JPH07111434B2 - Method for producing insoluble carrier particles on which antibody or antigen is immobilized - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing an immunological measuring reagent, and more specifically to a method for producing insoluble carrier particles having an antibody or an antigen immobilized thereon.

[Problems to be Solved by Prior Art and Invention]

Conventionally, a carrier particle in which an immunologically active substance such as an antibody or an antigen is immobilized by physical adsorption or covalent bond formation on the carrier particle (hereinafter, abbreviated as immobilized carrier particle) and a corresponding in a subject such as serum or urine There is known an immunological measurement method for measuring a corresponding antigen or antibody in a subject by observing an agglutination reaction or an aggregation inhibition reaction due to an antigen-antibody reaction with an antigen or an antibody. The measuring method using the above-mentioned immobilized carrier particles is widely used because it can quickly, highly accurately and easily measure a trace component contained in a subject.

In the old days, the above-mentioned immobilized carrier particles and a test substance were mixed on a reaction plate, and an agglutination or agglutination inhibition reaction based on an antigen-antibody reaction was performed to visually determine an agglutination image to qualify the corresponding antigen or antibody in the subject. Was being measured. In recent years, it has become possible to quantitatively measure an antigen or antibody in a subject by optically measuring the agglutination reaction product.

The most important thing in the immunological measurement method using such immobilized carrier particles is the uniformity of the particle diameter of the immobilized carrier particles.

The following phenomenon occurs in the process of mixing with carrier particles when immobilizing an antibody or an antigen. That is, in the process of immobilizing the antibody or antigen on the surface of the carrier particle, there is still an unimmobilized portion on the particle surface, and the immobilized antibody or antigen is further adsorbed on the carrier particle surface or Since covalent bonding is possible, when non-fixed portions of other carrier particles come into contact with each other, adsorption or covalent bonding also occurs there, and eventually the antibody or antigen serves as a cross-linking agent to cause aggregation of the carrier particles. In this way, under the immobilization condition in which the carrier particles are agglomerated with each other, immobilized carrier particles having a uniform particle size cannot be obtained, and the particle size is distributed. If the distribution of the particle size is widened, not only the quantitativeness is impaired particularly in the quantitative measurement method, but also in the qualitative measurement method, the difference between the presence and absence of aggregation is reduced and the determination becomes difficult. Furthermore, it becomes difficult to reproducibly produce immobilized carrier particles having the same distribution of aggregated particle size under such immobilization conditions.

Conventionally, as a method of immobilizing, a method of adding a solution of an antibody or an antigen to a suspension of carrier particles and immediately stirring and mixing has been performed. In this method, the amount of the antibody or antigen added is used in a large amount as compared with the surface area of the carrier particles that can be immobilized,
At the time of mixing, it is necessary to quickly cover the surface of each carrier particle with an antibody or an antigen to suppress the agglutination reaction between the carrier particles. Furthermore, because it is necessary to mix with stirring within a short time,
If the amount of immobilization is increased, the mixing will be insufficient and the resulting agglomeration of the immobilized carrier particles will increase. Therefore, in the case of producing a large amount, it is necessary to repeat the immobilization many times.

Contrary to the above, in the method of adding a suspension of carrier particles to a solution of an antibody or an antigen, the particles previously added immobilize many antibodies or antigens, and as the addition proceeds, the unfixed antibody in the solution Alternatively, the amount of antigen decreases, and carrier particles immediately before the end of addition have a drawback that aggregated particles are easily formed. Therefore, this method requires more rapid addition. As described above, the conventional method for producing insoluble carrier particles having an immobilized antibody or antigen has a problem that the immobilized carrier particles easily form aggregated particles. For this reason, the conventional method is not suitable for mass production, and it is difficult to keep the characteristics of the reagent obtained in each production constant.

[Means for solving problems]

The present inventors have earnestly studied a method for suppressing agglomeration of insoluble carrier particles at the time of immobilization and obtaining a large amount of an immunological measurement reagent having a constant performance.

As a result, as will be described later in detail, when the insoluble carrier particles having an antibody or an antigen immobilized thereon are produced, the ratio of the amount of the antibody or antigen and the amount of the insoluble carrier particle per unit time becomes constant. Thus, at least two liquid streams, a liquid stream containing an antibody or an antigen and a suspension flow containing insoluble carrier particles, are combined under conditions where mixing is achieved within an extremely short period of time. It was found that the aggregation of the insoluble carrier particles can be suppressed and an immunological measuring reagent with a certain performance can be mass-produced.

According to this production method, it was found that the immobilization conditions obtained on a laboratory test tube scale have a merit that can be directly used as immobilization conditions during mass production on an industrial scale, and the present invention has been completed. , Came to propose here.

