JP3886639B2 - Immunological agglutination reagent and method for suppressing prozone phenomenon using the same - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to an immunological agglutination reagent and a method for suppressing a prozone phenomenon using the same, and particularly dilutes a specimen even when the antigen or antibody to be measured is contained in a high concentration in the specimen. The present invention relates to an immunological agglutination reaction reagent that can be used as it is without being carried out, and a prozone phenomenon suppression method using the same.
[0002]
[Prior art]
In recent years, it has been widely used to detect immunologically active substances such as proteins appearing in living bodies in association with various diseases by using an antigen-antibody reaction for diagnosis. Measurement methods using such antigen-antibody reactions include radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), latex agglutination (LA), immunoturbidimetry ( Various methods such as TIA) have been put into practical use.
[0003]
In particular, LA is widely used because it is easy to operate and can be measured in a short time. In LA, latex particles in which an antibody (or antigen) corresponding to an antigen (or antibody) to be measured is bound to its surface are used. Such latex particles are suspended in a medium such as a buffer and mixed with the specimen.
[0004]
The antigen (or antibody) in the specimen and the antibody (or antigen) on the latex surface cause an antigen-antibody reaction to form an immune complex, and latex particles are cross-linked through the antigen (or antibody) in the specimen. Agglomerate. The degree of this aggregation can be easily measured by a change in absorbance or a change in intensity of scattered light. Since LA has fewer reaction steps than RIA and EIA that require B / F separation, LA has a simple measuring method and is suitable for an automatic analyzer.
[0005]
However, when an antigen (or antibody) contained in a sample at a high concentration, for example, C-reactive protein (CRP) contained in a serum at a relatively high concentration is measured using a conventional reagent for LA, the prozone In some cases, the phenomenon occurs, and the measured value is much lower than the actual value, so that accurate diagnosis cannot be made.
[0006]
It is difficult to judge this prozone phenomenon from the measured value, and although the means for judging the prozone from the rise of the reaction, that is, the rate of increase in absorbance, is mounted on a part of the automatic analyzer, the effect is not sufficient. .. The most reliable means is to add more CRP to the reaction mixture after completion of the measurement, and determine whether it is a prozone phenomenon depending on whether the measured value further increases or decreases. If the prozone phenomenon is determined, the sample must be diluted or the amount of sample collected should be reduced and measured again. In order to avoid such complicated operations, it has been desired to provide a reagent that does not cause a prozone phenomenon.
[0007]
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 dextran having a specific molecular weight is contained in an aqueous solvent for agglutination test (Japanese Patent Laid-Open No. 59-220646) , An immunoassay based on a latex agglutination method containing an amino acid in a mixture of a specimen and a medium (Japanese Patent Laid-Open No. Sho 62-272157), an immunological agent containing a surfactant such as polyethylene glycol as an additive Active substance measuring reagent (JP-A-8-285847, JP-A-9-54092, JP-A-9-96638), immunological assay method containing a specific amount of sodium chloride (JP-A-9-89894) No. Gazette) has been proposed.
[0008]
On the other hand, LA is known to cause non-specific aggregation or precipitation when immunoglobulin molecules are adsorbed or aggregated on the surface of hydrophilic materials when used in a widely used automatic analyzer. Yes. In order to solve this problem, LA using polybasic acids such as boric acid, oxalic acid, citric acid and phthalic acid has been proposed (Japanese Patent Laid-Open No. 2-61561).
[0009]
[Problems to be solved by the invention]
However, the method described in JP-A-2-61561 merely discloses a method for preventing non-specific aggregation and precipitation, and does not suggest any improvement on the prozone phenomenon. . In addition, in the methods described in the above-mentioned other publications, although improvement to the prozone phenomenon is certainly observed, if the antigen (or antibody) to be measured is contained in a high concentration in the sample, the prozone phenomenon The suppression effect is still insufficient, and a further improved method has been strongly desired.
[0010]
Therefore, the present invention has been made paying attention to such a conventional problem, and even if the antigen (or antibody) to be measured is contained in a sample at a high concentration, the prozone phenomenon is sufficiently achieved. It is an object of the present invention to provide an immunological agglutination reaction reagent that can be suppressed and a prozone phenomenon suppression method using the same.
