JP3851807B2 - Method for suppressing prozone phenomenon in immune reaction and reagent for measuring immune reaction - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a drug and method for suppressing a prozone phenomenon that becomes an obstacle when measuring a substance to be measured over a wide concentration range in an immune reaction such as an immunoaggregation reaction or an immunoprecipitation reaction, and an immunoassay method using the same And an immune reaction measurement reagent.
[0002]
[Prior art]
In recent years, various tests in fields such as clinical tests have been automated and shortened in measurement time. In these tests, a measurement method using an immune reaction is widely used to measure a trace amount substance in a biological sample. There are many immunoassay methods such as RIA method, EIA method, immunoturbidimetric method, latex agglutination method, metal colloid agglutination method, and immunochromatography method. Among them, latex agglutination method and metal colloid agglutination method are the reaction solutions. It is suitable for automation because it does not separate or wash.
[0003]
Conventionally, when immunologically measuring a substance to be measured in a biological sample, an antigen (or a substance to be measured) is added by adding a nonionic, anionic, cationic or amphoteric surfactant to the immune reaction solution. It has been known that measurement sensitivity and measurement accuracy can be improved in a low concentration region of (antibody) (Japanese Patent Laid-Open No. 58-187802). On the other hand, in an immune reaction such as an immunoprecipitation reaction or an immunoagglutination reaction, when an antigen (or antibody) is present in excess from the equivalent range, a precipitate or aggregate is less likely to be formed, resulting in a decrease in the amount of precipitate ( "Prozone phenomenon" is known). For this reason, compared to antibodies (or antigens) bound to insoluble carrier particles such as latex or metal colloids, such as when antigens (or antibodies), which are substances to be measured in biological samples, are present in high concentrations in immune reaction solutions. If there is an excessive amount of antigen (or antibody), the measurement value will be smaller when measured by the latex agglutination method or metal colloid agglutination method due to the prozone phenomenon. It was.
[0004]
As a method for solving such a problem, an immunological agglutination reagent containing sulfates and, if necessary, polyethylene glycol (Japanese Patent Laid-Open No. 11-344492), or a specific amount of dicarboxylic acid was contained. A measurement method using an immunological agglutination reagent (Japanese Patent Laid-Open No. 11-344494) has been proposed.
[0005]
However, although these methods can suppress the prozone phenomenon to some extent, the suppression effect is insufficient for use in homogeneous measurement systems suitable for automation such as latex agglutination and metal colloid agglomeration. A new technique for suppressing the zonal phenomenon has been eagerly desired.
[0006]
[Problems to be solved by the invention]
In the present invention, the prozone phenomenon is sufficiently suppressed based on the above background, compared with an antibody (or antigen) in which an antigen (or antibody) as a substance to be measured is bound to insoluble carrier particles in an immune reaction solution. In this way, it is possible to obtain an appropriate measurement value even if it is contained excessively, thereby enabling measurement of a substance to be measured over a wide concentration range from a low concentration to a high concentration, and the prozone phenomenon used in this method. It is an object to provide an inhibitor, a reagent for measuring an immune reaction using the same, a method for measuring an immune reaction, and a method for suppressing a prozone phenomenon.
[0007]
[Means for Solving the Problems]
As a result of studying for a new technique for suppressing the prozone phenomenon in immunoassay, the present inventors have found that a sulfonate type or sulfate ester among various surfactants in an immune reaction solution containing a substance to be measured. It was found that if a salt type anionic surfactant is contained, the prozone phenomenon can be suppressed unexpectedly, and the present invention has been completed.
[0008]
That is, the present invention provides a prozone phenomenon inhibitor for immune reaction measurement, comprising one or two or more compounds selected from the group consisting of sulfate ester-based and sulfonate-based anionic surfactants. To do. Here, preferred examples of sulfate ester and sulfonate anionic surfactants include alkyl sulfate ester, alkyl benzene sulfonate, alkyl naphthalene sulfonate, alkyl diphenyl ether sulfonate. , Polyoxyethylene alkyl sulfate ester, polyoxyethylene alkyl allyl sulfate, and alkane sulfonate anionic surfactants, but are not limited thereto.
[0009]
The present invention also provides a reagent for measuring an immune reaction, which contains these compounds as a prozone phenomenon inhibitor in an immune reaction. It is preferable that the reagent for measuring immune reaction contains the prozone phenomenon inhibitor in such an amount that the concentration of the prozone phenomenon inhibitor in the immune reaction solution is 0.008 to 4%. The immune reaction measurement reagent is more preferably a gold colloid agglutination reaction. More preferably, the reagent for measuring an immune reaction contains a reaction accelerator.
