JPH11344492A - Immunological agglutination reaction reagent and method for suppressing prozone phenomenon by using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To sufficiently suppress a prozone phenomenon even when an antigen or an antibody to be measured is included in a specimen at a high concentration by adding sulfates to an immunological agglutination reaction reagent. SOLUTION: Sulfates to be used can be appropriately selected from known sulfates, but sodium sulfate or ammonium sulfate is preferred. The sulfates are included by 1-10 w/v% to a total quantity of a reagent. When the amount of the included sulfates is smaller than 1.0 w/v%, a prozone phenomenon cannot be effectively suppressed. When the amount is larger than 10 w/v%, an agglutination reaction is obstructed and a measured value is lowered. If the prozone phenomenon is not suppressed sufficiently effectively, polyethylene glycol is added further to enhance the suppression effect. An addition amount is preferably 0.05-1.0 w/v% to the total quantity of the reagent. Insoluble carrier particles constituting the reagent and an antigen, an antibody immunized to the carrier particles can be selected from respective known ones. Any known agglutination test water medium is utilizable as a medium suspending the carrier particles.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological agglutination reagent and a method for suppressing the prozone phenomenon using the same, particularly when the antigen or antibody to be measured is contained in a sample at a high concentration. The present invention relates to an immunological agglutination reagent which can be used as it is without diluting a specimen without dilution, and a method for suppressing the prozone phenomenon using the reagent.

[0002]

2. Description of the Related Art In recent years, it has been widely used to detect immunologically active substances such as proteins appearing in a living body in connection with various diseases by utilizing an antigen-antibody reaction and to use them for diagnosis.
As a measurement method using such an antigen-antibody reaction,
Radioimmunoassay (RIA), enzyme immunoassay (EIA),
Fluorescence immunoassay (FIA), latex agglutination (L
A), various methods such as immunoturbidimetry (TIA) have been put to practical use.

In particular, LA is widely used because its operation is simple and can be measured in a short time. In LA, latex particles having an antibody (or antigen) corresponding to the antigen (or antibody) to be measured bound to its surface are used. Such latex particles are suspended in a medium such as a buffer and mixed with the analyte.

[0004] The antigen (or antibody) in the specimen and the antibody (or antigen) on the surface of the latex cause an antigen-antibody reaction to form an immune complex, and the antigen (or antibody) in the specimen is formed.
, The latex particles are crosslinked and aggregated. The degree of aggregation can be easily measured by a change in absorbance or a change in intensity of scattered light. This LA is B /
Since the number of reaction steps is smaller than that of RIA or EIA which requires F separation, the measurement method is simple and suitable for an automatic analyzer.

However, antigens (or antibodies) contained in a specimen at a high concentration, for example, C-reactive protein (CRP) contained at a relatively high concentration in serum, are conventionally used as L-proteins.
When measured with the reagent for A, a prozone phenomenon occurs,
In some cases, the measured value was considerably lower than the actual value, and the diagnosis could not be performed with high accuracy.

[0006] It is difficult to judge this prozone phenomenon from the measured values, and although the means for judging the prozone from the start of the reaction, that is, the rate of increase in the absorbance, is installed in a part of the automatic analyzer, the effect is not as good. Not enough .. The most reliable measure is to add CR to the reaction mixture after the measurement is completed.
P is added, and it is determined whether or not the phenomenon is a prozone phenomenon based on whether the measured value further increases or conversely decreases. If it is determined that this is a prozone phenomenon, the sample must be diluted or the amount of sample collected must be reduced, and the measurement performed again. In order to avoid such complicated operations, it has been desired to provide a reagent that does not cause a prozone phenomenon.

As a method for improving the prozone phenomenon, a method for lowering the ionic strength of an aqueous solvent used for measurement,
An immunoassay method in which a dextran having a specific molecular weight is contained in an aqueous solvent for an agglutination test (JP-A-59-220646), an immunoassay method based on a latex agglutination method in which an amino acid is contained in a mixture of a sample and a medium ( JP-A-62-2
No. 72157), a reagent for measuring an immunologically active substance containing a surfactant such as polyethylene glycol as an additive (JP-A-8-285847, JP-A-9-54).
No. 092, Japanese Patent Application Laid-Open No. 9-96638), an immunological assay containing a specific amount of sodium chloride (Japanese Patent Application Laid-Open No. 9-89894), and the like have been proposed.

[0008]

However, in the above-mentioned methods described in these publications, although an improvement in the prozone phenomenon is certainly observed, it is difficult to obtain a sample containing a high concentration of the antigen or antibody to be measured. However, the effect of suppressing the prozone phenomenon is still insufficient, and further improved methods have been strongly desired.

