure the radioactivity of the precipitate.
Bound suppression phenomenon on the high concentration side of FSH antigen (hook)
The amount of the suspension used is 4 μg / 100 μ so that no
1 (40 μg / ml). Therefore, 40 μg / ml was selected as the final concentration of the anti-FSH monoclonal antibody-bound fine particle suspension used for immunological measurement.

The measurement of FSH using the anti-FSH monoclonal antibody-bound fine particle suspension obtained above is performed as follows.

First, 100 μl of a test sample (or each concentration is standard FSH serum), 100 μl of an anti-FSH monoclonal antibody-bound fine particle suspension, and 100 μl of ¹²⁵ I-labeled anti-FSH monoclonal antibody were mixed at room temperature for 1 hour. The FSH antigen in the test sample (or standard serum at each concentration) is subjected to a sandwich reaction between the antibody in the fine particle suspension and the labeled antibody.

Next, 1 ml of 0.4% polyvinyl acetate resin was added, and the mixture was centrifuged at 2000 g for 30 minutes. The supernatant was removed, and the radioactivity of the precipitate was measured. Finally, a standard curve was prepared from the radiation dose of the standard serum, from which the FSH concentration in the test sample was determined. The relationship between the FSH concentration of the standard serum and the radiation dose for obtaining a standard curve is as follows.

[0090]

Example 4 Production of Anti-LH Monoclonal Antibody-Bound Fine Particles and Measurement of LH Concentration: In the same manner as in Example 1, (1), BγG
And the insolubilized substance (agglomerated mass) obtained from glutaraldehyde were washed several times with 20 mM phosphate buffer (pH = 6.5) to remove unreacted reagent. Next, 5 ml of an anti-FSH monoclonal antibody solution adjusted to 1 mg / ml is added to the insolubilized substance, and the mixture is left overnight. In this process,
Free aldehyde groups remaining after aggregation with BγG
The amino group of the H monoclonal antibody chemically bonds.

Next, after gently removing the supernatant, 50 ml of a 0.5 M glycine buffer (pH = 8.0) is added to the anti-LH monoclonal antibody-bound aggregate to block unreacted aldehyde groups. (This operation is repeated twice.) Further, the glycine buffer is removed, 15 ml of a new glycine buffer is added, and the anti-LH monoclonal antibody-bound aggregates are micronized by stirring by a pipette operation. Transfer to a centrifuge tube and centrifuge at 1000 g for 30 minutes.

The supernatant after centrifugation was removed, and the precipitate was
Resuspend in phosphate buffer (pH = 6.8) containing SA to bring the total volume to 25 ml. The antibody concentration of this anti-LH monoclonal antibody-bound fine particle suspension was 200 μg / ml.
It is.

After appropriately diluting this suspension with the above-mentioned phosphate buffer containing 1% -BSA, 100 μl each and L of each concentration were added.
¹²⁵ I-labeled anti-LH monoclonal antibody 1 which has a different LH recognition site from 100 μl of H antigen and the antibody used for microparticles 1
The mixture (00 μl) was allowed to react at room temperature for 1 hour, and after adding 1 ml of 0.4% -polyvinyl acetate resin, the mixture was centrifuged, and the radioactivity of the precipitate from which the supernatant was removed was measured.

As a result, the amount of the suspension used so as not to cause the bounce suppression phenomenon (hook) on the high concentration side of the LH antigen was 2.5 μg / 100 μl (25 μg / ml). Therefore, the final concentration of the anti-LH monoclonal antibody-bound fine particle suspension used for immunological measurement was 25 μg / m 2.
l was selected.

The measurement of LH using the suspension of microparticles conjugated with the anti-LH monoclonal antibody obtained above is performed as follows.

That is, first, 100 μl of a test sample (or standard LH serum at each concentration), 100 μl of an anti-LH monoclonal antibody-bound fine particle suspension, and 100 μl of ¹²⁵ I-labeled anti-monoclonal antibody were mixed and reacted at room temperature for 1 hour. And the L in the test sample (or standard serum of each concentration)
The H antigen is subjected to a sandwich reaction between the antibody in the particle suspension and the labeled antibody.

Then, 1 ml of 0.4% -polyvinyl acetate resin is added, and the mixture is centrifuged at 2000 g for 30 minutes. The supernatant is removed, and the radioactivity of the precipitate is measured. Finally, a standard curve was prepared from the radiation dose of the standard serum, and the LH concentration in the test sample was determined from this. The relationship between the LH concentration of the standard serum and the radiation dose for obtaining the standard curve is as follows.

