JP2006234831A - Reagent for immunoassay - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide immunoassay method using solid phase carriers, especially porous membrane, which inhibits any affect from causative agent of nonspecific reaction in a biological sample. <P>SOLUTION: This reagent is a washing solution to be one component of the reagent for enzyme immunoassay using porous membrane as a solid phase carrier, namely, a solid phase washing for enzyme immunoassay which contains alkyl glycoside system detergent or steroid-type detergent, and also the solid phase washing solution for enzyme immunoassay which contains polyoxyethylene system nonionic detergent. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention suppresses the influence of a substance that causes a nonspecific reaction in a biological sample that adversely affects the measured value in a method of immunologically measuring a biological sample component on a solid phase carrier, particularly a porous membrane. The present invention relates to a method enabling a more accurate measurement and a cleaning reagent therefor.

Conventionally, as a method for measuring various components in a biological sample, there is an immunological measurement method using an antigen-antibody reaction. Among such immunological assay methods are enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), fluorescence immunoassay (FIA), etc. There are measuring methods using antibodies or antigens labeled with enzymes such as horseradish peroxidase and alkaline phosphatase, radioactive isotopes (RI) such as iodine 125, luminescent substances such as acridinium compounds or fluorescent substances such as fluorescein isothiocyanate, Because of their high sensitivity, they are now widely used. As these measuring methods, immunological measuring methods using a solid phase carrier are widely used.

However, these immunological measurement methods can measure even a very small amount of components in a biological sample, but a non-specific reaction due to a non-measurement target component contained in a biological sample may also occur. In the case of measurement, the measurement result is greatly affected.

For example, if a substance with the same activity as a labeling substance such as an enzyme used in the detection system is present in the biological sample and the substance remains in the reaction system at the detection reaction stage, it is not related to a specific immune reaction. It is possible that a non-specific reaction occurs, and a sample that should originally be determined as negative is erroneously determined as positive.

In addition, if the free labeling substance itself that did not bind to the antigen or antibody during the immune reaction remains in the detection reaction system due to some influence, it causes a nonspecific reaction.

Thus, various methods have been conceived so far in order to suppress the influence of non-specific reaction-causing components derived from these biological samples. For example, as a method using a solid phase carrier, a method using a blocking agent such as casein, bovine serum albumin, gelatin or the like in order to prevent adsorption of these nonspecific reaction components to the solid phase carrier is known. As another method for suppressing non-specific reactions, a method of adding a surfactant or the like to the measurement reagent is known.

However, these methods may not prevent non-specific reactions derived from biological samples. For example, in an immunoassay using a solid phase carrier, particularly a porous membrane, it has the same activity as a labeling substance and cannot pass through the membrane because it is larger than the pores of the porous membrane. Free labeling substances that normally pass through the membrane, such as when a toxic substance (blood cells, etc.) is mixed in the biological sample, or soluble components in the biological sample are insolubilized, causing clogging of the porous membrane In addition, the blocking agent cannot prevent a substance in the biological sample having the same activity as the labeling substance from remaining on the membrane. In addition, in the method of adding a surfactant to the measurement reagent, depending on the type of surfactant to be added, antibodies or antigens that bind to the solid phase carrier may be detached from the carrier or the immune reaction may be inhibited. As a result, the specific signal may be extremely reduced.

The present inventors have reached the present invention as a result of intensive studies to suppress the influence of a causative substance of a nonspecific reaction in a biological sample in an immunological assay using a solid phase carrier.

That is, the present invention is a cleaning solution that is one of the components of an enzyme immunoassay reagent using a porous membrane as a solid phase carrier, and is used for enzyme immunoassay containing an alkyl glycoside surfactant or a steroid surfactant. Solid phase washing solution.

The present invention also provides a solid phase carrier comprising a porous membrane on which an antibody or antigen that immunologically reacts with a biological sample component is immobilized, an enzyme-labeled antibody or antigen that immunologically reacts with the biological sample component. A reagent for enzyme immunoassay comprising a solid phase washing solution for enzyme immunoassay containing a reagent to be contained and an alkylglycoside surfactant or a steroid surfactant.

