JPH06130060A - Method for increasing specific gravity of particles for particle immunoassay - Google Patents

Abstract

(57) [Summary] [Structure] Particles for immunoassay, such as animal red blood cells and artificial carriers, are treated with a metal salt solution to prepare high specific gravity particles. [Effects] When the highly-densified particles obtained in the present invention are used in a measurement method such as a microplate agglutination reaction method, it is possible to make a determination in a shorter time than conventional particles for particle immunoassay.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological test, and more particularly to a method for increasing the specific gravity of a carrier in a particle immunoassay, and an immunological test reagent using such particles to shorten the determination time.

[0002]

2. Description of the Related Art In diagnosing a disease, an immunoserologic test method utilizing an antigen-antibody reaction has been generally used because the amount of biological components to be tested is minute.
The greatest feature of this test method is that biological components secreted in body fluids and urine can be measured with extremely high sensitivity and specificity. One of such inspection methods is a particle immunoassay method. Generally, in the particle immunoassay, particles of an appropriate size are used as carriers, and antigens or antibodies are sensitized (adsorbed or bound) to the carriers, and the carriers sensitized by the presence of the corresponding antibodies or antigens, respectively. Is caused by aggregation. Conventionally, the carrier particles most commonly used in the above-mentioned particle immunoassay method include:
For example, erythrocytes of various animals such as sheep or chicken, or artificially synthesized polymer particles such as polystyrene latex particles. The former method using animal blood cells as a carrier has been performed as a so-called hemagglutination reaction for a long time, and this method has a wide range of antigens and antibodies that can be targeted, and when the microtiter method is used, it takes only 2-3 hours. It is an excellent item in that it can make a judgment. Furthermore, there is an advantage that the size of blood cells is constant depending on the type of animal.

On the other hand, conventional synthetic polymer particles such as polystyrene generally have a particle size of about 0.1 to 1 μm and are particularly useful as a carrier for a latex agglutination reaction, and have an antigenicity themselves. It is excellent in that it does not do so and that it can stably obtain a large amount of homogeneous products. Furthermore, recently, an insolubilized granular artificial carrier containing gelatin, a water-soluble polysaccharide and sodium polymetaphosphate and cross-linked with an aldehyde cross-linking agent (JP-A-57-153658, JP-A-57-160465, etc.),
So-called gelatin carrier particles and a polyamino acid artificial carrier mainly composed of polyamino acid have been developed and put to practical use as a substitute for animal blood cells. These artificial carriers have the same performance as animal erythrocytes, which were previously considered to be the best as carriers for immunoaggregation reactions, are chemically and physically homogeneous and stable, and have no antigenic activity. It has the advantage that it can easily be mass-produced with any particle size. However, particles made of conventional synthetic polymer substances (for example, polystyrene particles) have higher sensitivity and use the microtiter method for quantitative determination, compared with the case where blood cells are used as a carrier. However, there is a problem that it takes a long time for sedimentation, and quick determination cannot be performed. In addition, when using animal red blood cells or gelatin carriers, it is possible to make a determination in a relatively short time of 2-3 hours, but with the recent trend toward rapid and automated measurement in the field of clinical testing, agglutination test However, a quicker determination time has come to be desired.

In order to shorten the determination time, it is possible to increase the sedimentation rate of the carrier used. One way to increase the sedimentation rate is to increase the particle size of the carrier. However, increasing the particle size of the carrier results in a decrease in the total surface area, resulting in a decrease in the immobilized amount of the antigen or antibody, which cannot be said to be an appropriate means.
As another means for increasing the sedimentation speed, it is possible to use a carrier having a large specific gravity or a carrier having a high specific gravity. With this method, the sedimentation rate can be increased without reducing the total surface area. However, in order to produce a carrier with a large specific gravity, it is necessary to study from the raw materials for producing it, and even if a carrier with a large specific gravity is obtained, it is a carrier for particle immunoassay that has been conventionally used. In comparison with, there remains a question of whether or not the measurement system has the property of giving equal or higher sensitivity. On the other hand, in the case of increasing the specific gravity of the conventionally used carriers, it is most preferable that the specific gravity can be easily increased without impairing the characteristics of those carriers. In addition,
Although there was a known method for increasing the amount of silk by using tin salt (Ikuzo Sakaguchi, (Continued: Silk Structure, edited by Hojo et al., Published by Shinshu University, Faculty of Textile Science), p586-624 (1980)) Until now, no examples have been reported in which such a carrier has a high specific gravity.

