JP3920458B2 - Immunoassay - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring an antigen or antibody in a test sample, and more particularly to an immunoassay method with enhanced sensitivity or an expanded measurement range.
[0002]
[Prior art]
Conventionally, immunoassay methods based on an antigen-antibody reaction are known that utilize an agglutination reaction or that utilizes an antibody labeled with a detection enzyme. In these immunoassays, the amount of immune complex produced by a specific antigen-antibody reaction is measured visually or as an optical change. In particular, an agglutination reaction based on an antigen-antibody reaction between an antigen (or antibody) to be measured and an insolubilized particle (hereinafter abbreviated as “immobilized particle”) in which an antibody (or antigen) corresponding to the object to be measured is supported on an insoluble carrier. Alternatively, an antigen (or antibody) measurement method (hereinafter abbreviated as “aggregation method”) in a test sample using an agglutination-inhibiting reaction is widely used by using an automatic analyzer because the measurement can be automated. Yes.
[0003]
In many of the conventional aggregation methods, latex particles sensitized with polyclonal or monoclonal antibodies are reacted with target antigens in a test sample to form immune aggregates, and the degree of aggregation is determined. In these aggregation methods, the concentration range that can be measured is limited to a certain concentration range. Therefore, in order to acquire a wide measurement range from a low concentration range to a high concentration range, an attempt has been made to control an optical change or a change amount that reflects the formation of immune aggregates. Examples of such attempts include: (1) By reducing or increasing the particle size according to the concentration of the measurement system, the optical change rate corresponding to the same measurement target amount can be changed to the original particle size. (2) A method of simply increasing or decreasing the amount of immobilized particles used in one measurement, (3) Loading two different amounts of antibody (2) Measurement in a high concentration range by reacting immobilized particles and free antibodies with antigens competitively (2) Latex particles with different particle diameters (JP-A-55-15126) A method (JP-A-59-92353) intended to expand the range is known.
[0004]
[Problems to be solved by the invention]
However, the above conventional methods have the following drawbacks. In other words, in the method (1) of changing the particles of the insoluble carrier, the measurable range is generally limited by the particle diameter. In the method of changing the amount of immobilized particles used in (2), the relationship between the amount of immobilized particles used and the amount of optical change caused by immune aggregates may not change temporarily. The measurement range in the area may be inferior to the original one. The method of (3) was developed as an improved method of the methods of (1) and (2), but each time a reagent is prepared, the complexity of combining insoluble carriers having different antibody amounts and particle sizes is reduced. Accompany. In the method (4), two specific antibodies must be prepared, and there are problems such as a decrease in accuracy due to a decrease in the amount of optical change in the low value range.
[0005]
Accordingly, an object of the present invention is to expand the measurement range in an immunoassay method using agglutination caused by the reaction between a measurement object and immobilized particles without the above-described drawbacks.
[0006]
[Means for Solving the Problems]
In this situation, the present inventors have conducted intensive studies, and as a result, guanidines conventionally used for the purpose of eliminating non-specific reactions in the aggregation method (Japanese Patent Laid-Open Nos. 56-2556 and 56-158947). Unexpectedly, it was found that the measurable range was expanded or the measurement sensitivity was increased, and the present invention was completed.
[0007]
That is, the present invention adds an insoluble carrier particle carrying an antibody or antigen to the measurement target to a sample containing the antigen or antibody to be measured, and measures the degree of formation of an immune complex by the antigen-antibody reaction. An immunoassay that is so small that nonspecific reactions do not affect the measurement results (measured values and conditions under which no compound is used to avoid nonspecific reactions) Immunization characterized by coexisting 7.5 / 202 M to 75/202 M in the reaction system with guanidine, a guanidine salt or a derivative thereof It is intended to provide a method for expanding the measurement range in a mechanical measurement method.
The present invention also provides an immunoassay for measuring the degree of formation of an immune complex by an antigen-antibody reaction by adding an insoluble carrier particle carrying an antibody or antigen against the measurement target to a sample containing the antigen or antibody to be measured. Measurement method that is so small that non-specific reaction does not affect the measurement result (measured value and condition under which no compound is used to avoid non-specific reaction) Guanidine, guanidine salt, or a derivative thereof is allowed to coexist in the reaction system in a range of 24/324 M to 120/324 M). The present invention provides a method for increasing measurement sensitivity in an immunoassay.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
The above-mentioned conventional method attempts to control the optical change rate physically by changing the size and concentration of the insoluble carrier, or to control the immune reaction by a competitive reaction. The principle of the present invention is completely different from that of the present invention in which the concentration is not changed and a substance that performs an antigen-antibody reaction is not used.