Furthermore, the present invention comprises reacting the antibody or antigen with the biological sample component on a solid phase carrier comprising a porous membrane on which an antibody or antigen that immunologically reacts with the biological sample component is immobilized, and then reacting the antibody or antigen. And a reagent containing an enzyme-labeled antibody or antigen that immunologically reacts with the component, and then an enzyme immunoassay containing an alkylglycoside surfactant or a steroid surfactant This is an immunological assay characterized in that the solid phase carrier is washed with a solid phase washing solution for use and the labeling substance on the solid phase carrier is measured.

The present invention also provides an alkyl glycoside by reacting an antibody or antigen with a biological sample component on a solid phase carrier comprising a porous membrane on which an antibody or antigen that immunologically reacts with the biological sample component is immobilized. The solid phase carrier was washed with a solid phase washing solution for enzyme immunoassay containing a surfactant or a steroid surfactant, then reacted with the biological sample component, and an enzyme labeled immunologically reacting with the component A reagent containing an antibody or an antigen is reacted, and the solid phase carrier is washed with a solid phase washing solution for enzyme immunoassay containing an alkylglycoside surfactant or a steroid surfactant, and a labeling substance on the solid phase carrier is removed. It is an immunological assay characterized by measuring.

The present invention also provides a reaction of a biological sample component and an antibody or antigen that immunologically reacts with the component on a solid phase carrier comprising a porous membrane, and then alkylglycoside surfactant or steroidal surfactant. An immunological measurement method comprising a step of washing with a solid phase washing solution for enzyme immunoassay containing an agent.

The present invention suppresses the influence of a causative substance of a nonspecific reaction in a biological sample that adversely affects the measurement value in an immunological measurement method of a biological sample component that is detected on a solid phase carrier, particularly a porous membrane, The present invention provides a method and a measurement reagent that enable more accurate measurement. In addition, the improvement of the cleaning power also increases the effect of washing away the free labeling substance remaining on the solid phase carrier, particularly the porous membrane. As a result, an improvement in signal / noise ratio, that is, an improvement in detection power is also expected. it can.

In the present invention, the biological sample component is a component contained in blood, serum, plasma, lymph, urine, feces, ascites, pleural effusion, and the like, for example, CEA (carcinoembryonic antigen), AFP (α-fetoprotein) , CA19-9, CA125, tumor markers such as PSA (prostate specific antigen), CRP (C-reactive protein), IgA (immunoglobulin A), IgG (immunoglobulin G), inflammation such as IgM (immunoglobulin M) Markers, HIV (human immunodeficiency virus), HBV (hepatitis B virus), HCV (hepatitis C virus), markers related to infectious diseases such as toxoplasma, chlamydia, syphilis, and allergen-specific IgE (immunoglobulin E) Is done.

In the present invention, the antibody or antigen that immunologically reacts with a biological sample component is an antibody when the component is an antigen, and an antigen when the component is an antibody. Examples of antibodies that react with biological sample components (antigens) include polyclonal antibodies, monoclonal antibodies, or fragments thereof (Fab, Fab ', F (ab') 2, Fv, etc.).

The solid phase carrier used in the present invention is a solid phase carrier usually used in an immunological measurement method, and examples of the form thereof include a porous membrane, fine particles, beads, a microtiter plate, and a tube. In particular, a porous membrane is preferable. The material for the porous membrane used in the present invention may be any material, for example, polyethylene, polyethylene terephthalate, nylons, glass, polysaccharides such as cellulose or cellulose derivatives, or ceramics. Specifically, there are glass fiber filter paper and cellulose filter paper sold by manufacturers such as Toyo filter paper and Whatman. The fine particles include those made from a polymer such as polystyrene and polypropylene, and those made by coating them with magnetic particles such as iron oxide particles.