That is, the present invention provides a liquid flow containing antibody or antigen and a suspension flow containing insoluble carrier particles so that the ratio of the amount of antibody or antigen and the amount of insoluble carrier particles per unit time is constant. A method for producing insoluble carrier particles on which an antibody or an antigen is immobilized, characterized in that at least two liquid streams are joined under conditions where instantaneous mixing is achieved.

Further, as a preferred embodiment for carrying out the present invention, in the production of the insoluble carrier particles on which the above-mentioned antibody or antigen is immobilized, a mixer having a plurality of supply ports and a single outlet is used and a suspension containing insoluble carrier particles is used. An insoluble carrier on which an antibody or antigen is immobilized, characterized in that the suspension and the antibody or antigen-containing solution are separately supplied from the supply ports, both are instantaneously mixed to form a uniform flow, and then collected from the extraction port. A method for producing particles is provided.

In the present invention, the antibody or antigen immobilized on the insoluble carrier particles is not particularly limited, and known ones can be used.
Typical examples that can be suitably used are, for example, human albumin, anti-human albumin antibody, human immunoglobulin G (human IgG), anti-human IgG antibody, human immunoglobulin A (human IgA), anti-human IgA antibody, Human immunoglobulin M (human IgM), anti-human IgM antibody, human immunoglobulin E (human IgE), anti-human IgE antibody, human C-reactive protein (human CRP), anti-human CRP antibody, alphafetoprotein (AFP) ), Anti-AFP antibody, carcinoembryonic antigen (CEA),
Anti-CEA antibody, human ciliated gonadotrobin (HCG), anti-HCG
Antibodies, Inshiyurin, anti Inshiyurin antibody, B-type hepatitis surface antigen (HB _S), anti-HB _S antibody, complement Clq, anti-Clq antibody, complement
C ₃ , anti-C ₃ antibody, complement C ₄ , anti-C ₄ antibody, fibrinogen degradation product (FDP), anti-FDP antibody, denatured human γ-blobulin,
Rheumatoid factors and the like.

The insoluble carrier particles are insoluble carrier particles which are substantially insoluble in a liquid medium used for immobilization, storage and measurement, and fine particles having an average particle size of about 10 μm or less are preferably used, which will be described in detail later.

Various types of these particles are already known for use in the antigen-antibody reaction, and in the present invention, these known fine particles can be used without particular limitation. Examples of particularly preferably used ones include polystyrene and styrene-
Fine particles of organic polymer substance such as organic polymer latex obtained by emulsion polymerization such as butadiene copolymer, styrene-methacrylic acid copolymer, polyglycyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, or silica , Silica-alumina, inorganic oxides such as alumina, or inorganic particles obtained by introducing a functional group into the inorganic oxide by an operation such as silane coupling, and particles derived from organisms such as human O type red blood cells and sheep red blood cells. Is.

Regarding the particle size of the insoluble carrier particles, when the particle size is large, the amount of change in particle size due to aggregation is large but the aggregation reaction rate is slow, and when the particle size is small, Brownian mobility is active and the aggregation reaction rate is fast. Since the primary particle size is small, the amount of change in particle size due to the agglutination reaction is small. Therefore, the average particle size of the insoluble carrier particles used in the agglutination reaction is 10μ.
Insoluble carrier particles of about m or less, preferably 0.05 to 5.0 μm are suitably used.

When quantifying an antigen or antibody in a test substance by an optical measurement method, the wavelength of the light used in the measurement has a relatively large change in transmitted light or scattered light with the progress of the reaction and is highly sensitive, and usually coexists in the test sample. 400-1000 nm, preferably 500-95 nm, which has relatively little interference with chyle, hemoglobin, bilirubin, etc.
Wavelengths in the range of 0 nm are preferably used. In the case of optical measurement, the particle size to be used may be selected in consideration of the wavelength of light used, and insoluble carrier particles having an average particle size of 0.05 to 0.5 μm are particularly preferably used.

In the present invention, the method of immobilizing the antibody or antigen on the insoluble carrier particles may be either physical adsorption or formation of a chemical covalent bond, but a protein having a relatively high physical adsorption ability such as an antibody or a high molecular weight protein can be used. Physical adsorption is preferably used for immobilization. On the other hand, in the case of immobilizing a highly hydrophilic protein having poor physical adsorption ability, a low molecular weight antigen, or the like, or in the case of avoiding desorption, it is preferable to form a chemical covalent bond. Many methods have already been proposed for chemical covalent bonding, and it is advisable to select an immobilization method from known methods according to the characteristics of the antibody to be immobilized.

