JP6931673B2 - Immunoassay methods and reagents - Google Patents

Description

The present invention relates to immunoassay methods and reagents for them.

The immunoassay method is widely used in clinical tests of serum, plasma, urine, stool, cerebrospinal fluid, etc., and in recent years, since it is possible to perform simple and rapid measurement, the reaction to the measurement are automatically performed collectively. Analyzers are widely used.

The immunoassay method is a measurement method using an antigen-antibody reaction and is known to be a highly specific measurement method. However, there is a problem that a non-specific reaction such as false positive or false negative occurs depending on the sample. For example, a factor that recognizes and reacts to an antibody may be present in the sample, in which case a positive measurement will be obtained even if the antigen to be measured is not present in the sample. There may be a factor that interferes with the antigen-antibody reaction in the sample. In such a case, even if the antigen to be measured is present in the sample, the measured value will be negative, which is different from the true value. There was a problem of showing the value.

It has been known to add a human IgM natural antibody or a polymer obtained by polymerizing an aromatic monomer having a sulfone group or a salt thereof as a method for suppressing a non-specific reaction (see Patent Documents 1 and 2). However, these additives may be insufficient, and it has been difficult to suppress non-specific reactions, especially in the low concentration range. In the reagent with higher sensitivity, non-specific reaction was likely to occur because the reactivity of the antibody was improved.

Japanese Patent No. 4065600 Japanese Patent No. 4580180

An object of the present invention is to provide an immunoassay method capable of measuring an antigen with high sensitivity and accuracy in immunoassay, and a reagent for that purpose.

As a result of diligent research, the inventor of the present application facilitates even highly sensitive measurement by the presence of a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group in immunoassay. It was found that non-specific reactions can be suppressed.

That is, the present invention relates to the following 1) to 9).
1) Antigen-antibody reaction and / or measurement is performed in the presence of a polyoxyethylene alkyl ether surfactant (excluding polyoxyethylene lauryl ether) having 8 to 14 carbon atoms in the alkyl group, which improves specificity. Latex agglutination immunoassay method.
2) The method of 1), wherein the polyoxyethylene alkyl ether surfactant has an HLB of 15 to 19 as a nonionic surfactant.
3) The method according to 1) or 2), wherein the concentration of the polyoxyethylene alkyl ether surfactant in the reaction system and / or the measurement system in which the polyoxyethylene alkyl ether surfactant is present is 0.0001 to 1%.
4) The method according to any one of 1) to 3), wherein the process from the start of the antigen-antibody reaction to the completion of the measurement is carried out in the presence of the polyoxyethylene alkyl ether surfactant.
5 ) A latex agglutination immunoassay reagent used in the method 1) with improved specificity, which is a polyoxyethylene alkyl ether surfactant (polyoxyethylene) having an alkyl group having 8 to 14 carbon atoms. An immunoassay reagent comprising (excluding lauryl ether).
6 ) The reagent of 5) in which the HLB of the polyoxyethylene alkyl ether surfactant is 15 to 19.

According to the method of the present invention, highly sensitive immunoassay is performed by a simple means of allowing a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group to be present in the reaction and / or measurement system. In addition, the non-specific reaction can be effectively suppressed, and the antigen can be accurately measured in the immunoassay, and the specificity is improved.

The calibration curve (calibration curve) created in the following Examples and Comparative Examples is shown.

Hereinafter, the method of the present invention will be described. In addition, "%" in this specification means mass-based (w / v%) unless otherwise specified.

The immunoassay method of the present invention is an immunoassay method in which an antigen-antibody reaction is carried out using an immunoassay reagent that immunologically reacts with a substance to be measured in a sample, and the obtained reaction product is measured. It is characterized in that the reaction and / or measurement is carried out in the presence of a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms.

The immunoassay method itself is well known. The immunoassay method to which the method of the present invention is applied may be any known immunoassay method, but the immunoagglutination method is preferable, and the latex agglutination method using latex particles as insoluble carrier particles is particularly preferable. A method for detecting agglutination of sensitized particles in an immunoaggregation method is well known, and in the present invention, a well-known method such as a method for detecting absorbance or light scattering due to agglutination of sensitized particles can be used. Examples thereof include an immunoturbidimetry method (TIA method, latex agglutination method), a colorimetric method, an RPLA method, a CL method and an immunochromatography method, and the turbidimetry method and the colorimetric method having high sensitivity and good quantification accuracy are preferably used. Be done.

