JPH0875746A - Evaluation of agglomeration characteristics of carrier particles for immunological agglomeration reaction - Google Patents

Abstract

PURPOSE: To evaluate the agglomeration characteristics of carrier particles having a high sedimentation speed by premixing standard carrier particles and an agglomerating agent with water in various ratios to disperse them and together using the electric conductivities of respective mixed solns. and agglomeration decision by a microtiter method to calculate the lowest electric conductivity generating agglomeration. CONSTITUTION: As a standard carrier, org. and inorg. composite particles for microtitration are used to be well washed with pure water. Next, an agglomerating agent aq. soln. is prepared as an agglomerating agent by dissolving sodium chloride in pure water, for example, in an amt. of 20wt.% and mixed with the standard carrier particle dispersion in various ratios under stirring and the electric conductivities of the respective mixed dispersions are measured. The respective mixed dispersions after the completion of measurement are transferred to microwells to be allowed to stand and the presence of the agglomeration images of the bottoms of the microwells is observed and the lowest electric conductivity (agglomeration electric conductivity) generating agglomeration is calculated to be compared with the electric conductivity of a carrier to be evaluated to evaluate whether agglomeration is good.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for evaluating the agglutination characteristics of carrier particles used as a carrier for immunological agglutination reagents. In particular, it is useful for a method of evaluating the agglomeration characteristics of carrier particles having a large sedimentation rate, that is, carrier particles having a large particle size or a high specific gravity, which are used as a carrier for the microtiter method.

[0002]

2. Description of the Related Art In recent years, it has been widely practiced to measure biological components secreted in blood or urine for diagnosing diseases and determining prognosis. For example, viral infections such as hepatitis virus, AIDS, adult T-cell leukemia, rubella,
Examples include autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and Hashimoto's disease, candida, mycoplasma, streptococcus, bacterial infections such as syphilis, hormone tests for insulin, human chorionic gonadotropin, estrogen, and other tumor marker tests. To be

[0003] Diagnosis of these diseases is extremely difficult due to a small amount of biological components to be tested by conventional chemical analysis, and immunological tests using antigen-antibody reaction are generally performed. The most important feature of this test method is that biological components secreted in blood or urine can be measured with extremely high sensitivity and specificity.

An immunological agglutination reaction method is one of the immunological tests. Various particles such as animal red blood cells, bacterial cells, polystyrene latex particles, gelatin particles, inorganic substance particles, and organic-inorganic composite particles are used as carriers. The immunological agglutination reaction method is a method of mixing a reagent prepared by immobilizing an antigen or an antibody on carrier particles used as a carrier with a target sample, and aggregating carrier particles based on the resulting antigen-antibody reaction. It is a method of analyzing biological components in a target sample by measuring the size or strength of an object.

In using the reagent of this immunological agglutination method, it is extremely important to use particles having good agglutination characteristics as a carrier. That is, if particles with poor performance are used, the performance of the finally obtained reagent, for example, sensitivity, specificity, storage stability, or aesthetics of agglutination images will also be poor. In particular, a non-specific agglutination reaction may occur, resulting in an erroneous diagnosis, or the target sensitivity may not be reached, making it impossible to perform an accurate test and causing a very serious defect.

Therefore, in order to obtain a reagent for the immunological agglutination reaction method with good performance, it is extremely important to secure a homogeneous carrier having excellent agglutination properties and not different among lots. For that reason, carrier particles used as a carrier It is desired to construct an analytical means for accurately evaluating the performance of the above, especially the aggregation characteristics.

Conventionally, as a method for evaluating carrier particles, a measuring method such as a zeta potential for examining electrical characteristics or a contact angle for evaluating affinity with water is known. However, these conventional evaluation methods are not satisfactory in measuring carrier particles of the immunological agglutination reaction method with good reproducibility, and the carrier particle characteristics obtained from the measurement results are directly reflected in the reagent performance. However, there were still some issues to be improved.