In general, an antibody or antigen dissolved or suspended in a dispersion medium and a dispersion medium in which insoluble carrier particles are suspended and, if necessary, respective liquid streams such as a crosslinking agent may be combined and mixed. Known crosslinking agents such as glutaraldehyde and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride can be used.

The dispersion medium for immobilizing the antibody or the antigen on the insoluble carrier particles is not particularly limited, and a known dispersion medium is used. When the above-mentioned crosslinking agent is used, the components in the dispersion medium react with the crosslinking agent. It is necessary to use a dispersion medium that does not. A glycine-sodium hydroxide buffer solution is preferably used as the dispersion medium,
A buffer solution such as Tris-hydrochloric acid buffer solution, ammonium chloride-ammonia buffer solution, or phosphate buffer solution is preferably used. The concentration of the insoluble carrier particles in the dispersion medium at the time of immobilization is not particularly limited, but it is generally 0.05% by weight or more, preferably 0.2 to 2.0% by weight when mixed with the antibody or the antigen. It is preferable to select. The concentration of the antibody or antigen is not particularly limited, but generally 0.0005% by weight or more when mixed with the insoluble carrier particles, preferably
It is preferable to select 0.002 to 0.2% by weight. The concentration ratio of the insoluble carrier particles and the antibody or antigen is the particle size of the insoluble carrier particles, the ratio of the surface area that can be immobilized and the type of the antibody or antigen, and the dispersion liquid of the insoluble carrier particles and the antibody when mixing for immobilization. Alternatively, it may be determined in consideration of various factors such as the amount of the antigen-dissolved liquid.

In the present invention, as a preferred embodiment, it is convenient to use a mixer having a plurality of supply ports and a single discharge port. That is, it is necessary to allow the dispersion liquid of insoluble carrier particles and the dissolution liquid of an antibody or an antigen to be separately supported from the at least two supply ports by the amount of the insoluble carrier particles present in both liquids and the insoluble carrier particles. There is a method in which the amount of the antibody or the antigen is supplied so that the amount of the antibody or the antigen is sufficient, and the flows of the supplied liquids are instantaneously and uniformly mixed, and collected from the outlet. Examples of the mixer having the above function include a mixer in which Y-shaped tubes are combined and a mixer in which T-shaped tubes are combined. In the mixer of the present invention, it is more effective in consideration of the cross-sectional area of the mixing section. If the cross-sectional area of the mixing portion is too large with respect to the flow rate of the liquid to be supplied, the flow velocity of the liquid becomes small, and the turbulence of the liquid flow does not easily occur when the different liquids join, and sufficient mixing cannot be obtained, and the mixing according to the present invention It becomes difficult to exert the effect of the vessel. For this reason, it is generally preferable that the cross-sectional area of the mixing portion of the mixer according to the present invention is about 5 cm ² or less. In the present invention, the cross-sectional area of the mixing portion is the cross-sectional area at the positions shown by S1 and S2 in FIGS. 1 and 2 in the case of the Y-shaped tube and the T-shaped tube. More preferably, the cross-sectional areas S1 and S2 are
The mixer may be designed to have a size of 0.2 cm ² or less.

FIG. 3 shows a cross section in a direction perpendicular to the pipe diameter of a mixer having a double pipe structure as one embodiment of the present invention. The mixer is composed of an inner pipe 4 and an outer pipe 5, and one end of the inner pipe 4 serves as a supply port 2 to supply, for example, a dispersion liquid of insoluble carrier particles. On the other hand, for example, an antibody solution is supplied to the gap between the inner tube 4 and the outer tube 5. Therefore, the supply port 1 may be provided by sealing a part of the outer tube 5. An outlet 3 is provided at the other end of the outer tube 5. At this time, the third
The pipe diameter may be reduced as shown in the figure.

The cross-sectional area of the mixing portion of the above mixer corresponds to S1 and S2 in FIG. 4 showing the cross section in the direction horizontal to the pipe diameter of the above mixer.

Alternatively, a mixer may be used in which a hole for an outlet is provided in the middle of one tube and both ends are supply ports.

Furthermore, three or more supply ports are provided, and two supply ports are used for supplying the insoluble carrier and the solution of the antibody or antigen. From the remaining supply port, for example, a solution of the crosslinking agent or a buffer solution for diluting the immobilized carrier particles may be supplied.

The mixer according to the present invention may have a structure for promoting mixing of a streak plate or the like at its mixing portion.

In the present invention, the liquid supply rate is not limited. or,
Even if different liquids are intermittently supplied from two supply ports, the effect of the present invention can be obtained if the ratio of the amount of insoluble carrier particles to the amount of antibody or antigen supplied in the different liquids is substantially constant. . Generally, by reducing the cross-sectional area of the mixing part and increasing the liquid supply rate, different liquids are sufficiently mixed, so that the agglomeration of the immobilized carrier particles is suppressed.

