JP3966615B2 - 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, a C-reactive protein (CRP) contained in a serum at a relatively high concentration is measured with a conventional reagent for LA, the prozone phenomenon is observed. In some cases, the measured value was considerably lower than the actual value, and the diagnosis could not be made with high accuracy.
[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 the 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]
[Problems to be solved by the invention]
However, although the above-mentioned methods described in these publications certainly improve the prozone phenomenon, if the antigen or antibody to be measured is contained in a high concentration in the sample, the effect of suppressing the prozone phenomenon Is still inadequate, and a further improved method has been highly desired.
[0009]
Therefore, the present invention has been made paying attention to such a conventional problem, and sufficiently suppresses the prozone phenomenon even when the antigen or antibody to be measured is contained at a high concentration in the specimen. It is an object of the present invention to provide an immunological agglutination reagent that can be used and a method for suppressing the prozone phenomenon using the same.
[0010]
[Means for Solving the Problems]
As a result of diligent research to solve the above-mentioned problems, the present inventor obtained a high inhibitory effect on the prozone phenomenon by adding sulfates and, if necessary, polyethylene glycol to a conventional immunological agglutination reagent. The present invention has been reached.
[0011]
In the immunological agglutination reaction reagent comprising an insoluble carrier particle to which an antibody or an antigen is bound and a medium for suspending the insoluble carrier particle, the above-mentioned problem of the present invention is that sulfates and, if necessary, polyethylene glycol It was achieved by an immunological agglutination reaction reagent characterized in that it contained, and a prozone phenomenon suppression method using the same.
[0012]
Hereinafter, the present invention will be described in more detail.
[0013]
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.
[0014]
In the present invention, the 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% with respect to the total amount of the reagent. . If the sulfate content is less than 1.0 w / v%, the prozone phenomenon cannot be effectively suppressed. Conversely, if it exceeds 10 w / v%, the aggregation reaction is inhibited and the measured value decreases. To do.
[0015]
In the present invention, even when sulfates are added alone, if the effect of suppressing the prozone phenomenon is insufficient, the addition of polyethylene glycol in addition to sulfates can suppress the prozone phenomenon. The effect can be further improved.
[0016]
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 preferably contained so as to be 0.05 to 1.0 w / v% based on the whole reagent.
[0017]
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.
[0018]
Sensitization of the antigen or antibody to these insoluble carrier particles can be performed according to a known method. Specific examples thereof include chemical coupling methods and antigens using so-called coupling agents such as glutaraldehyde, bisdiazobenzidine, tolylene diisocyanate, difluoronitrobenzene, carbodiimides, quinones, chromium chloride, and tannic acid. 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.
[0019]
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.
[0020]
As the medium for suspending the carrier particles, any conventionally known aqueous medium for agglutination tests can be used. For example, water, physiological saline, various buffers (Good buffer, phosphate buffer, Tris-HCl buffer, Acid buffer, glycine buffer) and combinations thereof are exemplified.
[0021]
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.
[0022]
【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.
[0023]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by this.
[0024]
Example 1
0.1 M HEPES buffer containing 0, 2.5, 5.0, 7.5, 10, 12.5 w / v% sodium sulfate in 3 μl of physiological saline solution containing 0-125 mg / dl CRP ( pH 7.4) 200 μl was added. 200 μl of a latex particle suspension (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 and allowed to react at 37 ° C. Absorbance was measured at 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.
[0025]
The result is shown in FIG. As shown in FIG. 1, when 5.0, 7.5, 10 w / v% sodium sulfate was added, the prozone phenomenon due to antigen excess did not occur. On the other hand, when 2.5 w / v% sodium sulfate is added, although the suppression effect of the prozone phenomenon is recognized, it is still insufficient. It can be seen that the zone phenomenon occurs. It can also be seen that when 12.5 w / v% sodium sulfate is added, the agglutination reaction is inhibited and immunoassay cannot be performed with high accuracy.
[0026]
Example 2
Inhibition of prozone in the same manner as in Example 1 except that 0, 2.5, 5.0, 7.5, and 10 w / v% ammonium sulfate were used instead of sodium sulfate used in Example 1. The effect was investigated. The result is shown in FIG. As shown in FIG. 2, when 5.0, 7.5, 10 w / v% ammonium sulfate was added, the prozone phenomenon due to antigen excess did not occur. On the other hand, when 2.5 w / v% ammonium sulfate is added, although the suppression effect of the prozone phenomenon is recognized, it is still insufficient, and when ammonium sulfate is not included, the prozone is caused by excess antigen. It can be seen that the phenomenon occurs.
[0027]
Example 3
The inhibitory effect of the prozone phenomenon was examined in exactly the same manner as in Example 1 except that 2.5 w / v% sodium sulfate was added and 0.08 and 0.5 w / v% polyethylene glycol were used. . The result is shown in FIG. As shown in FIG. 3, when sodium sulfate and polyethylene glycol are combined, it can be seen that the suppression effect of the prozone is further improved as compared with the effect obtained in Example 1.
[Brief description of the drawings]
FIG. 1 is a graph showing the suppression effect of prozone phenomenon when sodium sulfate is used. FIG. 2 is a graph showing the suppression effect of prozone phenomenon when ammonium sulfate is used. FIG. 3 is a combination of sodium sulfate and polyethylene glycol. Graph showing the suppression effect of prozone phenomenon

Claims (8)

An antibody or latex particles conjugated to an antigen, in a latex agglutination reagent and a medium for suspending the latex particles, the latex agglutination reaction, characterized in that it contains 5~10.0w / v% sulfuric acid salts reagent. The latex agglutination reagent according to claim 1, wherein the sulfate is sodium sulfate and / or ammonium sulfate. The latex agglutination reagent according to claim 1 or 2, which contains polyethylene glycol. The latex agglutination reaction reagent according to claim 3, wherein the content of polyethylene glycol is in the range of 0.05 to 1.0 W / V%. Prozone phenomenon of an antibody or latex particles conjugated to an antigen, in a latex agglutination reaction and a medium for suspending the latex particles, characterized in that it contained 5~10.0w / v% sulfuric acid salts Suppression method.   The method for suppressing a prozone phenomenon according to claim 5, wherein the sulfate is sodium sulfate and / or ammonium sulfate.   The method for suppressing a prozone phenomenon according to claim 5 or 6 containing polyethylene glycol.   The method for suppressing a prozone phenomenon according to claim 7, wherein the content of polyethylene glycol is in the range of 0.05 to 1.0 W / v%.

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