[0010]
Further, the present invention is characterized in that an immune reaction is performed by suppressing a prozone phenomenon in an immune reaction by performing an immune reaction in a reaction solution containing the prozone phenomenon inhibitor for immune reaction measurement. An immune response measurement method is also provided. In the immune reaction measurement method, the content of the prozone phenomenon inhibitor for immune reaction measurement is preferably 0.008 to 4%. In addition, the immunoreaction measurement method in the present invention is particularly effective in the case of using an immunoagglutination reaction measurement method, and the gold colloid agglutination reaction measurement method is an example of a particularly preferred immune reaction agglutination measurement method. Moreover, it is more preferable to include a reaction accelerator in the reaction solution.
[0011]
The present invention is also characterized in that the immune reaction solution contains one or more compounds selected from the group consisting of sulfate-based and sulfonate-based anionic surfactants, A prozone phenomenon suppression method in an immunoassay is also provided. Here, preferred examples of sulfate ester and sulfonate anionic surfactants include alkyl sulfate ester, alkyl benzene sulfonate, alkyl naphthalene sulfonate, alkyl diphenyl ether sulfonate. , Polyoxyethylene alkyl sulfate ester, polyoxyethylene alkyl allyl sulfate, and alkane sulfonate anionic surfactants, but are not limited thereto. In the method for suppressing the prozone phenomenon, the concentration of the compound in the immune reaction solution is preferably 0.008 to 4%. The prozone phenomenon suppression method in the present invention is particularly effective in the immunoagglutination reaction measurement method, and the gold colloid agglutination reaction measurement method is an example of a particularly preferred immunoagglutination reaction measurement method. Moreover, it is more preferable to include a reaction accelerator in the reaction solution.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
The prozone phenomenon inhibitor of the present invention can be appropriately selected from known sulfonic acid salt type or sulfate ester type anionic surfactants, and in particular, alkylphenyl ether disulfonic acid sodium salt system [Example : Perex SSH; Kao Co., Ltd., Eleminol MON2; Sanyo Chemical Industries, Ltd.], alkylnaphthalene sulfonic acid sodium salt system [Example: Perex NBL; Kao Corporation], alkane sulfonic acid sodium salt system [Example right: Latemul PS; Kao Corporation], polyoxyethylene lauryl ether sulfate sodium salt system [Example: Emar E70C; Kao Corporation], higher alkyl ether sulfate ester system [Example: Sandet ENM; Sanyo Chemical Industries, Ltd.] Polyoxyethylene lauryl ether sulfate sodium salt type [Example: Sandet EN Sanyo Chemical Industries, Ltd.], alkyl sulfate ester salts, alkylbenzene sulfonic acid, polyoxyethylene alkyl aryl anionic surfactant of sulfate systems are preferred.
[0013]
The prozone phenomenon inhibitor of the present invention can be used by being added to a reagent used in an immunoassay. The reagent for measuring the immune reaction of the present invention may be any reagent that is usually used in a known immunoassay method, except that a prozone phenomenon inhibitor is used.
[0014]
The concentration of these anionic surfactants in the immune reaction solution is 0.008 to 4% (in the present invention, it represents% by weight), preferably 0.008 to 1%, more preferably 0.016 to 0.4%.
[0015]
Further, by adding a reaction accelerator to the reagent for measuring immune reaction, the measurement sensitivity can be increased while suppressing the prozone phenomenon. Any reaction accelerator may be used as long as it increases the measurement sensitivity, and examples thereof include polyethylene glycol, polyacrylic acid, chondroitin sulfate, carboxymethylcellulose, dextran, pullulan, polyvinyl alcohol, and polyvinylpyridone. Not limited to.
[0016]
For example, polyethylene glycol can be appropriately selected from known polyethylene glycols. The average molecular weight is 1000-50000, preferably 4000-20000. The concentration of polyethylene glycol in the immune reaction solution at the time of measurement has no specific upper limit for exerting the reaction promoting effect, but is usually 0.01 to 5.0%, preferably 0.1 to 3.0%. In particular, it may be added to the reagent for measuring the immune reaction so that it is preferably 0.2 to 2.0%.