In the present invention, as a method for supplying the liquid to the mixer, for example, the mixer and the syringe containing each liquid may be connected by a rubber tube or the like, and the liquid may be press-fitted. Further, as a method for taking out the immobilized carrier particles after mixing, it may be introduced to the receiver through a rubber tube or the like from the take-out port. When the receiver side is kept under reduced pressure, it is not necessary to press-fit from the supply side, and supply and mixing can be automatically performed.

In the present invention, the material of the mixer is not particularly limited, but a material containing a component soluble in the liquid used is not preferable. Typical examples that can be suitably used include, for example, polystyrene, polyethylene, polypropylene, polycarbonate, polyvinyl chloride, polyethylene terephthalate, polymethyl methacrylate, Teflon, and other synthetic resins, silicone rubber, urethane rubber, and other synthetic rubbers. , Glass, and metals such as stainless steel and aluminum.

The same materials may be used for the pipes and syringes connected to the mixer.

The shape and dimensions of the inlet, outlet, mixing portion, etc. of the mixer may be determined in consideration of the type, concentration, supply amount, etc. of the insoluble carrier and the antibody or antigen to be mixed.

[Action and effect]

The mixer of the present invention is characterized in that an insoluble carrier dispersion liquid and an antibody or antigen dissolution liquid are supplied from separate supply ports, and both are mixed and then collected from the extraction port.

Comparing the method for producing insoluble carrier particles having the antibody or antigen immobilized according to the present invention with the conventional method, the immobilized carrier particles obtained by the method according to the present invention show less aggregation and the immobilization obtained by repeated production. This is an industrially extremely effective method because the carrier particles have uniform properties and can be produced in large quantities. The present inventors believe that the reason for this is that the two liquids are uniformly mixed in an extremely short time in the confluent portion of both liquids in the mixer. On the other hand, the above 2
The conventional method of adding the other solution to one of the solutions requires more time for the two solutions to be mixed homogeneously as compared with the method of the present invention, resulting in the antibody or antigen to the insoluble carrier particles. The immobilization state becomes uneven for each particle, and particles whose surface is not sufficiently covered with the antibody or antigen are likely to be generated. Since this particle can further immobilize the antibody or antigen, it immobilizes the antibody or antigen immobilized on other particles,
As a result, aggregated particles are easily generated.

Furthermore, it is difficult to repeatedly reproduce the same mixed state, and the characteristics of the immobilized carrier particles are likely to vary from one production to another.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 (a) Preparation of modified human γ-globulin-immobilized latex dispersion Polystyrene latex particles (carrier particles) having an average particle diameter of 0.131 μm were mixed with 0.05 M glycine-sodium hydroxide buffer PH8.5 (hereinafter buffer). The dispersion A) was diluted to prepare a dispersion liquid having a latex concentration of 1% by weight. Then, human γ-globulin fractionated from human serum by salting out treatment,
After dialysis against saline and water, buffer A
It was dialyzed against. The obtained human γ-globulin solution was diluted with buffer A to prepare a protein concentration of 10 mg / ml. Then, the human γ-globulin solution was heated at 60 ° C. for 30 minutes to obtain a denatured human γ-globulin solution.

As a mixer, a T-shaped tube (S1 and S2 are about 0.03 cm ² ) in which glass tubes having an inner diameter of 2 mm are connected in a T-shape was manufactured.

The latex dispersion was sampled with a glass syringe to the position of the 1 ml scale, and was connected to one end of the T-tube using a silicone rubber tube. Similarly, 1 ml of the denatured human γ-globulin solution was collected with a glass syringe and connected to the other end of the T-shaped tube. The connection position is such that the angle between the supply port for the latex dispersion and the modified human γ-globulin solution is 180 °.
° It was arranged so that the angle between the outlet and each supply port was 90 °. The distance from the tip of the syringe to the mixing part of the T-shaped tube was 20 mm. Equal amounts of the latex dispersion and the denatured human γ-globulin solution were supplied to the mixer under the following conditions.

 1 ml was supplied all at once.

 0.5 ml each was supplied in two divided portions.

 Each 0.2 ml was supplied in 5 divided portions.

 0.05 ml was supplied in 20 batches.

No stirring or mixing was performed under any of the conditions. A centrifuge tube made of plastic was placed at the outlet, and the obtained reaction product was collected. Then, after centrifuging to remove the supernatant, buffer A containing 0.05% by weight of bovine serum albumin (hereinafter abbreviated as BSA) was added to the precipitate to redisperse it, and buffer A was further added to concentrate the latex concentration. Was prepared to be 0.05% by weight to obtain a modified human γ-globulin-immobilized latex dispersion (immunological measurement reagent).