Accordingly, the present invention has been made in view of such a conventional problem, and even when the antigen or antibody to be measured is contained in a sample at a high concentration, the prozone phenomenon is prevented. An object of the present invention is to provide an immunological agglutination reagent which can be sufficiently suppressed, and a method for suppressing the prozone phenomenon using the reagent.

[0010]

Means for Solving the Problems The present inventors have made intensive studies to solve the above problems, and as a result, by adding sulfates and, if necessary, polyethylene glycol to a conventional immunological agglutination reagent, The present inventors have found that a high effect of suppressing the prozone phenomenon can be obtained, and have reached the present invention.

[0011] The object of the present invention is to provide an immunological agglutination reagent comprising an insoluble carrier particle to which an antibody or an antigen is bound, and a medium for suspending the insoluble carrier particle, comprising a sulfate and, if necessary, a sulfate salt. The present invention has been attained by an immunological agglutination reagent comprising polyethylene glycol, and a method for suppressing the prozone phenomenon using the reagent.

Hereinafter, the present invention will be described in more detail.

The sulfates used in the present invention can be appropriately selected from known sulfates. Among these sulfates, sodium sulfate and / or ammonium sulfate are particularly preferred.

In the present invention, the amount of the sulfate is 1.0 to 10 w / v%, preferably 5.0%, based on the total amount of the reagent.
-10 w / v%, more preferably 7.5 w / v%. When the content of sulfates is less than 1.0 w / v%, the prozone phenomenon cannot be effectively suppressed. Conversely, when the content exceeds 10 w / v%, the agglutination reaction is inhibited and the measured value decreases. I do.

In the present invention, even when sulfates are added alone, if the effect of suppressing the prozone phenomenon is insufficient, polyethylene glycol is further added in addition to sulfates to improve the effect of prozone. The effect of suppressing the phenomenon can be further improved.

The polyethylene glycol used in the present invention may be appropriately selected from known ones.
Its average molecular weight is 1000-10000, preferably 6
000. It is preferable that the polyethylene glycol be contained in an amount of 0.05 to 1.0 w / v% based on the whole reagent.

The insoluble carrier particles used in the present invention can be appropriately selected from those conventionally used as carrier particles for immunological agglutination. Specific examples thereof include, for example, inorganic substance powder,
Organic polymer powders, microorganisms, blood cells, cell membrane fragments and the like can be mentioned. The inorganic substance is not particularly limited,
Examples include metals such as gold, titanium, iron, and nickel, metal oxides such as alumina and titania, and silica. Examples of the organic polymer include, but are not particularly limited to, a styrene polymer, a styrene-styrene sulfonate copolymer, a methacrylic acid polymer, an acrylic acid polymer, an acrylonitrile-butadiene-styrene copolymer, and vinyl chloride-acrylic acid. Examples thereof include an ester copolymer and a vinyl acetate-acrylate copolymer, and particularly preferred are latex particles obtained by uniformly suspending these polymer powders in water.

The sensitization of these insoluble carrier particles with an antigen or antibody can be carried out according to a known method. Specific examples thereof include, for example, a chemical bonding method using a so-called coupling agent such as glutaraldehyde, bisdiazobenzidine, tolylene diisocyanate, difluoronitrobenzene, carbodiimides, quinones, chromium chloride, and tannic acid, and an antigen. Alternatively, a physical adsorption method and the like in which an antibody and a carrier are brought into contact with each other in a water-soluble solvent (for example, water, physiological saline, various buffer solutions, and the like) may be used.

In the present invention, the antigen or antibody sensitizing the carrier particles is not particularly limited, and can be appropriately selected from known ones and used. Specific examples thereof include C-reactive protein (CRP),
Antibodies to plasma proteins such as rheumatoid factor (RF) and transferrin, thyroid stimulating hormone (TSH), triiodothyronine, thyroxine, thyroxine binding protein, thyroglobulin, insulin, estriol,
Antibodies to hormones such as human placental lactogen, carcinoembryonic antigen (CEA), α-fetoprotein (AF
Antibodies against tumor-related substances such as P), HBs antigen, HB
s antibody, HBe antigen, antibody to viral hepatitis antigen such as HBe antibody and antigen to antibody, virus to mumps, herpes, measles, rubella etc., antibody or antigen to various biological components, phenobarbital, acetaminophenone, salicylic acid, cyclosporine And other various drugs.

As a medium for suspending the carrier particles,
Any conventionally known aqueous medium for agglutination tests can be used, such as water, physiological saline, various buffers (Good buffer, phosphate buffer, Tris-HCl buffer, borate buffer, glycine buffer), and these. The solution which consists of a combination of is illustrated.