[0011]
[Means for Solving the Problems]
As a result of diligent research to solve the above-mentioned problems, the present inventor has made a conventional immunological agglutination reaction reagent contain a predetermined amount of dicarboxylic acids and, if necessary, sulfates and / or polyethylene glycol, The inventors have found that a high suppression effect of the prozone phenomenon can be obtained, and have reached the present invention.
[0012]
The above-described problem of the present invention is that an immunological agglutination reagent comprising insoluble carrier particles to which an antibody or antigen is bound and a medium in which the insoluble carrier particles are suspended is 1.0 to 20 w / v%. This was achieved by an immunological agglutination reagent characterized by containing dicarboxylic acids, and a method for suppressing the prozone phenomenon using the reagent.
[0013]
Hereinafter, the present invention will be described in more detail.
[0014]
The dicarboxylic acids used in the present invention can be appropriately selected from known dicarboxylic acids. Specific examples thereof include at least one selected from the group consisting of malic acid, glutaric acid, adipic acid and succinic acid. The dicarboxylic acid may be a salt such as Na, and may form an ester with alcohol or the like.
[0015]
In the present invention, the dicarboxylic acids are contained in an amount of 1.0 to 20 w / v%, preferably 5.0 to 15 w / v% based on the total amount of the reagent. If the amount of dicarboxylic acid added is less than 1.0 w / v%, the prozone phenomenon cannot be effectively suppressed.
[0016]
In the present invention, even when dicarboxylic acids are added alone, if the effect of suppressing the prozone phenomenon is insufficient, the addition of sulfates and / or polyethylene glycol further suppresses the prozone phenomenon. The effect can be further improved.
[0017]
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 preferable.
[0018]
In the present invention, sulfates are contained in an amount of 1.0 to 10 w / v%, preferably 5.0 to 10 w / v%, more preferably 7.5 w / v%, based on the total amount of the reagent. If the added amount of sulfates is less than 1.0 w / v%, the prozone phenomenon cannot be effectively suppressed, and conversely if it exceeds 10 w / v%, the agglutination reaction is inhibited even in a low concentration range. The
[0019]
The polyethylene glycol used in the present invention may be appropriately selected from known ones. The average molecular weight is 1000 to 10000, preferably 6000. Polyethylene glycol is contained so as to be 0.05 to 1.0 w / v% based on the whole reagent.
[0020]
Prozone phenomenon is insoluble via an antigen (or antibody) in the mixture when an excess of antigen (or antibody) is present in the mixture relative to the antibody (or antigen) bound to the surface of the insoluble carrier particles. This is considered to occur because the carrier particles are not cross-linked and the aggregation of insoluble carrier particles does not occur. Accordingly, the combination and addition amount of dicarboxylic acids, polyethylene glycol and sulfates may be appropriately selected according to the concentration range of the antigen or antibody contained in the sample to be measured.
[0021]
The insoluble carrier particles used in the present invention can be appropriately selected from those conventionally used as carrier particles for immunological aggregation reactions. Specific examples thereof include inorganic substance powders, organic polymer substance powders, microorganisms, blood cells, cell membrane fragments, and the like. The inorganic substance is not particularly limited, and examples thereof include metals such as gold, titanium, iron, and nickel, metal oxides such as alumina and titania, silica, and the like. The organic polymer is not particularly limited, but styrene polymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile-budadiene-styrene copolymer, vinyl chloride-acrylic acid. An ester copolymer, a vinyl acetate-acrylic acid ester copolymer, and the like can be mentioned. Latex particles in which these polymer powders are uniformly suspended in water are particularly preferable.
[0022]
Sensitization of the antigen or antibody to these insoluble carrier particles can be performed according to a known method. Specific examples thereof include, for example, chemical coupling methods using so-called coupling agents such as glutaraldehyde, bisdiazobenzidine, tolylene diisocyanate, difluoronitrobenzene, carbodiimides, quinones, chromium chloride, tannic acid, antigens, and the like. Alternatively, a physical adsorption method in which the antibody and the carrier are brought into contact with each other in a water-soluble solvent (for example, water, physiological saline, various buffers, etc.) can be used.