(B) Measurement of Absorbance Using an optical cell having an optical path length of 10 mm, the absorbance of the measuring reagent at a wavelength of 600 nm was measured. The absorbance of the various modified human γ-globulin-immobilized latex dispersions is
0.69, 0.70 in the case of conditions, 0.70 in the case of conditions, 0.71 in the case of conditions, and no change in the obtained absorbance was observed even when the mixing conditions were changed. The dispersion prepared by diluting the latex used as the raw material with Buffer A to 0.05% by weight had an absorbance of 0.60.
showed that. By the way, when the absorbance of 0.70 is converted into a particle size, it corresponds to about 0.14 μm.

(C) Evaluation of immunological measurement reagent The above-mentioned reagent on which modified human γ-globulin (antigen) is immobilized as an immunologically active substance can be used as a detection reagent for rheumatoid factor (antibody).

1 drop of this detection reagent was mixed with 1 drop of various rheumatoid factor positive serum or negative serum on a glass plate, and after 10 minutes, the state of aggregation of latex particles was visually judged. The results are shown in Table 1.

In the measuring reagent according to the present invention, no difference in the reagent characteristics depending on the supply conditions was observed.

All reagents showed (-) against negative serum and did not cause non-specific agglutination reaction.

Comparative Example 1 (a) Preparation of modified human γ-globulin-immobilized latex dispersion 1 ml of the same latex dispersion as in Example 1 was collected in a glass test tube. The same modified human γ-globulin solution as in Example 1 was collected with a glass syringe and added to the latex dispersion under the following conditions (1) to (4).

(1) 1 ml was added all at once.

(2) 0.5 ml was added in two divided portions.

(3) 0.2 ml was added in 5 divided portions.

(4) 0.05 ml was added in 20 batches.

When adding in portions, the contents of the test tube were mixed with a test tube mixer for each addition.

Then, after centrifugation to remove the supernatant, a 0.05% by weight modified human γ-globulin-immobilized latex dispersion was prepared in the same manner as in Example 1.

(B) Measurement of absorbance The absorbance of the obtained modified human γ-globulin-immobilized latex dispersion was measured in the same manner as in Example 1. Absorbance changes greatly depending on the addition conditions, and the number of divisions increases with the dispersion obtained under condition (1) being 0.75, condition (2) being 0.81, condition (3) being 1.20 and condition (4) being 1.78. Absorbance increased.

It should be noted that when the absorbance of 1.78 is converted to a particle size, it is approximately 0.24 μm.
In comparison with Example 1, it was shown that aggregated particles are more likely to occur during immobilization.

(C) Evaluation of immunological measurement reagents The above reagents were evaluated in the same manner as in Example 1. The results obtained are shown in Table 2.

As is clear from Table 2, when the mixer according to the present invention was not used, the characteristics of the obtained reagent changed greatly depending on the addition conditions. It was considered that the reagent obtained under the condition (4) in which the number of divisions of addition was largest was agglutinated even to the negative serum, and showed clinically undesirable nonspecific agglutination.

As described above, when the immunoactive substance was added to the carrier particles by using the mixer according to the present invention, no difference in the reagent characteristics depending on the supply conditions was observed. On the other hand, when the immunoactive substance was added to the carrier particles without using a mixer, the reagent characteristics changed significantly depending on the addition conditions.

Further, the reagents were prepared 10 times each by the method of the supply conditions of Example 1 and the addition conditions (1) of Comparative Example 1. The coefficient of variation obtained by dividing the standard deviation value by the average value of the absorbance of the obtained reagent is 1.5% in the case of Example 1 and 5.8% in the case of Comparative Example 1.
Tonatsuta. In the case of Comparative Example 1, it was shown that it is extremely difficult to completely reproduce the addition conditions of the modified human γ-globulin to be added and the diffusion after the addition for each reagent preparation.

Example 2 (a) Preparation of anti-human CRP antibody-immobilized latex dispersion A anti-human CRP antibody was prepared by salting out anti-human CRP serum obtained by immunizing a goat with human C-reactive protein (hereinafter, human CRP). Fractionated.

Then, the anti-human CRP antibody was diluted with 0.05 M ammonium chloride-ammonia buffer (hereinafter buffer B) to reduce the protein concentration.
A 2 mg / ml anti-human CRP antibody solution was prepared.