[0017]
Further, the present invention does not preclude blending a nonionic surfactant with the reagent for measuring immune reaction. What is necessary is just to select suitably from well-known nonionic surfactant, when mix | blending a nonionic surfactant. Particularly preferred are polyoxyethylene alkyl ether, polyoxyethylene alkyl allyl ether, polyoxyethylene fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene sorbitol fatty acid ester, glycerin fatty acid Ester-based, polyoxyethylene alkylamine-based, and alkyl alkanolamide-based nonionic surfactants. When a nonionic surfactant is added, the immune reaction measurement reagent is used so that the concentration in the immune reaction solution at the time of measurement is 0.001 to 3.0%, preferably 0.02 to 2.0%. What is necessary is just to mix | blend.
[0018]
An immunoassay method particularly suitable for using the reagent for measuring an immune reaction of the present invention involves an immunoaggregation reaction. Of these, the latex aggregation method and the metal colloid aggregation method are particularly preferable. The metal colloid used in the metal colloid aggregation method includes colloids such as gold, silver, and selenium, and any of them may be used, but gold colloid is preferable from the viewpoint of easy use.
[0019]
For example, when measuring a substance to be measured which is an antigen (or antibody), in latex agglutination method or metal colloid aggregation method, an antibody (or antigen) corresponding to the antigen (or antibody) which is a substance to be measured is used as a carrier latex. Or pre-bonded to metal colloids. As an example of the measurement, in the case of the gold colloid aggregation method, a labeled antibody (or labeled antigen) previously bound to a gold colloid aggregates via an antigen (or antibody) that is a substance to be measured. The color difference (color tone change) generated at that time is optically measured to measure the amount of antigen or antibody.
[0020]
Substances to be measured to be measured with the immune reaction measuring reagent of the present invention include proteins, lipids, and saccharides, and examples thereof include various antigens, antibodies, receptors, enzymes, and the like. Specifically, C-reactive protein (CRP), fibrin degradation product (FDP), hemoglobin, hemoglobin A1c, α-fetoprotein (AFP), cystatin C, carcinoembryonic antigen (CEA), CA19-9, prostate specific antigen (PSA), pepsinogen I and II, collagen, serum amyloid A (SAA), ferritin, transferrin, α1-microglobulin, α2-macroglobulin, β2-microglobulin, α1-antichymotrypsin (ACT), myoglobin Examples include proteins, antigens and antibodies related to infectious diseases such as hepatitis B virus, hepatitis C virus, human immunodeficiency virus, Helicobacter pylori, and antibodies to these. According to the present invention, the prozone phenomenon in the immunoassay reaction, particularly the prozone phenomenon due to the excess of antigen, is suppressed, and these substances to be measured can be measured over a wide concentration range.
[0021]
In the present invention, it is not preferable to perform the immune reaction under excessively acidic or alkaline conditions. The pH of the reaction solution is preferably in the range of 4.5 to 9.5, more preferably in the range of 5.5 to 8.5. For maintaining the pH, suitable buffers such as phosphate buffer, Tris-HCl buffer, succinate buffer, glycylglycine, MES (2- (N-monophorino) ethanesulfonic acid), HEPES (N 2-hydroxyethyl-piperazine-N′-ethanesulfonic acid), TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid), PIPES (piperazine-1,4-bis (2-ethanesulfonic acid) )), DIPSO (3- (N′N-bis (2-hydroxyethyl) amino) -2-hydroxyethylpropanesulfonic acid), Tricine (tris (hydroxymethyl) methylglycine), TAPS (N-tris (hydroxymethyl)) Good buffer solution such as (methyl-3-aminopropane sulfonic acid) is preferably used. The buffering agent may be used in the immune reaction measurement reagent so that the concentration in the immune reaction solution at the time of measurement is 5 to 1000 mM, and more preferably in the range of 20 to 500 mM. That's fine.
[0022]
In the reagent for immune reaction of the present invention, animal serum, γ-globulin, specific antibodies against human IgG and IgM, albumin, or a modified or decomposed product thereof, sodium chloride and other inorganic salts, saccharides, amino acids A chelating agent such as EDTA, an SH reagent such as DTT, sodium azide, and the like. These substances may be included in the concentration range normally used in this field.
[0023]
[Action]
When the reagent of the present invention is used, the antigen (or antibody), which is the substance to be measured, is present in the sample at a high concentration, compared to the antibody (or antigen) bound to insoluble carrier particles such as latex or metal colloid. Even in the presence of an excessive amount of antigen (or antibody), the prozone phenomenon can be suppressed, and a wide concentration range can be obtained without re-adjusting the concentration balance of antigen and antibody by dilution of the sample. It is possible to measure the immune reaction of the substance to be measured.
[0024]
【Example】
Hereinafter, the present invention will be described more specifically based on a colloidal gold agglutination reaction as a typical example, but it is not intended that the present invention be limited thereto.