[0099]

Example 5 Production of Anti-AFP Monoclonal Antibody-Bound Fine Particles and AF
Method for measuring P concentration: B was measured in the same manner as in Example 1 (1).
The insolubilized substance (aggregate) obtained from γG and glutaraldehyde was washed several times with a 20 mM phosphate buffer (pH = 6.5) to remove unreacted reagent. Next, 5 ml of an anti-FSH monoclonal antibody solution adjusted to 1 mg / ml is added to the insolubilized substance, and the mixture is left overnight. In this step, the amino group of the anti-AFP monoclonal antibody chemically bonds to the free aldehyde group remaining after aggregation with BγG.

Next, after gently removing the supernatant, 50 ml of 0.5M-glycine buffer (pH = 8.0) is added to the aggregates bound with the anti-AFP monoclonal antibody to block unreacted aldehyde groups. (This operation is repeated twice.) Next, the glycine buffer was removed, 15 ml of a new glycine buffer was added, and the anti-AF was stirred by pipetting.
After the P monoclonal antibody-bound aggregate has been micronized,
Transfer the microparticle suspension to a centrifuge tube and centrifuge at 1000 g for 30 minutes.

The supernatant after centrifugation was removed, and the precipitate was
Resuspend in phosphate buffer (pH = 6.8) containing SA to bring the total volume to 25 ml. The antibody concentration of this anti-AFP monoclonal antibody-bound fine particle suspension was 200 μg / m 2.
l.

The suspension was appropriately diluted with the above-mentioned phosphate buffer containing 1% -BSA, 100 μl each, AFP antigen of each concentration 100 μl, and the antibody used for the microparticles were AF
A mixture with 100 μl of ¹²⁵ I-labeled anti-AFP monoclonal antibody having a different recognition site for P was reacted at room temperature for 1 hour,
After adding 0.4 ml of polyvinyl acetate resin (1 ml), the mixture is centrifuged, and the supernatant is removed to measure the radioactivity of the precipitate.
As a result, the amount of the suspension used was 5 μg so as to prevent the occurrence of the bounce suppression phenomenon (hook) on the high concentration side of the AFP antigen.
/ 100 μl (50 μg / ml). Therefore,
50 μg / ml was selected as the final concentration of the anti-AFP monoclonal antibody-bound fine particle suspension used for the immunological measurement.

The measurement of AFP using the anti-AFP monoclonal antibody-bound fine particle suspension obtained above is carried out as follows. That is, first, 100 μl of a test sample (or standard AFP serum at each concentration), 100 μl of an anti-AFP monoclonal antibody-bound fine particle suspension, ¹²⁵ I-labeled anti-AFP
100 μl of the monoclonal antibody is mixed and reacted at room temperature for 1 hour, and the AFP antigen in the test sample (or the standard serum at each concentration) is subjected to a sandwich reaction between the antibody in the fine particle suspension and the labeled antibody.

Next, 1 ml of 0.4% polyvinyl acetate resin was added, and the mixture was centrifuged at 2000 g for 30 minutes. The supernatant was removed, and the radioactivity of the precipitate was measured. Finally, a standard curve was prepared from the radiation dose of the standard serum, and the AFP concentration in the test sample was determined therefrom. The relationship between the AFP concentration of the standard serum and the radiation dose for obtaining the standard curve is as follows.

[0106]

Example 6 Method for producing estradiol-bound fine particles and method for measuring estradiol: 9.9 mg of estradiol and 49 mg of diaminobutane were dissolved in water-soluble carbodiimide (E
(DCI) at 100 mg to prepare an estradiol derivative. Excess diaminobutane was separated by Sephadex G-10.

On the other hand, as in (1) of the first embodiment,
The insolubilized substance (aggregate) obtained from γG and glutaraldehyde was washed several times with a 20 mM phosphate buffer (pH = 6.5) to remove unreacted reagent. Next, 5 ml of an estradiol derivative solution adjusted to 2 μg / ml is added to the insolubilized substance, and the mixture is allowed to stand overnight. In this step, the amino group of the estradiol derivative is chemically bonded to the free aldehyde group remaining after aggregation with BγG.