[0008]

Of the immunoaggregation reaction methods, it is used in the case of the sedimentation agglutination reaction method (represented by the microtitre method) for measuring the image of tube bottom agglutination generated in microwells or test tubes. It has been extremely difficult to correctly evaluate the aggregation characteristics of carrier particles. This is because the carrier particles used have a large particle size or a high specific gravity and are prone to sedimentation, and it is because accurate measurement is extremely difficult.

Generally, a phenomenon in which particles agglomerate when a large amount of salt is added to a solution in which particles are dispersed is known as salting out. It is said that the addition of salt reduces the repulsive force between particles by canceling the surface charge of particles, and the dehydration of solvated water on the surface of particles reduces the dispersibility of particles in a solution, resulting in agglomeration of particles. There is. The lowest salt concentration at which this particle aggregation occurs is generally referred to as salt aggregation concentration.

Conventionally, the salt aggregation concentration has been measured by measuring the change in particle aggregation rate or turbidity using a spectrophotometer, and by tracking the change in particle sedimentation rate or sediment volume. However, in the case of a method using a spectrophotometer, it is difficult to agglomerate particles between particles having a large particle diameter or high specific gravity, and it is difficult to accurately capture the change in absorbance with the phenomenon of particle sedimentation.
It is not easy to obtain results with good reproducibility. Further, also in the case of measuring the change of the sedimentation velocity or the sedimentation volume, there is a problem that the operation is complicated, the measurement takes time, the analysis is difficult, and a special and expensive measuring device is required.

[0011]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to solve the above-mentioned problems, and as a result of verifying carrier particles having excellent agglomeration characteristics, The inventors have found that the electrical conductivity of the suspension in which the carrier particles are dispersed is involved, and completed the present invention.

That is, the present invention relates to a method for evaluating the agglutination characteristics of carrier particles for immunological agglutination reaction, in which standard carrier particles and aggregating agent are dispersed and mixed in water in various proportions in advance, and each mixture solution obtained. By measuring the electrical conductivity of and by determining the aggregation state of each mixed solution by the microtiter method,
The minimum electrical conductivity at which agglomeration occurs (aggregative electrical conductivity) is determined, and then the aggregative electrical conductivity is similarly examined using new carrier particles, and the aggregative properties of the new carrier particles are compared by comparing both agglomerative electrical conductivities. Is a method for evaluating aggregation characteristics of the carrier particles for immunological agglutination reaction.

The present invention will be described below, but the feature of the present invention lies in the evaluation of the agglutination property of carrier particles for immunological agglutination reaction, which is a combination of the measurement of electric conductivity and the microtiter method. Furthermore, it is characterized by an inexpensive evaluation method that can accurately measure the agglutination characteristics of carrier particles for immunological agglutination reaction, and that is simple, rapid, and does not require a large apparatus.

The electrical conductivity referred to in the present invention is defined by the reciprocal of the electrical resistance and its unit is S / cm, as described in the Electrochemical Handbook (4th edition, edited by The Electrochemical Society, Maruzen).
(= 1 / ohm). In general, the electrical resistance of a solution varies depending on the dissolved chemical species, and the rate of change of the electrical resistance varies, so it cannot be generally stated, but it varies depending on the amount of dissolved ions. The electric conductivity also changes depending on the ion concentration in the solution, the solution composition, or the solution temperature.

A widely known method is adopted for measuring the electric conductivity, and for example, a commercially available electric conductivity meter composed of a platinum electrode, a Wheatstone bridge and the like is used.
Preferably, an electric conductivity meter with automatic temperature compensation based on the gold electrode method is used because it can measure electric conductivity easily and accurately.

By the way, it has been found that when the coagulant concentration is simply used as an index instead of the electric conductivity, the tube bottom agglomeration image causes a great variation even if the coagulant concentration is intended to be constant.
Since the variation difference greatly exceeds the generally accepted error in the microtiter method (for example, if diluting by a factor of 2, the difference between the microwells is within 1 tube), and accurate measurement is extremely difficult. . On the other hand, the results of the tube bottom agglomeration image and the electrical conductivity correlate well, and therefore, as in the present invention, the electrical conductivity may be used as an index to evaluate the agglomeration characteristics of the carrier particles having excellent reproducibility. I understood.