In the examples, the following anionic surfactants were used.
・ Perex SSH: Sodium alkyldiphenyl ether disulfonate (Kao Corporation)
・ Perex NBL: Sodium alkylnaphthalene sulfonate (Kao Corporation)
・ Latemul PS: Sodium alkane sulfonate (Kao Corporation)
・ Emar E70C: Sodium polyoxyethylene lauryl ether sulfate (Kao Corporation)
・ Eleminol MON2: Sodium alkyldiphenyl ether disulfonate (Sanyo Chemical Industries, Ltd.)
・ Sandet ENM: Higher alkyl ether sulfate ester (Sanyo Chemical Industry Co., Ltd.)
・ Sandet EN: Polyoxyethylene alkyl ether sulfate sodium salt (Sanyo Chemical Industry Co., Ltd.)
[0025]
<Reference Example 1> Preparation of colloidal gold solution
To 1 L of distilled water at 95 ° C., 2 mL of 10% chloroauric acid solution was added with stirring, and after 1 minute, 10 mL of 2% sodium citrate solution was added, stirred for another 20 minutes, and then cooled to 30 ° C. After cooling, the pH was adjusted to 7.1 with 0.1 M potassium carbonate solution.
[0026]
Reference Example 2 Preparation of anti-cystatin C antibody-binding gold colloid reagent
Anti-cystatin C antibody (Dako Japan Co., Ltd.) was diluted with 10 mM HEPES pH 7.1 containing 0.05% sodium azide to adjust the concentration to 50 μg / mL. 100 mL of this solution was added to about 1 L of the colloidal gold solution prepared in Reference Example 1 and stirred for 2 hours under refrigerated conditions. Next, 110 mL of 10 mM HEPES pH 7.1 containing 5.46% mannitol, 0.5% BSA and 0.05% sodium azide was added thereto, followed by stirring at 37 ° C. for 90 minutes. After centrifuging at 8,000 rpm for 40 minutes and removing the supernatant, about 1 L of 5 mM HEPES pH 7.5 (A solution) containing 3% mannitol, 0.1% BSA and 0.05% sodium azide was added. After the antibody-bound gold colloid is dispersed, the mixture is centrifuged at 8,000 rpm for 40 minutes, the supernatant is removed, the antibody-bound gold colloid is dispersed with the solution A to a total volume of 160 mL, and the anti-cystatin C antibody-bound gold colloid reagent Was prepared.
[0027]
Reference Example 3 Preparation of anti-AFP antibody-bound colloidal gold reagent
Anti-AFP antibody (Dako Japan Co., Ltd.) was diluted with 10 mM HEPES pH 7.1 containing 0.05% sodium azide to adjust the concentration to 50 μg / mL. 100 mL of this solution was added to about 1 L of the colloidal gold solution prepared in Reference Example 1 and stirred for 2 hours under refrigerated conditions. Next, 110 mL of 10 mM HEPES pH 7.1 containing 5.46% mannitol, 0.5% BSA and 0.05% sodium azide was added thereto, followed by stirring at 37 ° C. for 90 minutes. After centrifuging at 8,000 rpm for 40 minutes and removing the supernatant, about 1 L of 5 mM HEPES pH 7.5 (A solution) containing 3% mannitol, 0.1% BSA and 0.05% sodium azide was added. After the antibody-bound gold colloid is dispersed, the mixture is centrifuged at 8,000 rpm for 40 minutes, the supernatant is removed, the antibody-bound gold colloid is dispersed with the solution A to a total volume of 160 mL, and the anti-AFP antibody-bound gold colloid reagent is added. Prepared.
[0028]
<Example 1>
One of the following anions is added to 3 μL of serum containing cystatin C at a concentration of 0 to 50 μg / mL, to 0.2 M MES buffer (pH 6.0) containing 0.2% EDTA · 2Na and 1% NaCl. 240 μL of each prepared R1 reagent solution was added by adding the active surfactant at the indicated concentration, or without adding any anionic surfactant.