[0009]
By the way, in order to avoid non-specific aggregation due to sample-derived components in the immunoaggregation reaction, a method in which a compound such as guanidine hydrochloride, iodide, thiocyanic acid or the like is allowed to coexist in the reagent is known (Japanese Patent Laid-Open No. 56). -2556, JP 56-158947). However, these are only for the purpose of avoiding non-specific aggregation due to blood-derived components in the sample, and antigen-antibody reactions in which a specific antigen-antibody reaction is observed regardless of the presence of these compounds, ie non-specific Only antigen-antibody reactions whose reaction does not affect the measurement results are targeted, and by adding guanidines to this system, the present invention, which enables highly sensitive or wide-range measurements, is achieved by the coexistence of compounds. The effects are completely different.
[0010]
In the present invention, “the non-specific reaction is so small that it does not affect the measurement result” means, for example, a compound (guanidine hydrochloride, iodide, thiocyanate, urea, which has been conventionally used for avoiding the non-specific reaction). The correlation function of the measured value derived under the condition where the compound is not present and the condition where the compound is present is 0.7 or more, or the ratio between the measured value when the compound is not present and the measured value when the compound is present is 0.7. This refers to the case where the consistency is within 1.3.
[0011]
Examples of the guanidine salt used in the present invention include guanidine hydrochloride, guanidine carbonate, guanidine thiocyanate, guanidine sulfate, guanidine nitrate, guanidine phosphate, guanidine sulfamate, and the like, Examples thereof include guanidinobenzoic acid, guanidinoglutaric acid, guanidinosuccinic acid, guanidinoacetic acid, guanidinopropionic acid, guanidinobenzimidazole and the like. The concentration of these substances in the measurement system is not particularly limited, but is preferably 1M or less, particularly preferably 0.001 to 1M.
[0012]
Although the mechanism of action of guanidines in the present invention is not clear, it does not react with antigens or antibodies in the measurement system, so it is considered that the rate of formation of immune complexes is affected.
[0013]
As the insoluble carrier used in the present invention, any known substance conventionally used for measuring antigens or antibodies using immobilized particles can be used without any limitation, for example, organic polymer substances, inorganic substances, cell membranes. , Blood cells, microorganisms and the like.
[0014]
As the organic polymer substance, for example, latex particles in which fine powders such as acrylic acid polymer, styrene polymer, and methacrylic acid polymer are uniformly suspended are preferable. Examples of the inorganic substance include fine particles such as silica and alumina. The shape of the insoluble carrier is not particularly limited, and the average particle size is preferably 0.02 to 1.6 μm, particularly preferably 0.03 to 1.2 μm.
[0015]
As for the method for immobilizing an antibody or antigen on an insoluble carrier, a conventional immobilization method such as physical adsorption, covalent bond, or immunological bond can be used. There are no particular restrictions on the solution that dissolves and suspends the substance that affects the formation rate of the immune complex and the immobilized particles, but in general, phosphate buffer, glycine buffer, Tris buffer, Good's buffer A buffer solution such as a liquid can be used, and additives such as sodium chloride can be added as necessary. The pH in the reaction is preferably 5 to 10, more preferably 6 to 9. The concentration of the immobilized particles in the reagent finally prepared is not particularly limited, but is preferably 0.1 to 10 mg / ml in the suspension.
[0016]
The antibody used in the present invention may be either a monoclonal antibody or a polyclonal antibody. Further, the antibodies may be used alone or as a mixture of plural kinds.
[0017]
The substance to be measured in the present invention is not particularly limited, and the present invention can be applied to any substance that is measured using an antigen-antibody reaction.
[0018]
【Example】
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.