The microtiter method referred to in the present invention is a type of immunoserologic measurement method based on the precipitation-aggregation reaction of carrier particles based on the principle of antigen-antibody reaction, which is a widely known measurement technique in medical tests. Is. It is especially used for mass screening of bacterial and viral infectious diseases because it is a highly sensitive measurement method that is easy to operate and does not require special equipment. The normal operation of the microtiter method is to mix a reagent and a sample in a reaction vessel to cause a sedimentation agglutination reaction, and visually observe the resulting tube bottom agglutination image or by a machine (a commonly used method is image processing). Semi-quantitatively determine the test substance in the sample by evaluating and measuring. This tube bottom agglomeration image is formed by a subtle rolling phenomenon due to electrostatic surface interaction between the bottom of the inclined reaction vessel and the carrier particles.

In the present invention, standard carrier particles or carrier particles to be evaluated and an aggregating agent are dispersed and mixed in water at various ratios, and the tube bottom agglomeration state of the mixed solution in the reaction vessel is determined.

This reaction vessel generally has a V-shaped or U-shaped bottom surface in order to efficiently cause the sedimentation and aggregation reaction. Usually a polystyrene resin microwell (for example, a 96-well microtiter plate),
Alternatively, glass test tubes are widely used.

The carrier particles for immunological agglutination reaction of the present invention may be particles which are insoluble in water and generally used in immunological agglutination reaction method. Preferred are particles having a particle size or specific gravity that causes spontaneous precipitation in water. The average particle diameter is not particularly limited, but is 0.0
5 μm to 200 μm, preferably 0.1 μm to 10 μm
Selected from the range of. When the average particle size is smaller than 0.1 μm, the sedimentation speed is low, and therefore a very long measurement time is required, which impairs the feature of the present invention of rapid measurement. On the other hand, when the average particle diameter is larger than 10 μm, the sedimentation speed is too fast, and the tube bottom agglomeration image resulting from the sedimentation agglutination reaction becomes poor, and accurate measurement may not be possible.

The specific gravity of the carrier particles for immunological agglutination reaction is from 1.001 g / cm 2 when the specific gravity is measured in water.
50 g / cm, preferably 1.01 g / cm to 10 g /
It is selected from the range of cm. When the specific gravity is less than 1.01 g / cm, there is a drawback that the measuring time is long because the sedimentation speed is low. If the specific gravity exceeds 10 g / cm, the sedimentation speed is too fast, and the tube bottom agglomeration image resulting from the sedimentation agglutination reaction tends to be poor.

Carrier particles for immunological agglutination reaction which satisfy these requirements are, for example, erythrocytes of animals, bacterial cells of bacteria,
Polystyrene latex particles, gelatin particles, inorganic substance particles, organic-inorganic composite particles (trade name "Immunoicles
"HDP", manufactured by Tokuyama Co., Ltd., and the like. In general, these carrier particles are often stored in water, a buffer solution, or an organic solvent containing a stabilizer such as a preservative or a surfactant after synthesis. In that case, before measurement,
The electrical conductivity of a suspension of carrier particles composed of the water and carrier particles (25
There is no problem as long as it is confirmed that (C) is 0.06 μS / cm or less.

As the standard carrier particles of the present invention, the above carrier particles for immunological agglutination reaction are used without any limitation. The carrier particles for immunological agglutination reaction, which are preferably used as the carrier particles for the reagent for immunological agglutination reaction of the microtiter method, are preferable as long as they have stable performance and can be continuously obtained. Specifically, carrier particles that can be stably obtained, for example, commercially available artificial carrier particles such as organic-inorganic composite particles (manufactured by Tokuyama Corporation, Immunotex HDP) or polystyrene latex particles can be preferably used.