Example 1 except that buffer B was used in place of buffer A, anti-human CRP antibody was used in place of denatured human γ-globulin, and the following supply conditions (1) and (2) were used instead of the supply conditions (1) to (3). Similarly, an anti-human CRP antibody-sensitized latex dispersion was prepared. The same mixer as in Example 1 was used.

2 ml each of the latex dispersion and the anti-human CRP antibody solution were supplied all at once.

5 ml each of the latex dispersion and the anti-human CRP antibody solution were supplied all at once.

The reaction product was put into a test tube from the outlet, heated to 37 ° C., and allowed to stand for 2 hours. Then, 2 ml of each reaction product was transferred to a centrifuge tube made of plastic and centrifuged to remove the supernatant. To the obtained precipitate, 2 ml of buffer solution B containing BSA at a concentration of 2 mg / ml was added and redispersed. Then, the mixture was heated to 37 ° C and left standing for 2 hours.
By this operation, the above-mentioned BSA was adsorbed on the portion of the carrier particles on which the antibody was not adsorbed to prevent nonspecific aggregation of the carrier particles.

Then, centrifugation was performed again for the purpose of removing unadsorbed antibody and excess BSA remaining in the first centrifugation operation, and the supernatant was removed. Buffer B was added to the obtained precipitate and redispersed to adjust the latex concentration to 0.05% by weight, and anti-human
CRP antibody-immobilized latex dispersion (immunoassay reagent)
Got

(B) Measurement of Absorbance In the same manner as in Example 1, the absorbance of the obtained immobilized latex dispersion was measured. The absorbance obtained was 0.75 for both conditions. When the absorbance of 0.75 was converted into a particle size, it corresponded to about 0.14 μm, and the generation of aggregated particles was obviously suppressed as compared with Comparative Examples described later.

(C) Evaluation of Immunological Assay Reagent The above-mentioned reagent having an anti-human CRP antibody immobilized as an immunologically active substance can be used as a quantitative reagent for human CRP.

The absorbance was measured by a device in which a temperature controller and a magnet type stirring device were attached to the photometric section of a Hitachi U-3200 type self-recording spectrophotometer. 2 ml of the above-mentioned reagent was dispensed into a glass optical cell having an optical path length of 10 mm, a cylindrical stirrer was put therein, and it was inserted into the photometric section and kept at 37 ° C.

Then, 20 μl of the sample was added while stirring the reagent in the container with the stirring device. The measurement of the absorbance was started at the same time as the addition. The absorbance was measured using a light beam having a wavelength of 580 nm. The stirring was stopped 10 seconds after the addition of the sample.
The sample used for measurement was purified human CRP with a human CRP concentration of 240 mg / dl.
The solution was diluted with human CRP-free serum that had been made to be substantially free of human CRP by absorption treatment with human CRP.
The concentration was adjusted to 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 mg / dl.

Of the obtained absorbances, the difference in absorbance for 1 minute, that is, the rate of increase in absorbance, was determined from the absorbances 1 minute and 2 minutes after the addition of the sample. The results are shown in Table 3.

Comparative Example 2 Example 2 was repeated except that the anti-human CRP antibody solution was collected in advance in a test tube instead of using the mixer of Example 2, and the latex dispersion was added all at once with a glass syringe.
An anti-human CRP antibody-immobilized latex dispersion was prepared in the same manner as in.

The conditions for adding the latex dispersion were the following conditions (5) and (6).

(5) 2 ml of the latex dispersion was added all at once to 2 ml of the anti-human CRP antibody solution.

(6) 5 ml of the latex dispersion was added all at once to 5 ml of the anti-human CRP antibody solution.

After the addition, the mixture was mixed with a test tube mixer, heated to 37 ° C., and allowed to stand for 2 hours. Then, the same procedure as in Example 2 is carried out to obtain anti-human CR.
A P-antibody-immobilized latex dispersion was obtained.

(B) Measurement of Absorbance In the same manner as in Example 1, the absorbance of the obtained immobilized latex dispersion was measured. The absorbance obtained is under condition (5)
It was 0.80 and 1.10 under the condition (6). Absorbance 0.80 and 1.10
Is converted to particle size of about 0.15μm and 0.17μm, respectively.
Equivalent to μm.

(C) Evaluation of immunological measurement reagents The above reagents were evaluated in the same manner as in Example 2. The results obtained are shown in Table 4.

The results of evaluation as the immunological measurement reagents of Example 2 and Comparative Example 2 are shown in the figure.