[0005]

In view of these problems, the present inventors have conducted intensive studies and, as a result, have found a method for easily increasing the specific gravity of conventional particles for immunoassay as described above. That is, by treating animal red blood cells and artificial carriers with a metal salt solution, the specific gravity can be easily increased,
As a result, we succeeded in producing a carrier that can be determined in a shorter time in a measurement method such as the microplate agglutination reaction method. That is, the present invention provides a method for increasing the specific gravity of particles for particle immunoassay, which comprises treating the particles with a metal salt solution. Also provided is a test reagent comprising particles for immunoassay having a high specific gravity in this manner. In the present invention, particles having a high specific gravity include red blood cells of various animals such as sheep or chicken which have been conventionally used as particles for particle immunoassay, or polystyrene latex particles, gelatin carrier, polyamino acid carrier, etc. Any of the artificial carriers can be used. The high specific gravity of the present invention is carried out by subjecting animal red blood cells and artificial carriers to stirring treatment with an aqueous metal salt solution, and then heat treating with a buffer solution. As the metal salt, salts of tin, lead, calcium, magnesium, zinc and the like, for example, hydrochlorides, sulfates, nitrates, acetates, chlorides and the like are used. Specifically, stannic chloride, aluminum sulfate, aluminum acetate, aluminum chloride, titanium sulfate, titanium acetate,
Examples thereof include titanium chloride, strontium sulfate, and strontium chloride. Among them, tin salts such as stannic chloride are effective.

The metal salt used for increasing the specific gravity of the present invention is usually used in a 20 to 60 w / w% aqueous solution. When it is 20 w / w% or less, it takes a long time to process, and when it is 60 w / w% or more, the operability is deteriorated. The amount of the metal salt used is usually 5 times the weight of the carrier or more. The treatment time varies depending on the type and amount of carrier, but is usually about 30 minutes to 2 hours. The treatment temperature can be in the range of 10 to 70 ° C, but usually room temperature, that is,
It is carried out at 15-25 ° C. After the above metal salt treatment, the carrier is separated and treated with a buffer solution. The buffer used is usually pH 6
A phosphate buffer solution of about 8 to 8 is used. Also, other commonly used buffers such as citric acid, acetic acid, borate buffers can be used. The treatment temperature is 20 to 90 ° C, preferably 50 to 80 ° C, and the treatment time is 15 minutes to 2
It's about time. After the buffer solution treatment, a carrier having a high specific gravity can be obtained by a separation operation such as filter-filtration, centrifugation, decantation and the like. To sensitize the particles prepared according to the present invention with an antigen or an antibody, conventional sensitization methods for animal red blood cells can be easily used. For example, the obtained carrier particles are treated with tannic acid by a conventional method, and then the target antigen or antibody is adsorbed. The antigen or antibody to be sensitized may be any one derived from a natural source corresponding to the antigen or antibody to be measured or derived from an artificial means such as gene recombination, cell fusion, chemical synthesis or the like.

[0007]

EFFECTS OF THE INVENTION According to the present invention, it is possible to very simply and easily increase the specific gravity of the carrier used in the conventional particle immunoassay. Further, the obtained sensitized carrier, as shown in the following examples, that the sedimentation rate is faster, that is, the judgment time can be shortened, and the judgment can be made, as compared with conventional animal blood cells or artificial carriers. It has the great advantage of not compromising the strength of time. Hereinafter, the present invention and its features will be specifically described with reference to examples.