The mode of the immunoassay method is not particularly limited to the insoluble carrier particles used, and may be well-known ones conventionally used for immunoassay reagents. For example, latex particles such as polyethylene and polystyrene, alumina particles, silica particles, colloidal gold, magnetic particles and the like can be mentioned. Among these insoluble carriers, latex particles, particularly polystyrene latex particles, are preferably used. The immunoagglutination method is well known as a method for optically detecting the aggregation of sensitized particles sensitized with an antigen or antibody, and the turbidimetry method or the colorimetric method is preferably used for the detection. For example, by irradiating light in the visible to near-infrared region from the outside of the cell, for example, light of usually 300 to 1000 nm, preferably 500 to 900 nm, and detecting a change in absorbance or a change in intensity of scattered light, the sensitized particles The degree of agglomeration is measured. Polystyrene latex particles are particularly preferably used as the latex particles. The size of the latex particles is not particularly limited, but the particle size is preferably 30 to 600 nm.

An antibody or an antigen-binding fragment thereof that immunoreacts with the antigen to be measured is immobilized on the above-mentioned latex particles. The method of immobilization is also well known, and is carried out by a well-known method such as physical adsorption or covalent bond. When the obtained suspension of sensitized particles and the test sample are mixed, the sensitized particles are aggregated by the substance (antigen) to be measured contained in the test sample, and the absorbance of the sensitized particle suspension changes. do. The amount of change in absorbance (endpoint method) or rate of change (rate method) is measured. A plurality of standard samples containing the antigen to be measured at various known concentrations are prepared, and the amount of change or the rate of change in absorbance is measured for them by the above method. A calibration curve is drawn by plotting the concentration of the antigen to be measured in the standard sample on the horizontal axis and the amount of change or rate of change in the measured absorbance on the vertical axis. The amount of change or rate of change in absorbance of an unknown test sample is measured by the same method, and the measurement result is applied to the calibration curve to quantify the antigen in the test sample.

Various automatic devices for performing such an immuno-aggregation method are commercially available, and can be easily and easily performed by using a commercially available automatic device for the immuno-aggregation method.

The substance to be measured by immunoassay in the present invention is not limited as long as it is a substance that can be measured by immunoassay, but when the substance to be measured is an antigen, for example, CRP (C-reactive protein), prostate-specific antigen, ferritin. , Β-2 microglobulin, myoglobin, hemoglobin, albumin, creatinine and other protein markers, IgG, IgA, IgM and other immunoglobulins, various tumor markers, LDL, HDL, TG and other lipoproteins, type A influenza virus, type B Influenza virus, RS virus (RSV), rhinovirus, rotavirus, norovirus, adenovirus, astrovirus, HAV, HBs, HCV, HIV, EBV and other viral antigens, Chlamydia trachomatis, lytic bacteria, pertussis, helicobacter pylori , Leptspira, Treponema paridam, Toxoplasma gondii, Borrelia, Regionella spp., Charcoal antigen, MRSA and other bacterial antigens, Bacterial toxins, Mycoplasma lipid antigens, Human villus gonadotropin and other peptide hormones, steroid hormones, etc. Examples thereof include, but are not limited to, steroids, physiologically active amines such as epinephrine and morphine, vitamins such as vitamin B, antibiotics such as prostaglandins and tetracyclines, pesticides, and environmental hormones. Preferred examples include antigens such as CRP, prostate-specific antigen, ferritin, β-2 microglobulin and hemoglobin.

When the substance to be measured is an antibody, the above protein markers, various tumor markers, lipoproteins, viral antigens, bacterial antigens, toxins produced by bacteria, peptide hormones, steroids, physiologically active amines, vitamins, antibiotics, pesticides , Antibodies that specifically react with antigens such as environmental hormones.

The sample used for immunoanalysis is not particularly limited as long as it contains the substance to be measured, but blood, serum, plasma, urine, stool, saliva, tissue fluid, spinal fluid, body fluid such as swab, or a dilution thereof. Blood, serum, plasma, urine, stool, spinal fluid or dilutions thereof are preferred.

As described above, in the method of the present invention, in the reaction system and / or the measurement system, the antigen-antibody reaction and the antigen-antibody reaction are carried out in the presence of a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group. / Or it is characterized by making measurements. The HLB of the polyoxyethylene alkyl ether surfactant is not particularly limited, but 15 to 19 is preferable.