Perex SSH: 0.03% (Figure 1)
Perex NBL ... 0.1% (Fig. 1)
Laterum PS ... 0.03% (Fig. 1)
Emar E70C ... 0.05% (Fig. 2)
Eleminol MON2 ... 0.03% (Fig. 2)
Laterum S-180A ... 0.1% (Fig. 2)
Sandet ENM 0.2% (Figure 3)
Sandet EN ... 0.2% (Fig. 3)
Then, after 5 minutes at 37 ° C., 60 μL of the anti-cystatin C antibody-conjugated gold colloid solution prepared in Reference Example 2 was added as an R2 reagent solution, reacted at 37 ° C., and 27 points and 50 points using Hitachi 7150 automatic analyzer. The difference in absorbance (main wavelength 546 nm, subwavelength 660 nm) was measured. The results are shown in FIGS. As is clear from the figure, no prozone phenomenon was observed in any of the reaction solutions prepared by adding these anionic surfactants. On the other hand, it was observed that the prozone phenomenon occurred in the reaction solution without an anionic surfactant.
[0029]
<Example 2>
A nonionic surfactant was added to the immune reaction solution, and the presence or absence of the effect of suppressing the prozone phenomenon was compared with the case of adding the anionic surfactant Perex SSH. Using Triton X100, Tween 80 and Rhedol-TW? L120 as nonionic surfactants, containing 0.2% EDTA · 2Na and 1% NaCl, each containing 0.2% EDTA. Cystatin C was measured in the same manner as in Example 1 using MES buffer (pH 6.0) as each R1 reagent solution. At the same time, 0.2M MES buffer solution (pH 6.0) containing 0.2% EDTA · 2Na and 1% NaCl to which a concentration of Perex SSH was added to 0.03% was used as an R1 reagent solution. Cystatin C was measured in the same manner as in Example 1. The result is shown in FIG. As is clear from the figure, the nonionic surfactant did not have an inhibitory effect on the prozone phenomenon.
[0030]
<Example 3>
Japanese Patent Application Laid-Open No. 11-344492 describes the effect of suppressing the prozone phenomenon by sulfate. Therefore, an immune reaction solution to which sodium sulfate was added was prepared and compared with an immune reaction solution to which the anionic surfactant Plex SSH containing a sulfate group was added for the effect of suppressing the prozone phenomenon. 0.2 M MES buffer (pH 6.0) containing 0.2% EDTA · 2Na and 1% NaCl containing sodium sulfate at a concentration of 0, 2.0, 5.0, or 9.0%, respectively. Were used as R1 reagent solutions, and cystatin C was measured in the same manner as in Example 1 using these solutions. At the same time, 0.2M MES buffer (pH 6.0) containing 0.2% EDTA · 2Na and 1% NaCl to which a concentration of Perex SSH was added to 0.03% was used as an R1 reagent solution. Cystatin C was measured in the same manner as in Example 1. The results are shown in FIG. As is clear from the figure, when sodium sulfate was added, the measured value of cystatin C at a high concentration of 50 μg / mL was lower than the measured value of cystatin C concentration of 4 μg / mL, and the prozone phenomenon was not eliminated at all. In contrast, when Perex SSH was added at 0.03%, the measured value of cystatin C at a high concentration of 50 μg / mL did not fall below the measured value at a lower concentration range, and the prozone phenomenon was completely eliminated.
[0031]
<Example 4>
0.2M MES buffer containing 0.2% EDTA · 2Na and 1% NaCl with Perex SSH or Emar E70C added to a concentration of 0, 0.01, 0.02 or 0.03% ( Cystatin C was measured in the same manner as in Example 1 using pH 6.0) as the R1 reagent solution. The results are shown in FIGS. As apparent from FIG. 6, it was recognized that when the concentration of Perex SSH was 0.01%, the measured value of cystatin C at a high concentration of 50 μg / mL increased as compared with the case of no addition. When Perex SSH is added at a concentration of 0.03%, the measured value of cystatin C at a high concentration of 50 μg / mL does not fall below the measured value of cystatin C at a lower concentration range, and the prozone phenomenon is completely eliminated. It was.
[0032]
Even when Emar E70C in FIG. 7 was added, it was observed that the measured value at a high concentration of 50 μg / mL of cystatin C increased with the addition of 0.01% compared to the case without addition. When Emul E70C is added at a concentration of 0.02%, the measured value of cystatin C at a high concentration of 50 μg / mL does not fall below the measured value of cystatin C at a lower concentration range, and the prozone phenomenon is completely eliminated. It was.
[0033]
From these, it can be seen that PELEX SSH or EMAL E70C has an effect of suppressing the prozone phenomenon even at a low concentration of 0.01%.