[0023]
In the present invention, the antigen or antibody to be sensitized to the carrier particles is not particularly limited and can be appropriately selected from known ones. Specific examples thereof include, for example, antibodies to plasma proteins such as C-reactive protein (CRP), rheumatoid factor (RF), transferrin, thyroid stimulating hormone (TSH), triiodothyronine, thyroxine, thyroxine binding protein, thyroglobulin. , Antibodies against hormones such as insulin, estriol, human placental lactogen, antibodies against tumor related substances such as carcinoembryonic antigen (CEA), α-fetoprotein (AFP), HBs antigen, HBs antibody, HBe antigen, HBe antibody, etc. Antibodies against human hepatitis antigens, antigens against antibodies, viruses such as mumps, herpes, measles, rubella, antibodies or antigens against various biological components, antibodies against various drugs such as phenobarbital, acetaminophenone, salicylic acid, cyclosporine It is.
[0024]
As the medium for suspending the carrier particles, any conventionally known aqueous medium for agglutination test can be used. For example, water, physiological saline, various buffer solutions (Good buffer solution, phosphate buffer solution, Tris-HCl buffer solution, boric acid solution) Buffer, glycine buffer) and combinations thereof.
[0025]
The immunological agglutination reagent of the present invention measures the degree of aggregation of carrier particles produced by mixing and reacting a specimen and a reagent according to a conventional method, and measuring the change in absorbance of the mixture and the change in intensity of scattered light. Can be used.
[0026]
【The invention's effect】
The present invention makes it possible to perform an immunoassay in a stock solution without diluting the sample even when the antigen or antibody to be measured is contained at a high concentration in the sample. Therefore, according to the present invention, time-consuming and labor-intensive work for accurately diluting the specimen can be omitted.
[0027]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by this.
[0028]
Example 1
To 3 μl of physiological saline solution containing 0 to 150 mg / dl CRP, 200 μl of 0.1 M HEPES buffer (pH 7.4) containing 0, 5.0 w / v% malic acid was added. A suspension of latex particles (0.2 w / v%) having a particle size of 0.08 μm bound with an anti-CRP antibody (animal name: goat) was added to the mixture, reacted at 37 ° C., and after 1 to 5 minutes. Absorbance was measured at a wavelength of 570 nm, 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.
[0029]
The result is shown in FIG. As shown in FIG. 1, when 5.0 w / v% of malic acid was added, the prozone phenomenon due to antigen excess did not occur. In contrast, when malic acid was not included, a prozone phenomenon due to an excess of antigen occurred.
[0030]
Example 2
The effect of suppressing the prozone phenomenon was examined in exactly the same manner as in Example 1 except that 0, 5.0 w / v% glutaric acid was used instead of malic acid used in Example 1. The result is shown in FIG. As shown in FIG. 2, when 5.0 w / v% glutaric acid was added, the prozone phenomenon due to an excess of antigen did not occur. On the other hand, when no glutaric acid was contained, a prozone phenomenon occurred due to an excess of antigen.
[0031]
Example 3
In place of malic acid used in Example 1, 0, 3.75, and 7.5 w / v% adipic acid were used, and the effect of suppressing the prozone phenomenon was examined in exactly the same manner as in Example 1. . The result is shown in FIG. As shown in FIG. 3, when 3.75 and 7.5 w / v% adipic acid was added, the prozone phenomenon due to antigen excess did not occur. On the other hand, when adipic acid was not included, a prozone phenomenon due to an antigen excess occurred.
[0032]
Example 4
The suppression effect of the prozone phenomenon was examined in exactly the same manner as in Example 1 except that 0, 5.0, 10 w / v% dimethyl glutaric acid was used instead of malic acid used in Example 1. The result is shown in FIG. As shown in FIG. 4, when 5.0 or 10 w / v% dimethylglutaric acid was added, the prozone phenomenon due to antigen excess did not occur. On the other hand, when dimethylglutaric acid was not included, a prozone phenomenon occurred due to an excess of antigen.