[0019]
Example 1
(1) Preparation of anti-CRP antibody 100 μg of purified human CRP (Chemicon) was used for one immunization. Freund's complete adjuvant was used for the first immunization, and incomplete adjuvant was used for the booster immunization. For each immunization, 200 μl of an emulsion prepared by mixing equal amounts of CRP and Freund's adjuvant was used and injected into the peritoneal cavity of BALB / c mice. Immunization was repeated 4 times at 2-week intervals.
[0020]
Antibody titers in blood collected from the mouse fundus vein were measured by ELISA, and mice with high antibody titers were selected for cell fusion. Two weeks after the fourth immunization, 100 μg of CRP dissolved in 200 μl of physiological saline was injected into the abdominal cavity of the mouse, and the spleen was removed 3 days later. The spleen was loosened in RPMI1640 medium and collected by centrifugation at 1500 rpm to collect splenocytes. After washing with the same medium, 2 ml of RPMI1640 medium containing 15% fetal calf serum was added to obtain a cell suspension. 10 After mixing ⁸ splenocytes and 10 ⁷ myeloma cells SP2 / O-AG14, the precipitate was collected by centrifugation at 1500 rpm, and the GKN solution (8 g sodium chloride, 0.4 g potassium chloride, 2 g glucose, phosphoric acid) A suspension of 1.41 g of disodium hydrogen and 0.78 g of sodium dihydrogen phosphate dihydrate dissolved in purified water to make 1 liter) was washed by centrifugation and the precipitate was recovered. This was placed in 30 ml of RPMI1640 medium containing 15% fetal bovine serum, and HAT medium and feeder cells were placed in 200 μl per well of three 96-well microplates. The cells were cultured in a gas incubator.
[0021]
The presence of the anti-CRP antibody in the culture supernatant was evaluated by an ELISA method in which CRP was immobilized. After 10 days, proliferation of the fused cells was confirmed in all wells. Specifically, 100 μl of 10 mM phosphate buffer (pH 7.2; hereinafter abbreviated as PBS) containing 150 mM sodium chloride containing CRP at 10 μg / ml was dispensed into a 96-well microplate and left at 4 ° C. overnight. . After leaving it, it was discarded, and then it was washed 3 times with 300 μl of PBS containing 0.05% Tween20 and 1% bovine serum albumin, 50 μl of each culture supernatant was added and left at room temperature for 1 hour. After washing three times with PBS containing 0.05% Tween 20, 50 μl of peroxidase-labeled anti-mouse antibody (Daiichi Chemical) was added and left at room temperature for 1 hour. This was washed 3 times with PBS containing 0.5% Tween20, 50 μl of citrate buffer solution (pH 5) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added, allowed to stand at room temperature for 15 minutes, and then 50 μl of 4.5 N sulfuric acid was added. In addition, the reaction was stopped, the absorbance at a wavelength of 492 nm was measured, and a well having a high absorbance was selected.
[0022]
Monocloning was performed by limiting dilution. That is, 0.1 ml each of a 96-well microplate in which 106 ⁶ thymocytes of BALB / c mice were dispensed as feeder cells was diluted to 10 hybrids in a positive well. The medium was cultured for 10 days at 37 ° C. in a 5% carbon dioxide medium using RPMI1640 containing 15% fetal calf serum for the first time and HT medium for the first time. Anti-CRP monoclonal antibody-producing cells (hybridoma 08204; deposited as FERM P-16765 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology) by repeating the selection of positive wells by ELISA and the single cloning operation by limiting dilution three times each. Got. About 10 ⁵ cells were administered to the abdominal cavity of mice pretreated with pristane, and the generated ascites was collected. After removing insolubles by centrifugation, an equal amount of saturated ammonium sulfate solution was added, and the mixture was allowed to stand overnight with stirring, and then the precipitate was collected by centrifugation. The precipitate was dissolved in 20 mM Tris buffer (pH 8) and dialyzed. The dialysis contents were adsorbed on a DEAE-Sepharose column equilibrated with the same buffer, then eluted with a sodium chloride 0-0.3M gradient in the same buffer, and the IgG fraction was dialyzed against 0.05 M glycine buffer. The purified antibody was obtained.