[0034]
<Example 5>
The effects of anionic surfactants in the presence of nonionic surfactants on the prozone phenomenon were investigated. To 0.2M MES buffer (pH 6.0) containing 0.2% EDTA · 2Na and 1% NaCl with Tween 80 added to a concentration of 0.2% as a nonionic surfactant, Perex NBL was added as an ionic surfactant so that the concentrations would be 0, 0.2, 0.3, 0.4, and 0.5%, respectively, to obtain an R1 reagent solution. Cystatin C was measured using these in the same manner as in Example 1. The result is shown in FIG. When the concentration of Perex NBL was 0.3%, the measured value of cystatin C at a high concentration of 50 μg / mL did not fall below the measured value of cystatin C in the lower concentration region, and the prozone phenomenon was completely eliminated. When the concentration of Perex NBL was increased from 0.3%, a decrease in the measured value was observed as a whole, but the prozone phenomenon remained suppressed. From this, it can be seen that even if an anionic surfactant is used together with a nonionic surfactant, the effect of suppressing the prozone phenomenon of the anionic surfactant is not impaired.
[0035]
<Example 6>
When the concentration of Perex NBL was added to 0.5% in Example 5, the measured value of cystatin C was significantly decreased as a whole (FIG. 5). Therefore, the effect of suppressing the prozone phenomenon of an anionic surfactant in the presence of a reaction accelerator was investigated. R1 containing 0.5% of Plex NBL of Example 5 so that polyethylene glycol 20000 is used as a reaction accelerator and the concentration is 0, 0.5, 0.75, 1.0, or 1.25%, respectively. Cystatin C was measured in the same manner as in Example 1 by adding to the reagent solution. At the same time, polyethylene glycol 20000 was added to the R1 reagent solution without Plex NBL at 0.5%, and cystatin C was measured in the same manner as in Example 1. The results are shown in FIG. When polyethylene glycol 20000 is added to the R1 reagent solution without Plex NBL added, the measured value increases due to the reaction promoting effect of polyethylene glycol. However, since it simply increases uniformly over the entire concentration range of cystatin C, In addition, the suppression effect of the prozone phenomenon is not recognized. On the other hand, when polyethylene glycol 20000 was added to the R1 reagent solution containing 0.5% PELEX NBL, the measured value increased while suppressing the prozone phenomenon. Moreover, the effect of this polyethylene glycol was consistently increased corresponding to the concentration tested, and no clear upper limit concentration at which the effect peaked was found. From this, even if the measured value decreases when the prozone phenomenon is suppressed by adding an anionic surfactant, the prozone phenomenon can be reduced by adding an appropriate amount of a reaction accelerator such as polyethylene glycol. It can be seen that the measurement sensitivity can be freely set by increasing the measured value while suppressing it.
[0036]
<Example 7>
The inhibitory effect of anionic surfactants on the prozone phenomenon was examined for AFP measurement reagents other than cystatin C. Add 0.2 M MES buffer (pH 6.0) containing 0.2% Triton X100, 0.2% EDTA · 2Na and 1% NaCl to 3 μL of serum containing AFP at a concentration of 0 to 250 μg / mL as R1 reagent. 240 μL was added as a solution. Then, after 5 minutes at 37 ° C., 60 μL of the anti-AFP antibody-bound gold colloid solution prepared in Reference Example 3 was added as an R2 reagent solution, reacted at 37 ° C., and absorbance at 27 and 50 points using Hitachi 7150 automatic analyzer. The difference (main wavelength 546 nm, subwavelength 660 nm) was measured. Perex SSH was added to the R1 reagent solution as an anionic surfactant at a concentration of 0.1%, AFP was measured in the same manner, and the effect of inhibiting the prozone phenomenon of the anionic surfactant was examined. The results are shown in FIG. Also in the AFP measurement reagent, the effect of suppressing the prozone phenomenon was recognized by the addition of the anionic surfactant Perex SSH.
[Brief description of the drawings]
FIG. 1 is a graph (cystatin C) showing the prozone phenomenon inhibitory effect of an anionic surfactant.
FIG. 2 is a graph (cystatin C) showing the effect of inhibiting the prozone phenomenon of an anionic surfactant.
FIG. 3 is a graph (cystatin C) showing the prozone phenomenon inhibitory effect of an anionic surfactant.
FIG. 4 is a graph showing the prozone phenomenon with addition of a nonionic surfactant (cystatin C).
FIG. 5 is a graph (cystatin C) showing a comparison between sodium sulfate and an anionic surfactant in suppressing prozone phenomenon.
FIG. 6 is a graph (cystatin C) showing the relationship between the concentration of Perex SSH and the effect of suppressing the prozone phenomenon.
FIG. 7 is a graph showing the relationship between the concentration of Emar 70C and the effect of suppressing the prozone phenomenon (cystatin C).