The mixed solution of the present invention is obtained by dispersing and mixing carrier particles and an aggregating agent in water at various ratios.
Water as used herein means a chemical formula H ₂ O having a chemical weight of 1 and a molecular weight of 1 described in the Chemical Handbook (Basic Edition I, The Chemical Society of Japan, Maruzen).
It is a colorless and transparent liquid which is 8.02, tasteless, odorless and colorless. Preferably A. S. T. M. (The America
nSociety for Testing and
Materials that meet the water quality standards set forth in Materials) may be used. That is, the electric conductivity (25 ° C.) is 0.06 μS / cm or less, and the specific resistance (25 ° C.) is 16.6.
Pure water, distilled water or ion-exchanged water of MΩ / cm or more is desirable.

The concentration of carrier particles in the mixed solution is usually 10% by weight to 0.01% by weight, preferably 2.5% by weight to 0.05% by weight with respect to water. When it is more than 2.5% by weight, the number of carrier particles is large and it becomes difficult to stably obtain the tube bottom aggregation image obtained by sedimentation aggregation. In addition, a considerably large amount of coagulant is required to change the tube bottom agglomeration image. on the other hand,
If the concentration of the carrier particles is lower than 0.05% by weight, the carrier particles agglomerate rapidly due to a slight change in the electrical conductivity, and it is difficult to control the sedimentation and agglomeration and the reproducibility tends to deteriorate. In addition, since the number of carrier particles is small, the tube bottom agglomeration image is not clear, and its determination tends to be difficult.

The aggregating agent used in the present invention is a substance which is soluble in water and does not react with water, and which dissociates into ions to cause a change in electrical conductivity, and is not limited as long as it causes agglomeration of carrier particles. . Examples of flocculants are electrolyte substances that decompose into ions when dissolved in water, and include sodium hydroxide, calcium oxide, sodium chloride, ammonium chloride, sodium nitrate, aluminum sulfate, sodium aluminate, aluminum chloride, and primary sulfate. Examples thereof include inorganic salts such as iron, ferric sulfate and ferric chloride, organic salts such as sodium glutamate and sodium aspartate, and polymer electrolytes such as sodium polyacrylate and polyacrylamide. Further, these electrolyte substances may be used in a mixture of two or more kinds.

As the coagulant, sodium chloride is preferred, which is easy to control the coagulation of carrier particles.
Electrolytes with valent cations are desirable. The reason is 2
When an electrolyte having ions with a valence or more is used, the force for aggregating the carrier particles is very strong and it may be difficult to control the agglomeration. Also, among high molecular weight polyelectrolytes,
In some cases, the solution viscosity of the carrier particle dispersion liquid is increased and the tube bottom agglomeration image is disturbed to make accurate measurement impossible.

The method for preparing the mixed solution in the present invention is not particularly limited, and water, a flocculant and carrier particles may be mixed.
Preferably, a method of preparing carrier particle dispersion liquid in which carrier particles are previously dispersed in water and a coagulant aqueous solution in which a coagulant is dissolved in water separately, and mixing the necessary amounts of both are operationally convenient. . Further, in order to improve the mixing efficiency and make the electric conductivity of the mixed solution quickly constant, it is desirable to stir the mixed solution with a stirrer or the like.

The concentration of the coagulant in the mixed liquid of the present invention is usually 50% by weight to 0.0002% by weight, preferably 10% by weight to 0.001% by weight, based on water. If it is more than 10% by weight, the carrier particles agglomerate rapidly and the sedimentation and agglomeration cannot be easily controlled, resulting in poor reproducibility. on the other hand,
If it is less than 0.001% by weight, the agglomeration of carrier particles becomes extremely weak, and the tube bottom agglomeration image obtained as a result of sedimentation agglomeration tends to be poor, and discrimination tends to be difficult.