[0023]
Monoclonal antibodies (hereinafter referred to as “anti-CRP monoclonal antibodies”) that cause immunoaggregation by the use of a single type were selected as follows. To 5 ml of a solution in which the purified antibody is mixed with 0.05 M glycine buffer (pH 8) at a concentration of 1.4 mg / ml, 5 ml of 5% suspension of polystyrene latex (manufactured by Sekisui Chemical Co., Ltd.) with an average particle size of 0.1 μm is added. Stir for 2 hours at 4 degrees Celsius. After removing the supernatant by centrifugation, 0.05 M glycine buffer (pH 8) containing 2% bovine serum albumin was added to the precipitate, and the mixture was stirred overnight at 4 degrees Celsius. After collecting the precipitate by centrifugation, the suspension was suspended in 0.05 M Tris buffer (pH 7.5) containing 2% bovine serum albumin so that the absorbance at 600 nm was 2 OD, and each anti-CRP antibody immobilized particle A suspension was prepared. Add 2 μl of CRP-containing sample solution to 150 μl of 0.02 M Tris buffer (pH 8.5) containing 0.2 M sodium chloride, warm at 37 degrees Celsius for 5 minutes, and then add 50 μl of anti-CRP antibody-immobilized particle suspension. In addition, the amount of change in absorbance at a wavelength of 600 nm for 1 to 5 minutes after stirring was measured, and an antibody having a change in absorbance was selected.
[0024]
(2) Preparation of anti-CRP antibody-immobilized particle suspension Polystyrene latex with an average particle size of 0.1 μm in 5 ml of a mixture of anti-CRP monoclonal antibody mixed with 0.05 M glycine buffer (pH 8) at a concentration of 1.4 mg / ml. 5 ml of 5% suspension was added and stirred at 4 degrees Celsius for 2 hours. After removing the supernatant by centrifugation, 0.05 M glycine buffer (pH 8) containing 2% bovine serum albumin was added to the precipitate, and the mixture was stirred overnight at 4 degrees Celsius. After collecting the precipitate by centrifugation, the suspension was suspended in 0.05 M Tris buffer (pH 7.5) containing 2% bovine serum albumin so that the absorbance at 600 nm was 2 OD, and the anti-CRP antibody-immobilized particles were suspended. A suspension was prepared.
[0025]
(3) Preparation of guanidine hydrochloride solution Guanidine hydrochloride (manufactured by Kishida Chemical Co., Ltd.) is mixed with 0.02 M Tris buffer (pH 8.5) containing 0.2 M sodium chloride at a concentration of 0.1 to 1 M, and the guanidine hydrochloride solution is mixed. Prepared.
[0026]
(4) Measurement of CRP To 150 μl of guanidine hydrochloride solution, add 2 μl of a sample solution containing CRP, warm at 37 ° C. for 5 minutes, add 50 μl of anti-CRP antibody-immobilized particle suspension, and stir The change in absorbance at a wavelength of 600 nm for 5 minutes was measured. The relationship between the obtained absorbance and the CRP concentration is shown in FIG.
[0027]
Comparative Example 1
Using 150 μl of 0.02 M Tris buffer (pH 8.5) containing 0.2 M sodium chloride in place of the guanidine hydrochloride solution, CRP was measured in the same manner as (4) in Example 1, and the obtained absorbance The relationship of CRP concentration is shown in FIG.
[0028]
Example 2
(1) Preparation of anti-Lp (a) antibody-immobilized particle suspension Monoclonal antibody (industrial industry) that produces immunoaggregation by using a single kind of purified human apo (a) as an immunogen, obtained from mice by a conventional method Produced by Hybridoma 28205 (FERM BP-3755) deposited at the Institute of Biotechnology, National Institute of Biotechnology, hereinafter referred to as “anti-Lp (a) monoclonal antibody”] at a concentration of 1.4 mg / ml, 0.05 M 5 ml of a 5% polystyrene latex (manufactured by Sekisui Chemical Co., Ltd.) having an average particle size of 0.1 μm was added to 5 ml of a solution mixed with glycine buffer (pH 9), and the mixture was stirred at 4 ° C. for 2 hours. After removing the supernatant, 0.05M glycine buffer solution (pH 9) containing 2% bovine serum albumin was added to the precipitate, and the mixture was stirred overnight at 4 ° C. The precipitate was collected by centrifugation and collected into 2% bovine. 0.05M glycine buffer (pH 9) containing serum albumin In absorbance at a wavelength of 600nm is suspended so that the 2 OD, was prepared anti Lp (a) antibody-immobilized particle suspension.