FIG. 8 is a graph (cystatin C) showing the prozone phenomenon inhibitory effect of an anionic surfactant in the presence of a nonionic surfactant.
FIG. 9 is a graph (cystatin C) showing the prozone phenomenon inhibitory effect of an anionic surfactant in the presence of a nonionic surfactant and a reaction accelerator.
FIG. 10 is a graph (AFP) showing the effect of suppressing the prozone phenomenon of an anionic surfactant.
[Explanation of symbols]
Abs * 1000 = absorbance × 1000
Cys. C = cystatin C

Claims (23)

Sodium alkylphenyl ether disulfonate, sodium alkylnaphthalene sulfonate, sodium alkane sulfonate, sodium polyoxyethylene lauryl ether sulfate, higher alkyl ether sulfate and sodium polyoxyethylene lauryl ether sulfate A prozone phenomenon inhibitor for immune reaction measurement, comprising one or more compounds selected from the group consisting of salt-based anionic surfactants. Alkylphenyl ether disulfonate sodium anionic surfactant is sodium alkyldiphenyl ether disulfonate, alkyl naphthalene sulfonic acid sodium salt anionic surfactant is alkyl naphthalene sodium sulfonate, alkane sulfonic acid sodium salt. Anionic surfactant is sodium alkane sulfonate, polyoxyethylene lauryl ether sodium sulfate anionic surfactant is polyoxyethylene lauryl ether sodium sulfate, higher alkyl ether sulfate ester anionic surfactant Higher alkyl ether sulfate ester or polyoxyethylene lauryl ether sulfate sodium salt anionic surfactant is polyoxyethylene alkyl ether sulfate Immunoassay for prozone phenomenon inhibitor according to claim 1 which is ester sodium. Sodium alkylphenyl ether disulfonate, sodium alkylnaphthalene sulfonate, sodium alkane sulfonate, sodium polyoxyethylene lauryl ether sulfate, higher alkyl ether sulfate and sodium polyoxyethylene lauryl ether sulfate A reagent for measuring an immune reaction, comprising one or more compounds selected from the group consisting of a salt-based anionic surfactant as a prozone phenomenon inhibitor in an immune reaction. Surfactant of alkyl phenyl ether disulfonic acid sodium salt or alkane sulfonic acid sodium salt in a concentration of 0.03 to 4%, alkyl naphthalene sulfonic acid sodium salt, higher alkyl ether sulfate ester or polyoxyethylene lauryl ether sulfate It is characterized by containing an ester sodium salt surfactant in a concentration of 0.1 to 4%, or a polyoxyethylene lauryl ether sulfate sodium surfactant in an amount of 0.02 to 4%. The reagent for measuring immune reaction according to claim 3 . Alkylphenyl ether disulfonate sodium anionic surfactant is sodium alkyldiphenyl ether disulfonate, alkyl naphthalene sulfonic acid sodium salt anionic surfactant is alkyl naphthalene sodium sulfonate, alkane sulfonic acid sodium salt. Anionic surfactant is sodium alkane sulfonate, polyoxyethylene lauryl ether sodium sulfate anionic surfactant is polyoxyethylene lauryl ether sodium sulfate, higher alkyl ether sulfate ester anionic surfactant Higher alkyl ether sulfate ester or polyoxyethylene lauryl ether sulfate sodium salt anionic surfactant is polyoxyethylene alkyl ether sulfate Immunoassay reagent according to claim 3 or 4 is ester sodium. The reagent for measuring an immune reaction according to any one of claims 3 to 5 , wherein the immune reaction is an immune agglutination reaction. The reagent for measuring immune reaction according to any one of claims 3 to 6 , wherein the immune reaction is a colloidal gold agglutination reaction. The reagent for measuring an immune reaction according to any one of claims 3 to 7 , further comprising a reaction accelerator. The reagent for measuring immune reaction according to claim 8, wherein the reaction accelerator is selected from the group consisting of polyethylene glycol, polyacrylic acid, chondroitin sulfate, carboxymethylcellulose, dextran, pullulan, polyvinyl alcohol and polyvinylpyridone . Alkyl phenyl ether disulfonic acid sodium salt, Arukirunafu Tarensuruhon sodium salts, alkanesulfonic acid sodium salt, polyoxyethylene lauryl ether sulfate sodium salt, higher alkyl ether ester salts, and polyoxyethylene lauryl ether sodium sulfate ester sulfate By incorporating one or more compounds selected from the group consisting of salt-based anionic surfactants into the immune reaction solution as a prozone phenomenon inhibitor for immune reactions, the prozone phenomenon in the immune reaction is reduced. A method for measuring an immune