[0033]
Example 5
In place of malic acid used in Example 1, 0, 2.5, 5.0, 10, 15 w / v% dipotassium succinate was used in exactly the same manner as in Example 1 except for the prozone phenomenon. The inhibitory effect was investigated. The result is shown in FIG. As shown in FIG. 5, when 5.0, 10, 15 w / v% dipotassium succinate was added, the prozone phenomenon due to the excess of antigen did not occur. On the other hand, when 2.5 w / v% dipotassium succinate is added, although the suppression effect of the prozone phenomenon is recognized, it is still insufficient, and when dipotassium succinate is not included, the antigen Prozone phenomenon due to excess occurred.
[0034]
Example 6
The inhibitory effect of the prozone phenomenon was examined in exactly the same manner as in Example 1 except that 2.5 w / v% succinic acid was added and 2.5 and 5.0 w / v% sodium sulfate was further added. . The result is shown in FIG. As shown in FIG. 6, it can be seen that when succinic acid and sodium sulfate are combined, the effect of suppressing the prozone phenomenon is further improved as compared with the effects obtained in Examples 1-5.
[0035]
Example 7
The inhibitory effect of the prozone phenomenon was examined in exactly the same manner as in Example 1 except that 2.5 w / v% succinic acid was added and 0.08 and 0.5 w / v% polyethylene glycol were further added. . The result is shown in FIG. As shown in FIG. 7, when succinic acid and polyethylene glycol are combined, it is recognized that the effect of suppressing the prozone phenomenon is further improved as compared with the effects obtained in Examples 1-5.
[Brief description of the drawings]
FIG. 1 is a graph showing the suppression effect of prozone phenomenon when malic acid is used. FIG. 2 is a graph showing the suppression effect of prozone phenomenon when glutaric acid is used. FIG. 3 is a case where adipic acid is used. Graph showing the suppression effect of the prozone phenomenon of Fig. 4 Fig. 4 Graph showing the suppression effect of the prozone phenomenon when dimethyl glutaric acid is used Fig. 5 The suppression effect of the prozone phenomenon when dipotassium succinate is used Fig. 6 is a graph showing the suppression effect of the prozone phenomenon when succinic acid and sodium sulfate are combined. Fig. 7 is a graph showing the suppression effect of the prozone phenomenon when succinic acid and polyethylene glycol are combined.

Claims (14)

And latex particles conjugated to an antibody or antigen, in immunological latex agglutination reagent and a medium for suspending the latex particles, immunology, characterized in that it contains 7.5 ~ 15W / V% of the dicarboxylic acid Latex agglutination reagent . 2. The immunological latex agglutination reagent according to claim 1, wherein the dicarboxylic acid is at least one selected from the group consisting of malic acid, glutaric acid, adipic acid and succinic acid, or a salt or ester thereof. The immunological latex agglutination reagent according to claim 1 or 2, which contains sulfates. 4. The immunological latex agglutination reagent according to claim 3, wherein the sulfate content is in the range of 1 to 10 W / V%. The immunological latex agglutination reagent according to claim 3 or 4, wherein the sulfate is sodium sulfate and / or ammonium sulfate. 3. The immunological latex agglutination reagent according to claim 1 or 2, comprising polyethylene glycol. 7. The immunological latex agglutination reagent according to claim 6, wherein the polyethylene glycol content is in the range of 0.05 to 1.0 W / V%. Prozone to the latex particles conjugated with an antibody or antigen, in immunological latex agglutination reaction and a medium for suspending the latex particles, characterized in that it contained 7.5 ~ 15W / V% of the dicarboxylic acid How to suppress the phenomenon.   9. The method for suppressing a prozone phenomenon according to claim 8, wherein the dicarboxylic acid is at least one selected from the group consisting of malic acid, glutaric acid, adipic acid and succinic acid, or a salt or ester thereof.   10. The method for suppressing a prozone phenomenon according to claim 8 or 9, wherein sulfates are contained.   11. The method for suppressing a prozone phenomenon according to claim 10, wherein the sulfate content is in the range of 1.0 to 10 W / V%.   12. The method for suppressing a prozone phenomenon according to claim 10 or 11, wherein the sulfate is sodium sulfate and / or ammonium sulfate.   10. The method for suppressing a prozone phenomenon according to claim 8 or 9, wherein polyethylene glycol is contained.   14. The method for suppressing a prozone phenomenon according to claim 13, wherein the polyethylene glycol content is in the range of 0.05 to 1.0 W / V%.

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