The mixing ratio (weight ratio) of the carrier particles and the aggregating agent in the mixed liquid is usually 100 to 0.01, preferably 20 to.
It is set to 0.05. If the mixing ratio exceeds 20, aggregation tends to be weak and the tube bottom agglomeration image tends to be poor, making discrimination difficult. On the other hand, if it is less than 0.05, agglomeration of carrier particles may occur rapidly, making control difficult and making accurate measurement difficult.

The evaluation method of the present invention will be specifically described below as a typical method. First, an operation of determining the lowest electric conductivity at which the agglomeration of standard carrier particles occurs, that is, the agglomerated electric conductivity is performed. The purpose of this operation is to know the aggregation electric conductivity which is an index in evaluating the aggregation characteristics. It is also to confirm that the operation is always performed correctly by measuring the agglomerated electric conductivity of the standard carrier particles.

The standard carrier particles are arbitrarily selected, but there is no limitation even if two or more kinds are selected for detailed examination. The standard carrier particles are thoroughly washed with water and then redispersed in water to prepare a standard carrier particle dispersion having a predetermined concentration. on the other hand,
A flocculant selected from a specific electrolyte substance is dissolved in water to prepare an aqueous flocculant solution.

Next, the two solutions are mixed to prepare a mixed solution containing standard carrier particles and an aggregating agent. For example, a beaker containing a stirrer is charged with an arbitrary amount of water in advance, and a predetermined amount of the standard carrier particle dispersion is added to the beaker and mixed with stirring. The concentration of the standard carrier particles at this time may be within the above range. Then, a predetermined weight of an aqueous coagulant solution is added to this beaker with a pipette and mixed well by stirring to finally obtain a mixed solution of standard carrier particles and a coagulant. Then, the electric conductivity of this mixed liquid is measured using an electric conductivity measuring device (CM-11P manufactured by Toa Denpa Kogyo Co., Ltd.). At this time, the concentration of the aggregating agent and the mixing ratio of the standard carrier particles and the aggregating agent may be within the above range.

The method for preparing the mixed solution is not limited at all.
For example, using one container to fix the amount of standard carrier particles,
A method of changing both mixing ratios of the standard carrier particles and the aggregating agent may be performed by sequentially adding the aggregating agent. Alternatively, a separate container may be used to prepare a series of mixed liquid systems in which the mixing ratio of the standard carrier particles and the aggregating agent is changed. On the contrary, a method of fixing the amount of the aggregating agent and changing the amount of the standard carrier particles can be adopted.

Next, the microtiter method is used to obtain a tube bottom aggregation image of the mixed solution. A predetermined volume of the mixed solution is sampled with a dispenser such as a micropipette and added into a reaction container (for example, a microwell of a 96-well V-type microtiter plate) prepared in advance. After the reaction container is left standing and a certain reaction time has elapsed, the tube bottom aggregation image formed in the reaction container is determined. There is no limitation on the method of determining the tube bottom agglutination image, and a method widely used by the microtiter method may be generally adopted. In other words, whether the tube bottom agglutination image for each microwell is an agglutination image (a spot does not form with an image that spreads over the entire area) based on the preset criteria for the tube bottom agglutination image, or It is evaluated whether the image is an aggregated image (a spot is formed).
Then, the lowest electric conductivity of the mixed solution corresponding to the microwell in which the tube bottom agglutination image shows an agglutination image is determined as the agglomeration electric conductivity of the standard carrier particles.

The tube bottom agglomeration image may be evaluated by the naked eye, mechanical image processing, or the like without any limitation. In order to obtain a faster result, in order to carry out the sedimentation-aggregation reaction in a short time, for example, centrifugation or the like may be performed to increase the sedimentation speed of the standard carrier particles.