In FIG. 5, the horizontal axis shows the CRP (antigen) concentration in the sample, and the vertical axis shows the rate of increase in absorbance in each reagent. The results of the conditions of Example 2 are indicated by (◯) and (Δ), and the results of conditions (5) and (6) of Comparative Example 2 are indicated by (●) and (▲), respectively.
Indicated by. The plotted points were connected by a straight line to obtain a calibration curve. In the figure, the straight line 1 shows the calibration curve of the conditions of Example 2, and the straight lines 2 and 3 show the conditions (5) of Comparative Example 2, respectively.
The calibration curve of (6) is shown.

As is clear from Tables 3, 4 and 5, when the mixer according to the present invention is used, the characteristics of the immunological measuring reagent obtained do not change depending on the amount of the liquid supplied, but the mixer according to the present invention. When is not used, the characteristics of the immunological measurement reagent greatly change depending on the amount of the immobilized liquid. In the case of the reagents obtained in Comparative Examples, the upper limit of the calibration curve tended to decrease as the amount of liquid to be immobilized increased. In addition, from the results of the above-mentioned absorbance measurement, it was confirmed that when the amount of the liquid to be immobilized increased, the degree of aggregation of the immobilized latex carrier particles increased.

From the above results, in the conventional method, in order to increase the amount of reagent to be obtained, it is not preferable to increase the immobilization amount per time, and it is necessary to increase the number of times of immobilization. On the other hand, the method according to the present invention is characterized in that a reagent having the same performance can be obtained even if the immobilized amount per time is increased.

Example 3 (a) Preparation of anti-human CRP antibody-immobilized latex dispersion A polypropylene Y-tube (S1 of about 0.05 m ² ) with an inner diameter of 2 mm was used as a mixer, and two polypropylene syringes for 25 ml and urethane rubber were used. Connected with the tube. Into the syringe, 25 ml each of the same antibody solution and latex dispersion as in Example 2 were collected in advance, and the mixture was supplied all at once to the mixer, and 50 ml of the reaction product was taken from the outlet into a plastic centrifuge tube. The mixture was heated to ℃ and left standing for 2 hours. Then, 0.01 g of bovine serum albumin (BSA) is added for the purpose of preventing the carrier particles from being pressure-bonded, which tends to occur during high-speed centrifugation, and centrifugation is performed. After removing the supernatant, add 2 mg / m of BSA to the precipitate.
50 ml of Buffer B containing 1 was added and redispersed. Then 37 ° C
The mixture was warmed up and left to stand for 2 hours. After centrifugation again to remove the supernatant, buffer solution B was added and redispersed to adjust the latex concentration to 0.05% by weight.

(B) Measurement of Absorbance The absorbance of the obtained immobilized latex dispersion was measured in the same manner as in Example 1, and it was 0.76, which was in agreement with Example 2.

(C) Evaluation of immunological measurement reagent Evaluation was performed in the same manner as in Example 2. The rate of increase in absorbance is CR
T concentration is 0, 31, 61, 93, 123, 154, 180 (10 ^-3 / mm) for 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 (mg / dl), showing the same reagent characteristics as in Example 2. It was

Example 4 (a) Preparation of anti-human CRP antibody-immobilized latex dispersion A polypropylene Y-shaped tube (S1 of about 1.1 cm ² ) having an inner diameter of 1 cm was used as a mixer and was connected to two separating funnels for use. Into the separating funnel, the same antibody solution and the same latex dispersion as in Example 2 were collected in advance, one by one, and supplied all at once while applying nitrogen pressure to the separating funnel. The obtained immobilized latex dispersion was dispensed into a 50 ml plastic centrifuge tube, and an anti-human CRP antibody-immobilized latex dispersion was obtained in the same manner as in Example 3.

(B) and (c) Measurement of absorbance and evaluation of immunological measurement reagent When the absorbance was measured in the same manner as in Example 1, the absorbance of the obtained reagent was 0.76, which was consistent with that of Examples 2 and 3.

The immunological measurement reagents were evaluated in the same manner as in Example 2.
The rate of increase in absorbance was 0,1.0,2.0,3.0,4.0,5.0,6.0 when the CRP concentration was
(Mg / dl) 0, 30, 60, 92, 121, 150, 178, (10
^-3 / mm), showing the same reagent characteristics as in Example 2.

Example 5 (a) Synthesis of carrier particles forming covalent bond 95 g of glycidyl methacrylate (hereinafter GMA) having highly reactive epoxy group in the side chain, 3 g of methacrylic acid (hereinafter MA),
2g of ethylene glycol dimethacrylate (hereinafter referred to as EGDM) was subjected to soap-free emulsion polymerization in an aqueous medium at 70 ° C together with 1.4l of water and 0.4g of potassium persulfate to obtain latex particles having an average particle diameter of 0.25µm. After the latex particles were purified by dialysis, etc., they were reacted with ε-aminocaproic acid, which reacts only with the epoxy groups in the surface layer, and the epoxy groups in the surface layer were quantified to show 1.4 × 10 ^-4 mol / g-latex. It was Next, 50 ml of 5% latex dispersion and diethylenetriamine
After mixing with 50 ml of a 2 mol / l aqueous solution, the mixture was purified by dialysis or the like to obtain aminated latex particles. Results of nitrogen analysis 4.5 × 10 ^-4 mol
Nitrogen in / g-latex was detected. When the amount of reacted diethylenetriamine was calculated from this nitrogen amount, it corresponded to 1.07 times the epoxy group concentration of the surface layer.