In the method of the present invention, the polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group can be used from the start of the antigen-antibody reaction to the end of detection and quantification of the amount of the antigen-antibody reaction. At this stage, a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group may be contained in the reaction and / or measurement system (also referred to as “reaction / measurement system”). Is preferably contained from the start of the antigen-antibody reaction to the detection and quantification. Therefore, the polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group is preferably added into the reaction system before or at the same time as the start of the antigen-antibody reaction. Specifically, it may be added when the sample is diluted, or when the antibody or antigen is mixed with the sample.

Further, various reagents used for immunoanalysis may contain a polyoxyethylene alkyl ether surfactant having an alkyl group having 8 to 14 carbon atoms in advance, and the present invention has such an alkyl group having 8 to 14 carbon atoms. It also provides an immunoassay reagent containing 8 to 14 polyoxyethylene alkyl ether surfactants. Here, as various reagents used for immunoanalysis, for example, a sample diluent, an antibody / antigen diluent, an immobilized antibody / antigen, a sensitized particle suspension, a washing solution, an enzyme solution, a substrate solution, and a calibration line preparation are used. Examples of the immunoassay reagent containing a polyoxyethylene alkyl ether surfactant having an alkyl group having 8 to 14 carbon atoms include the standard solution of the test substance in the above, and these reagents have an alkyl group carbon atom. A reagent containing a polyoxyethylene alkyl ether surfactant having a number of 8 to 14, for example, a buffer for diluting a sample, a reagent containing an antibody or an antigen, etc., has an alkyl group having 8 to 14 carbon atoms. Examples thereof include those containing a polyoxyethylene alkyl ether surfactant.

Further, for example, a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms of an alkyl group is added to an immunoaggregating reagent containing latex particles (sensitized particles) on which an antibody or antigen is immobilized (sensitized). It can be contained. In this case, the concentration of the sensitized particles in the immunoassay reagent is not particularly limited, but is preferably 0.01 to 0.5%. The amount of antibody and the amount of antigen in the sensitized particle suspension may be as usual and are not particularly limited. For example, in the case of antibody-sensitized latex, the amount of antibody is 0.01 to 2.0 mg / in the latex suspension. It is preferably mL.

The concentration of the polycarboxylic acid-type surfactant in the reaction / measurement system is preferably 0.0001 to 1%, more preferably 0.001 to 0.5%, from the viewpoint of suppressing non-specific reactions. Therefore, when the immunoassay reagent contains a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group in advance, the concentration in the reaction and / or measurement system becomes the above concentration. It may be contained in the immunoassay reagent so as to be.

The blank sample used for immunoassay is not particularly limited as long as it does not contain the substance to be measured, but purified water, physiological saline, buffer solution, negative sample or a dilution thereof is preferable.

As described in the examples below, when a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group is present in the reaction and / or measurement system, the non-specific reaction is suppressed. Will be done. Then, the specificity is significantly improved as compared with the case where the polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms of the alkyl group is not present. Therefore, by using the method of the present invention, it is possible to improve the reagent performance as compared with the conventional one, especially in the reagent with high sensitivity in which a non-specific reaction is likely to occur.

Hereinafter, the present invention will be described in more detail based on Examples and Comparative Examples. However, the present invention is not limited to the following examples.

Examples 1-6, Comparative Example 1
(1) Preparation of Reagents Using an antibody against ferritin, a reagent for measurement by the immunoagglutination method was prepared as follows.
i) Sensitive particles in which 0.03 mg of an anti-ferritin antibody is carried in 1 mL of a polystyrene latex suspension having an average particle size of 300 nm are suspended in a buffer solution (Tris, pH 8.0) so as to be 0.04%. , Latex buffer was prepared.
ii) In a buffer solution (Tris, pH 8.5), a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms of an alkyl group (“Emargen 1118S-70” (trade name, commercially available from Kao Corporation) ), HLB16.4) was added to prepare the following reagents A to F (Examples 1 to 6). As Comparative Example 1, a reagent G to which nothing was added was prepared.

(2) Measurement by automatic analyzer The automatic analyzer used the Hitachi 7180 automatic analyzer to perform automatic measurement by the endpoint method.
Using the above-mentioned reagents A to G, two false positive samples and one normal sample known to cause a non-specific reaction were measured. To 15.0 μL of the sample solution, 100 μL of the buffer solution prepared above for Reagents A to G was added, and the mixed solution was stirred and mixed at 37 ° C. After standing for 5 minutes, 100 μL of the latex suspension was added, and the mixture was further stirred and mixed at 37 ° C. The agglutination reaction for about 5 minutes was measured as the amount of change in absorbance, and the ferritin concentration of each sample was calculated from the calibration curve.