Following the above-described series of operations for determining the cohesive electrical conductivity of the standard carrier particles, an operation for determining the cohesive electrical conductivity of the carrier particles to be evaluated is carried out. All you have to do is perform the operation. Finally, by comparing the agglomeration electric conductivities of the measurement carrier particles and the standard carrier particles, the agglomeration characteristics of the carrier particles to be measured can be evaluated. However, it is necessary to compare the standard carrier particles and the carrier particles to be evaluated using the same kind of particles. Therefore, if the carrier particles to be evaluated are changed, it is necessary to re-measure the agglomerated electric conductivity using standard carrier particles of the same type.

In the comparison and evaluation, for example, the agglomeration characteristics of the carrier particles to be evaluated and the standard carrier particles are compared to determine their agglomeration characteristics as follows. If the difference in the coagulation electric conductivity of the standard carrier particles is usually within 0.5 μS / cm, it is evaluated that the standard carrier particles have the same coagulation characteristics. Then, if the value is higher than the coagulation electrical conductivity of the standard carrier particles, it can be seen that the coagulation characteristics of the target carrier particles are superior to the standard carrier particles in dispersion stability, and conversely if the value is low, the dispersion stability is Can be judged to be inferior.

Further, in order to have excellent agglutination properties as carrier particles for a reagent for the microtiter method, it is preferable that the agglomeration electric conductivity is in the range of 1 μS / cm to 30 μS / cm, and the microtiter method is based on this criterion. It can be evaluated whether or not the carrier can be suitably used as a carrier. When the agglutination electric conductivity is lower than 1 μS / cm, the carrier particles are very rich in dispersion stability, and the tube bottom agglutination image shows a phenomenon of almost no agglutination, resulting in extremely low sensitivity of the microtiter reagent. On the other hand, in the case of carrier particles exceeding 30 μS / cm,
The dispersion stability is very poor, the hydrophobicity is strong, and it becomes difficult to handle the carrier particles, and the tube bottom agglutination image of the microtiter reagent is also very poor, which causes a considerable amount of nonspecific agglutination. Values for this preferred cohesive electrical conductivity range are:
Mixing ratio of carrier particles and aggregating agent in mixture (weight ratio)
If the above condition is satisfied, the condition is constant without being affected by the conditions such as the type of carrier particles and the type of aggregating agent.

The present invention can exert a particularly remarkable effect in the evaluation of the agglomeration characteristics of carrier particles used as a carrier in the microtitre method based on the principle of sedimentation agglomeration, but other known immunological agglutination reactions such as qualitative It can be used without any limitation for the evaluation of carrier particles for immunological agglutination reaction used in the flat plate method as a quantitative test method or the turbidimetric method / scattering method as a quantitative test method and a particle measurement method.

As the biological substance to be immobilized on the carrier particles in the immunological agglutination reaction using the carrier particles selected by the evaluation method of the present invention, known substances are used without particular limitation. Preferably, immunologically active substances such as antigens, haptens and antibodies obtained by immunizing these are used. For example, immunoglobulin, albumin, fibrinogen, α-fetoprotein, C-reactive protein, β2-
Microglobulin, myoglobin, carcinoembryonic antigen, hepatitis virus antigen, human chorionic gonadotropin, human placental lactogen, insulin, digoxin, protein A, protein G, DNA, RNA and other proteins, hormones, drugs, etc., and their immunity The antibody etc. obtained in.

As a method for immobilizing the biological substance on the carrier particles, a known immobilization method may be adopted. For example, it is preferable to bring the above-mentioned protein into contact with the carrier particles in an aqueous medium, and generally the mixture is allowed to stand after mixing, but it may be stirred or shaken to enhance the contact efficiency. After the contact, the protein in the supernatant may be removed by centrifugation and washing with an aqueous medium if necessary.

[0043]

According to the evaluation method of the present invention thus obtained, the agglutination characteristics of carrier particles for immunological agglutination reaction can be evaluated accurately and reproducibly, and further the agglutination characteristics affect the performance of immunological agglutination reagent. It has characteristics that reflect it very well. In particular, it is useful as a method for evaluating the agglutination characteristics of carrier particles for immunological agglutination reaction, which have a high sedimentation rate and are used in a microtiter method based on sedimentation agglomeration.