[0029]
(2) Preparation of guanidine hydrochloride solution Guanidine hydrochloride (manufactured by Kishida Chemical Co., Ltd.) was mixed with 0.05 M glycine buffer (pH 9) containing 0.2 M sodium chloride at a concentration of 0.1 to 1 M to prepare a guanidine hydrochloride solution. .
[0030]
(3) Measurement of Lp (a) Add 4 μl of sample solution containing Lp (a) to 240 μl of guanidine hydrochloride solution, heat at 37 degrees Celsius for 5 minutes, and then suspend anti-Lp (a) antibody-immobilized particles. 80 μl of the solution was added and the change in absorbance at a wavelength of 600 nm was measured for 1 to 5 minutes after stirring. The relationship between the obtained absorbance and the Lp (a) concentration is shown in FIG.
[0031]
Comparative Example 2
In place of guanidine hydrochloride solution, 240 μl of 0.05 M glycine buffer (pH 9) containing 0.2 M sodium chloride was used, Lp (a) was measured in the same manner as (3) of Example 2, and the resulting absorbance was obtained. FIG. 2 shows the relationship between Lp (a) concentration and Lp (a) concentration.
[0032]
Example 3
(1) Preparation of anti-myoglobin antibody-immobilized particle suspension Anti-myoglobin-rabbit antibody (manufactured by Oriental Yeast Co., Ltd.) is mixed with 0.05M glycine buffer (pH 8) at a concentration of 1.4 mg / ml in an average particle size of 5 ml. 5 ml of a 5% suspension of polystyrene latex (manufactured by Sekisui Chemical Co., Ltd.) having a diameter of 0.2 μm was added and stirred at 4 degrees Celsius for 2 hours. After removing the supernatant by centrifugation, 0.05 M glycine buffer (pH 8) containing 2% bovine serum albumin was added to the precipitate, and the mixture was stirred overnight at 4 degrees Celsius. The precipitate was collected by centrifugation, suspended in 0.05 M Tris buffer (pH 7.5) containing 2% bovine serum albumin so that the absorbance at 600 nm was 2 OD, and the anti-myoglobin antibody-immobilized particle suspension. Was prepared.
[0033]
(2) Preparation of guanidine hydrochloride solution A guanidine hydrochloride solution was prepared by mixing guanidine hydrochloride (manufactured by Kishida Chemical Co., Ltd.) in 0.05 M Tris buffer (pH 7.5) containing 0.15 M sodium chloride at a concentration of 250 mM.
[0034]
(3) Measurement of myoglobin Add 2 μl of a sample solution containing myoglobin to 150 μl of guanidine hydrochloride solution, warm it at 37 degrees Celsius for 5 minutes, add 50 μl of the anti-myoglobin antibody-immobilized particle suspension, and then add 1 to The change in absorbance at a wavelength of 600 nm for 5 minutes was measured. The relationship between the obtained absorbance and myoglobin concentration is shown in FIG.
[0035]
Comparative Example 3
In place of guanidine hydrochloride solution, 150 μl of 0.05 M Tris buffer (pH 7.5) containing 0.15 M sodium chloride was used, and myoglobin was measured in the same manner as in (3) of Example 3. The relationship of myoglobin concentration is shown in FIG.
[0036]
Example 4
(1) Preparation of anti-CRP antibody-immobilized particle suspension An anti-CRP antibody-immobilized particle suspension was prepared in the same manner as in Example 1.
[0037]
(2) Preparation of guanidine salt solution Guanidine carbonate (manufactured by Sigma) was mixed with 0.02 M Tris buffer (pH 8.5) containing 0.2 M sodium chloride at a concentration of 0.05 to 0.25 M to prepare a guanidine carbonate solution. .
[0038]
(3) Measurement of CRP To 150 μl of guanidine carbonate solution, add 2 μl of sample solution containing CRP, warm at 37 degrees Celsius for 5 minutes, add 50 μl of anti-CRP antibody-immobilized particle suspension and stir 1 to The change in absorbance at a wavelength of 600 nm for 5 minutes was measured. The relationship between the obtained absorbance and the CRP concentration is shown in FIG.