[0008]

【Example】

Example 1 Preparation of artificial carrier with high specific gravity 34.12 g of water was added to 22.6 g of stannic chloride pentahydrate (manufactured by Wako Pure Chemical Industries, Ltd.) and dissolved by stirring to prepare an aqueous stannic chloride solution. 30 ml of an aqueous suspension of a polyamino acid artificial carrier (Sample 1) obtained by the method described in JP-A-2-103470 (carrier content 12.5
The carrier (v / v%) was separated using a glass filter (No. 4) and then washed with 3 ml of an aqueous stannic chloride solution. Further, 15 ml of an aqueous stannic chloride solution was added, and the mixture was stirred at 25 ° C for 60 minutes. After completion, after separating the carrier with a glass filter (No.4), phosphate buffer solution (Na concentration = 0.2 mol / l, pH = 7.5) 50
ml was added, and the mixture was stirred and heated at a liquid temperature of 60 to 65 ° C for 30 minutes. After the completion, the artificial carrier was separated by decantation, and a physiological saline solution was used to perform centrifugal sedimentation (700 to 3000 rpm, 3 to 5 minutes) for 4, 5
Repeatedly washed. Finally, add about 50 ml of physiological saline, leave it at 5 ° C overnight, remove excess metal salt on the surface of the carrier by sonication, disperse the carrier, and decant the saline. After separation, physiological saline was further added to obtain 50 ml of a highly specific gravity artificial carrier suspension (Sample 2). The carrier content was 7.5 v / v%.

Example 2 Preparation of highly specific density sheep blood cells A suspension aqueous solution containing 10 v / v% sheep red blood cells (sample 3)
The same treatment as in Example 1 was carried out using 30 ml to obtain 50 ml of a hyperspecific gravity-treated blood cell suspension (sample 4). The carrier content was 6 v / v%. Example 3 Specific gravity measurement The specific gravity of the carrier was measured by a cesium chloride density gradient centrifugal equilibrium method. Divide 10 ml of cesium chloride (Katayama Chemical Industry cesium chloride for density gradient centrifugation) aqueous solution into centrifuge tubes,
0.2 ml of a 1 v / v% carrier dispersion was layered and centrifuged (centrifuge: Hitachi 70P-72, rotor: Hitachi RPS40T, rotation speed: 30,000 rpm, centrifugation time: 72 hours). After centrifugation, the part in which the sample carrier was suspended was collected, the refractive index was measured with a refractometer (ATAGO Model 3), and the specific gravity was calculated by the mathematical formula 1. Table 1 shows the specific gravity measurement results of various carriers.

[Formula 1] Specific gravity = Refractive index × 10.2402-12.6483

[0010]

[Table 1] Table 1 Cesium chloride Refractive index Specific gravity aqueous solution concentration Untreated artificial carrier 50w / w% 1.3753 1.4350 (Sample 1) High specific gravity artificial carrier 52.4w / w% 1.3928 1.6143 (Sample 2) Untreated blood cells 40w / w% 1.3619 1.2978 (Sample 3) High specific gravity blood cells 50w / w% 1.3867 1.5518 (Sample 4)

Example 4 Sensitization Method Each of the sensitizing materials (antigen or antibody) shown in Table 2 below was prepared in Example 1 and the high specific gravity carrier (Sample 2) obtained according to the present invention and not yet used as a reference. The treated polyamino acid carrier (Sample 1) was sensitized with tannic acid according to a conventional method.
Human positive sera and human negative sera corresponding to each sensitizing antigen and antibody were used as samples. For example, in the case of the test for HBs antigen-sensitized carrier, HBs
Antibody-positive human serum was used, and HBs antibody-negative human serum was used as a negative sample. However, bovine serum albumin (BSA)
As a sensitizing carrier, anti-BSA rabbit immune serum was used as a positive sample. The sensitization conditions were as follows. First, PB
2.5 v / v% carrier in S (pH 7.2) and 1 volume of 80,000-fold diluted tannic acid in PBS were mixed, treated at 37 ° C. for about 30 minutes, centrifuged, and washed with PBS. Then, 1 volume of the obtained tannic acid-treated carrier 2.5 v / v% PBS suspension and 1 of each sensitizing material
The mixture was mixed with 1 volume of 0 μg / ml solution, treated at 37 ° C. for 30 minutes, centrifuged, washed with PBS, and suspended in a storage medium at a concentration of 1 v / v%. Microtiter method agglutination test A carrier agglutination test was carried out by the usual microtiter method using the obtained sensitized carrier. First, 25 μl each of a predetermined dilution is dropped onto a microtiter plate, and then 25 μl of a sample is placed in the first well to perform 2-fold serial dilution. Next, 25 μl of each sensitized carrier suspension (1 v / v%) was added dropwise, mixed well, and allowed to stand at room temperature. did. The results are shown in Table 2 below, and the carrier obtained according to the present invention showed a titer equivalent to that of the untreated carrier.