reaction, characterized by suppressing an immune reaction. Surfactant of alkyl phenyl ether disulfonic acid sodium salt or alkane sulfonic acid sodium salt in a concentration of 0.03 to 4%, alkyl naphthalene sulfonic acid sodium salt, higher alkyl ether sulfate ester or polyoxyethylene lauryl ether sulfate surfactant concentration from 0.1 to 4 percent of the ester sodium salt, or polyoxyethylene lauryl ether surfactant sodium sulfate salt and is intended to be contained at a concentration of 0.02 to 4%, claim 10 The method for measuring an immune reaction according to 1. Alkylphenyl ether disulfonate sodium anionic surfactant is sodium alkyldiphenyl ether disulfonate, alkyl naphthalene sulfonic acid sodium salt anionic surfactant is alkyl naphthalene sodium sulfonate, alkane sulfonic acid sodium salt. Anionic surfactant is sodium alkane sulfonate, polyoxyethylene lauryl ether sodium sulfate anionic surfactant is polyoxyethylene lauryl ether sodium sulfate, higher alkyl ether sulfate ester anionic surfactant Higher alkyl ether sulfate ester or polyoxyethylene lauryl ether sulfate sodium salt anionic surfactant is polyoxyethylene alkyl ether sulfate Immunoassay method according to claim 10 or 11, which is a ester sodium. It is by immune agglutination assays, immunoassay method according to claim 10 to 12. It is due to colloidal gold agglutination assays, immunoassay method according to any one of claims 10 to 13. Further characterized in that to incorporate a reaction accelerator in the reaction solution, immunoassay method according to any one of claims 10 to 14. The immune reaction measuring method according to claim 15, wherein the reaction accelerator is selected from the group consisting of polyethylene glycol, polyacrylic acid, chondroitin sulfate, carboxymethylcellulose, dextran, pullulan, polyvinyl alcohol and polyvinylpyridone . In the immune reaction solution, sodium alkylphenyl ether disulfonate, sodium alkylnaphthalene sulfonate, alkane sulfonate, sodium polyoxyethylene lauryl ether sulfate, higher alkyl ether sulfate and polyoxy A method for suppressing a prozone phenomenon in an immunoassay, comprising one or more compounds selected from the group consisting of ethylene lauryl ether sulfate sodium salt-based anionic surfactants. In the immune reaction solution , the concentration of alkylphenyl ether disulfonic acid sodium salt type or alkanesulfonic acid sodium salt type surfactant is 0.03 to 4%, alkyl naphthalene sulfonic acid sodium salt type, higher alkyl ether sulfate ester type Alternatively, the concentration of the polyoxyethylene lauryl ether sulfate sodium salt surfactant is 0.1 to 4%, or the concentration of the polyoxyethylene lauryl ether sulfate sodium surfactant is 0.02 to 4% . The method for suppressing a prozone phenomenon in an immunoassay according to claim 17 . Alkylphenyl ether disulfonate sodium anionic surfactant is sodium alkyldiphenyl ether disulfonate, alkyl naphthalene sulfonic acid sodium salt anionic surfactant is alkyl naphthalene sodium sulfonate, alkane sulfonic acid sodium salt. Anionic surfactant is sodium alkane sulfonate, polyoxyethylene lauryl ether sodium sulfate anionic surfactant is polyoxyethylene lauryl ether sodium sulfate, higher alkyl ether sulfate ester anionic surfactant Higher alkyl ether sulfate ester or polyoxyethylene lauryl ether sulfate sodium salt anionic surfactant is polyoxyethylene alkyl ether sulfate According to claim 17 or 18 which is a ester sodium, method of inhibiting prozone phenomenon. 20. The method for suppressing a prozone phenomenon in an immunoassay according to claim 17 to 19 , wherein the immunoassay is based on an immunoagglutination assay. The method for suppressing a prozone phenomenon in an immunoassay according to any one of claims 17 to 20 , wherein the immunoassay is based on a colloidal gold agglutination assay. The method for suppressing a prozone phenomenon in an immunoassay method according to any one of claims 17 to 21 , wherein a reaction accelerator is further contained in the immune reaction solution. The prozone in the immunoassay method according to claim 22, wherein the reaction accelerator is selected from the group consisting of polyethylene glycol, polyacrylic acid, chondroitin sulfate, carboxymethylcellulose, dextran, pullulan, polyvinyl alcohol and polyvinylpyridone. How to suppress the phenomenon .

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