[0039]
Comparative Example 4
Using 150 μl of 0.02 M Tris buffer (pH 8.5) containing 0.2 M sodium chloride in place of the guanidine carbonate solution, CRP was measured in the same manner as in (3) of Example 4, and the absorbance obtained was The relationship of CRP concentration is shown in FIG.
[0040]
Example 5
(1) Preparation of anti-CRP antibody-immobilized particle suspension An anti-CRP antibody-immobilized particle suspension was prepared in the same manner as in Example 1.
[0041]
(2) Preparation of guanidine salt solution Guanidine thiocyanate (manufactured by Sigma) is mixed with 0.02M Tris buffer (pH 8.5) containing 0.2M sodium chloride at a concentration of 0.05 to 0.25M. A solution was prepared.
[0042]
(3) Measurement of CRP To 150 μl of guanidine / thiocyanate solution, add 2 μl of sample solution containing CRP, warm at 37 degrees Celsius for 5 minutes, and then add 50 μl of anti-CRP antibody-immobilized particle suspension and stir. The amount of change in absorbance at a wavelength of 600 nm for 1 to 5 minutes was measured. The relationship between the obtained absorbance and the CRP concentration is shown in FIG.
[0043]
Comparative Example 5
In place of the guanidine / thiocyanate solution, 150 μl of 0.02 M Tris buffer (pH 8.5) containing 0.2 M sodium chloride was used, and CRP was measured in the same manner as (3) of Example 5 to obtain. The relationship between absorbance and CRP concentration is shown in FIG.
[0044]
Example 6
(1) Preparation of anti-CRP antibody-immobilized particle suspension An anti-CRP antibody-immobilized particle suspension was prepared in the same manner as in Example 1.
[0045]
(2) Preparation of guanidine hydrochloride solution Guanidine hydrochloride (manufactured by Kishida Chemical Co., Ltd.) was mixed with 0.02 M Tris buffer (pH 8.5) containing 0.2 M sodium chloride at a concentration of 100 mM to prepare a guanidine hydrochloride solution.
[0046]
(3) Measurement of CRP To 150 μl of guanidine hydrochloride solution, add 2 μl of serum containing CRP, warm at 37 degrees Celsius for 5 minutes, add 50 μl of anti-CRP antibody-immobilized particle suspension and stir 1-5 The amount of change in absorbance at a wavelength of 600 nm was measured. In addition, using 150 μl of 0.02M Tris buffer (pH 8.5) containing 0.2M sodium chloride instead of guanidine hydrochloride solution, measure the same, and calculate the CRP concentration from the amount of change in absorbance of a sample with a known CRP concentration. Correlation was evaluated. The result is shown in FIG.
At the same time, CRP concentrations in 25 cases of serum were measured using a commercially available latex immunoturbidimetric reagent (CRP latex “Seiken” manufactured by Denka Seiken Co., Ltd.), and the correlation was evaluated. The results are shown in FIG.
[0047]
Example 7
(1) Preparation of anti-Lp (a) antibody-immobilized particle suspension An anti-Lp (a) antibody-immobilized particle suspension was prepared in the same manner as in Example 2.
[0048]
(2) Preparation of guanidine hydrochloride solution Guanidine hydrochloride (manufactured by Kishida Chemical Co., Ltd.) was mixed with 0.05 M glycine buffer (pH 9) containing 0.2 M sodium chloride at a concentration of 100 mM to prepare a guanidine hydrochloride solution.
[0049]
(3) Measurement of Lp (a) 4 μl of serum containing Lp (a) is added to 240 μl of guanidine hydrochloride solution, heated at 37 degrees Celsius for 5 minutes, and then anti-Lp (a) antibody-immobilized particle suspension. 80 μl was added, and the change in absorbance at a wavelength of 600 nm was measured for 1 to 5 minutes after stirring. In addition, instead of guanidine hydrochloride solution, 240 μl of 0.05 M glycine buffer solution (pH 9) containing 0.2 M sodium chloride was used for the same measurement, and Lp (a) was calculated from the amount of change in absorbance of a sample with known Lp (a) concentration. The results of calculating the concentration and evaluating the correlation are shown in FIG.