(3) Comparison of Sensitivity with Existing Reagents The measurement sensitivities of Examples 1 to 6 and Comparative Example 1 and the existing commercially available latex reagent FER-Latex X2 "Seiken", which is known to suppress non-specific reactions. We compared the sensitivity with CN (Denka Seiken) (hereinafter, "existing reagent"). The results are shown in FIG.

As shown in FIG. 1, in the methods of Examples 1 to 6 and Comparative Example 1, the amount of change in absorbance was large at the same ferritin concentration as compared with the method using the existing reagent, and Example 1 It can be seen that the methods of ~ 5 and Comparative Example 1 have higher measurement sensitivities.

(4) Measured value of false positive sample Leather The measurement results of Examples 1 to 6 and Comparative Example 1 were compared. The results are shown in Table 2.

（単位：ng/mL）

(Unit: ng / mL)

Comparing the measurement results of Examples 1 to 6 with the measurement results of Comparative Example 1, the polyoxyethylene alkyl ether surface activity in which the number of carbon atoms of the alkyl group is 8 to 14 in the highly sensitive immunoanalytical method. It was shown that the addition of the agent suppressed the non-specific reaction, and the measurement results of the normal specimens did not fluctuate and the measured values of the false positive specimens decreased.

Examples 7 and 8, Comparative Examples 2 and 3
(1) Preparation of Reagents Using an antibody against ferritin, a reagent for measurement by the immunoagglutination method was prepared as follows.
i) Sensitive particles in which 0.03 mg of an anti-ferritin antibody is carried in 1 mL of a polystyrene latex suspension having an average particle size of 300 nm are suspended in a buffer solution (Tris, pH 8.0) so as to be 0.04%. , Latex buffer was prepared.
ii) A polyoxyethylene alkyl ether surfactant (“Emargen 1118S-70” (trade name, commercially available from Kao Corporation) in which the number of carbon atoms of the alkyl group is 8 to 14 in a buffer solution (Tris, pH 8.5). ), HLB16.4) and human IgM natural antibody were added to prepare the following Reagent H (Example 7) and Reagent I (Example 8). As Comparative Example 2, Reagent J to which nothing was added and Reagent K to which only human IgM natural antibody was added were prepared.

(2) Measurement by automatic analyzer Using the reagents HK described above, two false positive samples known to cause a non-specific reaction were measured. To 15.0 μL of the sample solution, 100 μL of the buffer solution prepared above for Reagents HK was added, and the mixed solution was stirred and mixed at 37 ° C. After standing for 5 minutes, 100 μL of the latex suspension was added, and the mixture was further stirred and mixed at 37 ° C. The agglutination reaction for about 5 minutes was measured as the amount of change in absorbance, and the ferritin concentration of each sample was calculated from the calibration curve.

(4) Comparison with existing reagents The measurement results of Examples 7 and 8 and Comparative Examples 2 and 3 were compared. The results are shown in Table 4.

（単位：ng/mL）

(Unit: ng / mL)

Comparing the measurement results of Examples 7 and 8 with the measurement results of Comparative Examples 2 and 3, the non-specific reaction of the human natural IgM antibody known to suppress the non-specific reaction in the highly sensitive immunoassay method However, by adding a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group, the non-specific reaction can be suppressed and the measurement result of the false positive sample can be deteriorated. Shown.

Claims (6)

Antigen-antibody reaction and / or measurement in the presence of a polyoxyethylene alkyl ether surfactant (excluding polyoxyethylene lauryl ether) having 8 to 14 carbon atoms in the alkyl group, a latex with improved specificity. Aggregate immunoassay method. The method according to claim 1, wherein the polyoxyethylene alkyl ether surfactant has an HLB of 15 to 19 as a nonionic surfactant. The method according to claim 1 or 2, wherein the concentration of the polyoxyethylene alkyl ether surfactant in the reaction system and / or the measurement system in which the polyoxyethylene alkyl ether surfactant is present is 0.0001 to 1%. The method according to any one of claims 1 to 3, wherein the process from the start of the antigen-antibody reaction to the completion of the measurement is carried out in the presence of the polyoxyethylene alkyl ether surfactant. A latex agglutination immunoassay reagent used in the method according to claim 1, which has improved specificity, and is a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in an alkyl group (polyoxyethylene lauryl). An immunoassay reagent comprising (excluding ether). The reagent according to claim 5 , wherein the HLB of the polyoxyethylene alkyl ether surfactant is 15 to 19.

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