Although the reason for this is not always clear, attention was paid to the measurement of the cohesive electrical conductivity of the mixed solution of the carrier particles and the coagulant in place of the salt coagulation concentration, and the reaction actually used in the microtiter method. Adopting a method that examines the agglutination characteristics of carrier particles in a container, i.e., a microwell, makes it possible to evaluate the agglutination characteristics of carrier particles for immunological agglutination reactions more accurately and with better reproducibility than conventional methods. It is considered that the reason is.

Further, the present invention has an excellent feature in that it can measure various carrier particles for immunological agglutination reaction at the same time, and the amount to be used for the measurement can be extremely small, which is simple and inexpensive.

[0046]

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

Example 1 1) Measurement of Aggregate Electrical Conductivity Using Standard Carrier Particles A. Preparation of Standard Carrier Particle Dispersion and Flocculant Aqueous Solution As the standard carrier particles, organic-inorganic composite particles manufactured as carrier particles for the microtiter method (hereinafter, standard A: Immunotakes HDP, manufactured by Tokuyama Corporation, average particle The diameter was 1.8 μm and the specific gravity was 2.0). This standard carrier particle is a carrier particle used in a commercially available rheumatoid factor assay reagent (trade name: Immunotakes RF).

First, the standard carrier particles are thoroughly washed with pure water having an electric conductivity (25 ° C.) of 0.03 μS / cm obtained by Millipore's ultrapure water device Milly Q Lab. Washing by centrifugation was repeated until the electric conductivity of the supernatant became 0.03 μS / cm or less, and the standard carrier particles were dispersed in pure water so that the final concentration of the standard carrier particles was 5% by weight. A liquid was prepared.

On the other hand, sodium chloride (Wako Pure Chemical Industries, Ltd. special grade reagent) which is an electrolyte substance was used as the coagulant.
A 20% by weight coagulant aqueous solution was prepared by dissolving 10 g of sodium chloride in 40 g of pure water (electrical conductivity: 0.03 μS / cm).

B. Preparation of mixed solution of standard carrier particles and aggregating agent, and measurement of electric conductivity 28.5 g of 0.03 µS / cm pure water was added to a polypropylene resin 100 mL beaker containing a stirrer. . To this beaker, 1.5 g of the above 5 wt% standard carrier particle dispersion is added and stirred at 1,000 RPM. Next, 180 μg of the flocculant aqueous solution was added to the beaker with a pipette and rapidly stirred at 1,000 RPM to prepare a mixed liquid of standard carrier particles and the flocculant, and then an electric conductivity measuring device (Toa Denpa Kogyo Co., Ltd. The electric conductivity of the obtained mixed liquid was measured using a company CM-11P). The electric conductivity at this time was 2 μS / cm. Also,
The concentration of standard carrier particles and coagulant is 0.25 wt% each
And 0.12% by weight, and the mixing ratio of the standard carrier particles and the aggregating agent is 2.1 times.

C. Sedimentation and aggregation reaction of the mixed solution A 96-well V-type microtiter plate (Immunotails plate V manufactured by Tokuyama Corporation) was prepared in advance. In this microwell, B. 50 μL of the mixed solution obtained in step 1 was dropped with a micropipette. Simultaneous trials were performed in 3 wells to study reproducibility. The 96-well V-type microplate was covered with a lid to prevent water evaporation, and allowed to stand in a horizontal place so that sedimentation and aggregation were correctly performed. Furthermore, a series of these B. And C. This operation was repeated 12 times. Table 1 shows the weight concentration and electric conductivity of the coagulant corresponding to the cumulative weight of the coagulant aqueous solution.

D. Determination of cohesive electrical conductivity C. The tube bottom agglutination image of the 96-well V-type microtiter plate obtained by the above operation was observed with the naked eye after 40 minutes, and the agglutination image (a spread image without spots)
And a non-aggregation image (a spot is formed) was determined.
The lowest electric conductivity of the mixed solution corresponding to the microwell in which the tube bottom agglutination image showed an agglutination image was taken as the agglomeration electric conductivity of the standard carrier particles. However, when there was a difference between the three wells for the purpose of examining reproducibility, it was concluded that agglutination was imaged in two wells and non-aggregation was observed in two wells.