At the same time, the Lp (a) concentration of 20 sera was measured using a commercially available immunoturbidimetric reagent (Lp (a) Latex “Daiichi” manufactured by Daiichi Chemicals) to evaluate the correlation. The results are shown in FIG.
[0050]
As is clear from the evaluation diagrams 1 to 5, in Examples 1 to 5, the rate of change in absorbance depending on the antigen concentration is influenced by the presence of guanidine salt, and the absorbance is higher than that in Comparative Examples 1 to 5 or up to a high concentration range. The measurable range of change has been expanded. That is, in FIGS. 1, 4 and 5, the comparative example without using a guanidine salt reached a saturation at a CRP concentration of 20 mg / dl, and measurement in a high concentration range exceeding this cannot be performed. In the example, even if it exceeds 20 mg / dl, the absorbance further increases, and measurement in a higher concentration range is possible. 2 and 3, the example using the guanidine salt enables measurement with higher sensitivity as compared with the comparative example not using the guanidine salt.
[0051]
Also, FIGS. 6 to 9 of Examples 6 and 7 show that the effect of the guanidine salt in the present invention is different from the avoidance of nonspecific aggregation derived from the serum sample in the prior art, and there is no difference in the specificity of the antigen-antibody reaction. It shows no effect.
[0052]
【The invention's effect】
According to the method of the present invention, an agglutination immunoassay with high sensitivity or a wide measurement range can be achieved. In addition, guanidine, guanidine salts and derivatives thereof used in the present invention are stable chemical substances, and in principle have characteristics that do not affect the accuracy of antigen-antibody reaction and measurement system, and the production method is simple. Cost is also cheap.
[Brief description of the drawings]
FIG. 1 is a graph showing the results of CRP measurement in the presence (Example 1) and absence (Comparative Example 1) of guanidine hydrochloride.
FIG. 2 shows the results of Lp (a) measurement in the presence (Example 2) and absence (Comparative Example 2) of guanidine hydrochloride.
FIG. 3 shows the results of measuring myoglobin in the presence (Example 3) and absence (Comparative Example 3) of guanidine hydrochloride.
FIG. 4 shows the results of CRP measurement in the presence (Example 4) and absence (Comparative Example 4) of guanidine carbonate.
FIG. 5 is a view showing the results of CRP measurement in the presence (Example 5) and absence (Comparative Example 5) of guanidine thiocyanate.
FIG. 6 is a diagram showing the correlation when CRP is measured in the presence and absence of guanidine hydrochloride.
FIG. 7 is a diagram showing the correlation between the measurement of CRP in the presence of guanidine hydrochloride and the measurement of CRP using a reagent by a commercially available latex immunoturbidimetric method.
FIG. 8 is a diagram showing the correlation when Lp (a) is measured in the presence and absence of guanidine hydrochloride.
FIG. 9 is a diagram showing the correlation between the measurement of Lp (a) in the presence of guanidine hydrochloride and the measurement of Lp (a) using a commercially available latex immunoturbidimetric reagent.

Claims (3)

This is an immunoassay method in which an insoluble carrier particle carrying an antibody or antigen for the measurement target is added to a sample containing the antigen or antibody to be measured, and the degree of formation of an immune complex due to an antigen-antibody reaction is measured. The amount of non-specific reaction does not affect the measurement result (the measured value in the condition where the compound used for avoiding the non-specific reaction does not exist and the measured value in the existing condition) In an immunoassay characterized by coexisting 7.5 / 202 M to 75/202 M in the reaction system with guanidine, a guanidine salt or a derivative thereof) How to expand the range. This is an immunoassay method in which an insoluble carrier particle carrying an antibody or antigen for the measurement target is added to a sample containing the antigen or antibody to be measured, and the degree of formation of an immune complex due to an antigen-antibody reaction is measured. The amount of non-specific reaction does not affect the measurement result (the measured value in the condition where the compound used for avoiding the non-specific reaction does not exist and the measured value in the existing condition) in ratio refers to the case indicating a match to within 0.7 to 1.3), guanidine, in immunological assay, characterized in that the coexistence 24/324 M~ 120/324 M guanidine salt or derivative thereof in the reaction system Method for increasing measurement sensitivity.   The method according to claim 1 or 2, wherein the diameter of the insoluble carrier particles is 0.02 to 1.6 µm.

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