(B) Immobilization of immunoactive substance by formation of covalent bond The above aminated latex dispersion was diluted with 0.015 mol / l of phosphate buffer PH = 7.4 (hereinafter abbreviated as buffer C) to prepare 1% by weight. did. Glutaraldehyde aqueous solution (70% by weight)
Was diluted with water to prepare 7 × 10 ⁻³ mol / l. Rabbit IgG obtained by purifying normal rabbit serum by column treatment or the like was diluted with buffer C to prepare 1 mg / ml. A glass tube with an inner diameter of 2 mm was combined in a cross shape to produce a mixer. Then, 25 ml of the aminated latex dispersion prepared above, 25 ml of an aqueous solution of glutaraldehyde, and 5 ml of an aqueous solution of rabbit IgG were collected into three glass syringes, and the mixer and each syringe were connected with a silicone rubber tube.

The above liquids were supplied for 10 seconds at a constant supply rate for each liquid. The obtained immobilized carrier particles are dispersion stable,
Rabbit IgG was immobilized at 60 mg / g.

Comparative Example 3 25 ml of the aminated latex dispersion prepared in Example 4 was placed in a beaker and stirred with a magnetic stirrer while the above Example 4 was used.
25 ml of the aqueous solution of glutaraldehyde prepared in 1. was added at a constant rate for 10 seconds. After the addition was completed, the latex particles aggregated to form a precipitate. Further, 5 ml of the bovine IgG solution prepared in Example 4 was added, but the obtained rabbit IgG-immobilized carrier particles formed aggregates and were unsuitable as immunological measurement reagents.

[Brief description of drawings]

FIG. 1 shows a cross section of a mixer in which Y-shaped tubes are combined, which is one embodiment of the mixer of the present invention. It is composed of supply ports 1 and 2 and an outlet 3. S1 and S2 indicate cross-sectional areas of the mixing portion at the positions shown in the figure. FIG. 2 shows a cross section of a mixer in which tubes are combined in a T shape, which is one embodiment of the mixer of the present invention. It is composed of supply ports 1 and 2 and an outlet 3. S1 and S2 indicate cross-sectional areas of the mixing portion at the positions shown in the figure. FIG. 3 shows a vertical cross section of a mixer having a double pipe structure composed of an inner pipe 4 and an outer pipe 5, which is one embodiment of the mixer of the present invention. It is composed of supply ports 1 and 2 and an outlet 3. FIG. 4 shows a horizontal cross section at the AA 'portion of the double pipe shown in FIG. 3, and S1 and S2 show cross sectional areas of the mixing portion at the positions shown in the drawing. In FIG. 5, the horizontal axis represents the concentration of human CRP (antigen) in the subject, and the vertical axis represents the wavelength 58 during the agglutination reaction of the latex dispersion (immunological measurement reagent) on which anti-human CRP antibody was immobilized.
The amount of increase in absorbance at 0 nm for 1 minute, that is, the rate of increase in absorbance is shown. In the figure, (◯) and (Δ) show the case of the embodiment using the mixer of the present invention (Example 2), and the straight line 1 shows the calibration curve. Figures (●) and (▲) show the case of using the conventional method without a mixer (Comparative Example 2).
Indicates each calibration curve.

Claims (2)

[Claims] 1. At least a liquid flow containing an antibody or an antigen and a suspension flow containing an insoluble carrier particle so that the ratio of the amount of the antibody or antigen per unit time to the amount of the insoluble carrier particle becomes constant. A method for producing insoluble carrier particles on which an antibody or an antigen is immobilized, characterized in that the two liquid streams are combined under the condition that mixing is instantaneously achieved. 2. A suspension containing insoluble carrier particles, which is prepared by using a mixer having a plurality of supply ports and a single outlet when producing the insoluble carrier particles having the antibody or the antigen according to claim 1 immobilized thereon. And an antibody or an antigen-containing liquid are separately supplied from the supply ports, and both are instantaneously mixed to obtain a uniform flow, which is then collected from the extraction port of the insoluble carrier particles having the antibody or antigen immobilized thereon. Production method.