[0012]

[Table 2] Table 2 Potency Sensitization Material Positive Specimen Negative Specimen HBc Antigen Sensitized to Sample 2 ⁹ 2 ¹ (from recombinant yeast ) Sensitized to Sample 2 ⁹ 2 ¹ HBs antigen Sensitized to Sample 1 2 ⁷ 2 ¹ (derived from plasma) Sample 2 sensitized 2 ⁶ 2 ¹ HBs antigen Sample 1 sensitized 2 ⁷ 2 ¹ (from recombinant yeast ) Sample 2 sensitized 2 ⁷ 2 ¹ HBs antibody Sample 1 sensitized 2 ⁸ 2 ¹ (guinea pig) Sample Sensitization to 2 2 ⁸ 2 ¹ BSA Sensitization to sample 1 2 ⁹ 2 ¹ Sensitization to sample 2 2 ⁹ 2 ¹

Example 5 Determination of Judgment Time 25 μl of a predetermined dilution was dropped on a microtiter plate, and then each sensitized polyamino acid carrier suspension (1 v / v) was added.
%), And the mixture was allowed to stand at room temperature, and the diameter (mm) of the negative image thereof was measured every hour. The results are shown in Table 3.

[0014]

[Table 3] Table 3 Time BSA sensitization BSA sensitization HB _C antigen sensitization HB _C antigen sensitization Untreated carrier High specific gravity carrier Untreated carrier High specific gravity carrier (min) (mm) (mm) (mm) (mm) 10 Not measurable 0.33 Not measurable 0.33 20 Not measurable 0.50 Not measurable 0.50 30 0.33 0.66 0.33 0.65 40 0.50 0.83 0.50 0.81 50 0.67 0.92 0.66 0.90 60 0.81 1.00 0.81 0.98 090 0.92 1.00 0.92 0.98 120 1.00 1.00 1.00 0.98 900 1.03 1.02 1.02 0.99

In general, the determination of a negative sample by the microtiter method can be performed if a negative image of 80% or more is obtained with respect to the diameter (usually about 1 mm) of the negative image obtained after leaving for a sufficient time. . Therefore, it took about 40 to 60 minutes for the carrier of the present invention, whereas the untreated carrier required about 60 to 90 minutes to make a sufficient judgment.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshitaka Imagawa 4-16-2205, Higashimachi, Kumamoto City, Kumamoto Prefecture (72) Inventor Takenori Hirai 2080-10 Suya, Nishigoshi Town, Kikuchi District, Kumamoto Prefecture

Claims (9)

[Claims] 1. A method for increasing the specific gravity of particles for immunoassay of particles, which comprises treating the particles with a metal salt solution. 2. The method of increasing specific gravity according to claim 1, wherein the metal salt solution is a stannic chloride solution. 3. The concentration of the stannic chloride solution is 20-60 w /
The high specific gravity method according to claim 2, wherein w is w%. 4. The method of increasing specific gravity according to claim 1, wherein the particles are artificial carriers. 5. The method of increasing specific gravity according to claim 4, wherein the artificial carrier is a polyamino acid carrier. 6. The method of increasing specific gravity according to claim 4, wherein the artificial carrier is polystyrene latex particles. 7. The artificial carrier is a gelatin carrier.
High specific gravity method described. 8. The method of increasing specific gravity according to claim 1, wherein the particles are animal red blood cells. 9. A test reagent comprising particles for particle immunoassay obtained by the method of increasing specific gravity according to any one of claims 1 to 8.