That is, from the relationship between the electric conductivity shown in Table 1 and the tube bottom agglomeration image result, the agglomeration electric conductivity of the standard carrier particles was determined to be 8 μS / cm.

[0054]

[Table 1]

2) Measurement of Aggregation Electric Conductivity of Various Carrier Particles and Evaluation of Aggregation Properties Six kinds of carrier particles (HDP1,
2, 3, 4, 5, and 6: organic-inorganic composite particles manufactured by Tokuyama Corporation, average particle diameter is 1.8 μm, specific gravity is 2 g / c
m, each surface treatment is different)
The above A. , B. , C.I. , And D. It was decided according to the operation of. The results are shown in Table 2. An immunological agglutination reagent was prepared using these carrier particles and a performance test was conducted.

E. Preparation and performance test of immunological agglutination reagent Human Cohn FII fraction (manufactured by Sigma) is 1/150 M
Dissolve in 10mg / ml of phosphate buffered saline (pH = 7.2, hereinafter abbreviated as PBS) to give 1 mg at 60 ° C.
Heat-denatured γ-globulin was obtained by heating for 0 minutes. This heat-denatured γ-globulin was diluted 50 times with PBS, 1 ml of an antigen solution, and 1 ml of a carrier solution, in which carrier particles were diluted with PBS to 1% by weight, were sensitized for 2 hours while stirring at room temperature. The supernatant is then centrifuged and the unbound heat-denatured γ-of the supernatant is
2m containing 0.5% by weight rabbit serum, excluding globulin
An immunological agglutination reagent for rheumatoid factor detection was prepared by redispersion in 1 PBS.

The following operations were carried out to examine the performance of the immunological agglutination reagent thus obtained. A rheumatoid patient serum diluted solution is prepared using a stock solution obtained by diluting a pooled serum of a rheumatic patient 20 times with PBS. A 96-well V-type microtiter plate (Immunotails plate V manufactured by Tokuyama Corp.) was prepared for carrying out the antigen-antibody reaction, and 25 μl of the rheumatism patient serum diluted solution was added dropwise to each microwell. 25 μl of the immunological agglutination reagent thus prepared was added thereto, and the mixture was shaken for 5 minutes and allowed to stand at room temperature. The tube bottom agglutination image based on the antigen-antibody reaction was visually observed to examine the performance of the immunological agglutination reagent. The results are shown in Table 2.

[0058]

[Table 2]

Comparative Example 1 With respect to the three types of carrier particles of the standard A, HDP1 and HDP6 of Example 1, the concentration of the aggregating agent in the lowest mixed liquid at which the tube bottom agglomeration image gives an agglutination is examined, not the agglomeration electric conductivity. It was The operation is the same as in Example 1 except that the electric conductivity is not measured.

The results are shown in Table 3. From this, it was found that the concentration of the aggregating agent was not reproducible and the aggregating property of the carrier particles could not be evaluated.

[0061]

[Table 3]

[0062]

Claims (1)

[Claims] 1. A method for evaluating the aggregating property of carrier particles for immunological agglutination reaction, wherein standard carrier particles and aggregating agent are previously dispersed and mixed in water at various ratios, and the electrical conductivity of each resulting mixed liquid is obtained. The minimum electrical conductivity at which aggregation occurs (aggregation electrical conductivity) is determined by measuring the degree of aggregation and determining the aggregation state of each mixed solution by the microtiter method, and then using the new carrier particles, the aggregation electrical conductivity is similarly measured. The method for evaluating the agglutination property of carrier particles for immunological agglutination reaction, characterized in that the agglutination property of new carrier particles is judged by comparing the two agglutination electric conductivities.