Next, after the supernatant was gently removed, the estradiol-bound aggregate was added to a 0.5 M glycine buffer solution (pH = 8.
0) Add 50 ml to block unreacted aldehyde groups. (This operation is repeated twice.) Then, the glycine buffer was removed, 15 ml of a new glycine buffer was added, and the estradiol-bound aggregates were micronized by stirring by pipetting, and the microparticle suspension was transferred to a centrifuge tube. Transfer and centrifuge at 1000 g for 30 minutes. The supernatant after centrifugation was removed, and the precipitate was resuspended in a glycine-phosphate buffer (pH = 6.4) to reduce the total volume to 25 m.
l. This estradiol-bound fine particle suspension has an estradiol concentration of 400 ng / ml.

After appropriately diluting this suspension with the above-mentioned glycine-phosphate buffer, a mixture of 100 μl each and 100 μl of ¹²⁵ I-labeled anti-estradiol antibody was reacted at 25 ° C. for 2 hours to obtain 0.4% polyvinyl acetate. After adding 1 ml of the resin, the mixture was centrifuged and the supernatant was removed. The radioactivity of the precipitate was measured, and the estradiol concentration of the estradiol-bound fine particle suspension showing about 50% of the total radioactivity was determined. The estradiol concentration was about 8 ng / ml (0.8 ng / 100 μl). Therefore, 8 ng / ml was selected as the final concentration of the estradiol-bound fine particle suspension used for the immunological measurement.

Measurement of estradiol using the estradiol-bound fine particle suspension obtained above is performed as follows. That is, first, 100 μl of the test sample (or standard serum of each concentration), 100 μl of estradiol-bound fine particle suspension, ¹²⁵ I-labeled anti-estradiol antibody 1
After mixing with 100 μl, the mixture is reacted at 25 ° C. for 2 hours, and estradiol in the test sample (or standard serum at each concentration) and estradiol-bound fine particles are reacted on ¹²⁵ I-labeled anti-estradiol antibody.

Next, in order to increase the separation efficiency, 0.4%
-Add 1 ml of polyvinyl acetate resin, and 2,000 g
After centrifugation for 0 minutes, the supernatant is removed, and the radioactivity of the precipitate is measured. Finally, a standard curve was prepared from the radiation dose of the standard serum, and the estradiol concentration in the test sample was determined therefrom. The relationship between the estradiol concentration of the standard serum and the radiation dose for obtaining the standard curve is as follows.

[0113] that's all

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int.Cl. ⁶ , DB name) G01N 33/544 G01N 33/543

Claims (12)

(57) [Claims] An agglutinated mass is obtained by reacting a soluble substance having two or more amino groups with a compound having a functional group that reacts with two or more amino groups, and then chemically binding an antibody or antigen thereto. Fine particles for biochemistry. 2. The biochemical fine particles according to claim 1, wherein the chemical bond is a chemical bond between an aldehyde group of the aggregate and an amino group of the antibody or antigen. 3. The biochemical fine particles according to claim 1, wherein the particle diameter is in a range of 0.5 to 200 μm. 4. The antigen to be bound is LH, FSH, TS
The biochemical fine particles according to any one of claims 1 to 3, wherein the fine particles are selected from H, TPA, renin, prolactin, AFP, thyroxine, triiodothyronine, and steroids. 5. The antibody which binds to LH, FSH, TS
The biochemical microparticle according to any one of claims 1 to 3, wherein the microparticle is an antibody against an antigen selected from H, TPA, renin, prolactin, AFP, thyroxine, triiodothyronine, and steroids. 6. The following steps a to c, a. Reacting a soluble substance having two or more amino groups with a compound having a functional group that reacts with two or more amino groups to obtain an aggregate; b. Binding antibodies or antigens to the aggregates, c. A method for producing antibody or antigen-bound fine particles, comprising a step of inactivating the remaining functional groups of the aggregates and then dispersing them. 7. The method according to claim 6, wherein the soluble substance is selected from proteins, peptides, and polysaccharides. 8. The method according to claim 6, wherein the protein is selected from albumin or gamma globulin. 9. The method according to claim 6, wherein the compound having a functional group that reacts with two or more amino groups is glutaraldehyde. 10. The method according to claim 6, which is a method for producing fine particles to which LH, FSH, TSH, TPA, renin, prolactin, AFP, thyroxine, triiodothyronine, steroids or their antibodies are bound. . 11. An immunological measurement method using the fine particles according to claim 1. 12. The measurement target is LH, FSH, TSH, T
The method according to claim 11, which is PA, renin, prolactin, AFP, thyroxine, triiodothyronine, a steroid or a free part thereof.