The immunological agglutination reagent of the present invention measures the degree of agglutination of carrier particles produced by mixing and reacting a specimen and a reagent according to a conventional method, by measuring the change in absorbance of the mixture and the intensity of scattered light. It can be used by measuring change.

[0022]

According to the present invention, even when the antigen or antibody to be measured is contained at a high concentration in the sample, the immunoassay can be carried out without dilution of the sample without any dilution. Therefore, according to the present invention, it is possible to omit the time-consuming and laborious work of accurately diluting the sample.

[0023]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto.

Example 1 3 μm of physiological saline solution containing 0 to 125 mg / dl CRP
1, 0, 2.5, 5.0, 7.5, 10, 12.5 w /
200 μl of a 0.1 M HEPES buffer (pH 7.4) containing v% sodium sulfate was added. 200 μl of a suspension (0.2 w / v%) of latex particles having a particle size of 0.08 μm to which an anti-CRP antibody (animal name: goat) was bound was added to the mixture,
The reaction was carried out at 37 ° C., and the wavelength was 570 nm after 1 to 5 minutes.
Was used to measure the absorbance, and the amount of change in absorbance between each measurement point was determined. For the measurement, a fully automatic analyzer Hitachi 7070 (manufactured by Hitachi, Ltd.) was used.

FIG. 1 shows the results. As shown in FIG. 1, when 5.0, 7.5, and 10 w / v% of sodium sulfate were added, no prozone phenomenon due to excess antigen occurred. On the other hand, when 2.5 w / v% of sodium sulfate was added, although the effect of suppressing the prozone phenomenon was observed, the effect was still insufficient, and when sodium sulfate was not contained, the effect of excess antigen caused by pro-antigen. It can be seen that the zone phenomenon has occurred. Also, when 12.5 w / v% sodium sulfate was added, it was found that the agglutination reaction was inhibited and the immunoassay could not be performed accurately.

Example 2 Instead of the sodium sulfate used in Example 1, 0, 2,.
Except for using 5, 5.0, 7.5, and 10 w / v% ammonium sulfate, the inhibitory effect of prozone was examined in exactly the same manner as in Example 1. The result is shown in FIG. As shown in FIG. 2, when 5.0, 7.5, and 10 w / v% ammonium sulfate were added, the prozone phenomenon due to excess antigen did not occur. On the other hand, when 2.5 w / v% of ammonium sulfate was added, although the effect of suppressing the prozone phenomenon was observed, the effect was still insufficient. It turns out that the phenomenon has occurred.

Example 3 2.5 w / v% sodium sulfate was added, and 0.0
The suppression effect of the prozone phenomenon was examined in exactly the same manner as in Example 1, except that 8, 0.5 w / v% polyethylene glycol was used. The result is shown in FIG. As shown in FIG. 3, when sodium sulfate and polyethylene glycol were combined, it was found that the effect of suppressing prozone was further improved as compared with the effect obtained in Example 1.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of suppressing the prozone phenomenon when sodium sulfate is used.

FIG. 2 is a graph showing the effect of suppressing the prozone phenomenon when ammonium sulfate is used.

FIG. 3 is a graph showing the effect of suppressing the prozone phenomenon when sodium sulfate and polyethylene glycol are combined.

Claims (10)

[Claims] An immunological agglutination reagent comprising an insoluble carrier particle to which an antibody or an antigen is bound, and a medium for suspending the insoluble carrier particle, wherein the immunological agglutination reagent contains sulfates. Agglutination reagent. 2. The immunological agglutination reagent according to claim 1, wherein the content of sulfates is in the range of 1.0 to 10 w / v%. 3. The immunological agglutination reagent according to claim 1, wherein the sulfate is sodium sulfate and / or ammonium sulfate. 4. The immunological agglutination reagent according to claim 1, which comprises polyethylene glycol. 5. A polyethylene glycol content of 0.0
The immunological agglutination reagent according to claim 4, which is in a range of 5 to 1.0 w / v%. 6. A prozone containing sulfates in an immunological agglutination reaction using an insoluble carrier particle to which an antibody or an antigen is bound and a medium for suspending the insoluble carrier particle. How to control the phenomenon. 7. The method according to claim 6, wherein the concentration of sulfates is in the range of 1.0 to 10 w / v%. 8. The method according to claim 6, wherein the sulfates are sodium sulfate and / or ammonium sulfate. 9. The method for suppressing the prozone phenomenon according to claim 6, which comprises polyethylene glycol. 10. The polyethylene glycol content of 0.1.
The method for suppressing the prozone phenomenon according to claim 9, wherein the content is in the range of 0.05 to 1.0 w / v%.