The method for immobilizing an antibody or antigen on a solid phase carrier may be any method as long as it is generally used for preparing an immunological reagent. The antibody or antigen is physically adsorbed or chemically bound to the solid phase carrier. Therefore, it is sufficient that they are connected directly or indirectly.

The labeled antibody or antigen is a substance obtained by directly or indirectly binding a labeling substance to the antibody or antigen. Examples of the labeling substance include enzymes such as horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, iodine 125 And the like, luminescent materials such as acridinium compounds, luminol, fluorescent materials such as fluorescein isothiocyanate or europium (III) chelate, and metal colloids are known.

In the present invention, the causative substance of the nonspecific reaction in the biological sample is a substance that is contained in a biological sample such as blood and urine and has the same activity as a labeling substance such as an enzyme used in the detection system, For example, when using antibodies or antigens labeled with horseradish peroxidase in the detection system, substances having peroxidase-like activity in blood (animal peroxidases such as myeloperoxidase or substances having pseudoperoxidase activity such as hemoglobin and catalase), etc. Is included. In addition, a substance in which a soluble component in a biological sample is insolubilized due to a biological change such as the working of a blood coagulation system or a physical change such as freezing and thawing also causes a non-specific reaction.

The alkylglycoside surfactant used in the present invention is a surfactant having a saccharide as a hydrophilic group and an alkyl group as a hydrophobic group, both ionic and nonionic, which are ether- or thioether-bonded. Point to. Examples of such surfactants include n-octyl-β-D-glucopyranoside, n-nonyl-β-D-maltopyranoside, n-decyl-β-D-glucopyranoside, n-dodecyl-β-D-glucopyranoside, n-decyl-β-D-maltopyranoside, n-heptyl-β-D-thioglucopyranoside, n-octyl-β-D-thioglucopyranoside, n-dodecyl-β-D-maltopyranoside, n-nonyl-β-D- Examples include thiomaltopyranoside, octyl-β-D-thiogalactopyranoside, and in particular, n-octyl-β-D-glucopyranoside, n-heptyl-β-D-thioglucopyranoside or n-octyl-β -D-thioglucopyranoside is preferred.

Further, the steroidal surfactant used in the present invention refers to a surfactant having a steroid skeleton in a hydrophobic group regardless of whether it is ionic or nonionic, such as cholate, deoxycholate, Dehydrocholate, taurocholate, glycocholate, 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonic acid, 3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxy Examples include propanesulfonic acid, N, N-bis (3-D-gluconamidopropyl) coleamide, and steroid saponins such as digitonin. Among them, sodium deoxycholate, 3-[(3-cholamidopropyl) dimethyl Ammonio] propanesulfonic acid and the like are preferable.

In addition, the said surfactant may be used independently or may be used in combination of 2 or more types. The surfactant is preferably in a concentration range of 0.01 (w / v)% to 2.0 (w / v)%, particularly preferably 0.1 (w / v)% to 0.5 (w / v)%. Use.

In addition to the above surfactant, the solid phase cleaning liquid used in the present invention may contain a buffer usually used as a cleaning liquid component such as a phosphate buffer, a Tris buffer, or a Good buffer. Further, the pH of the cleaning liquid is not particularly limited, but is preferably in the range of pH 5-9. Furthermore, additives such as salts such as sodium chloride and preservatives usually used as cleaning liquid components may be added as appropriate.

In addition to the alkylglycoside surfactants or steroidal surfactants, the solid phase cleaning solution used in the present invention is usually an interface that is usually used in an immunoassay and does not adversely affect the measurement system. Activators (polyoxyethylene nonionic surfactants such as polyoxysorbitan monolaurate, polyoxyethylene mono-p-isooctylphenyl ether, etc.) are usually added in concentrations within the range used in this field May be.

The solid phase washing solution used in the present invention can be used for immunological assay methods that require washing of the solid phase, that is, heterogeneous immunoassay in general. It may be a law.

One embodiment of the immunological measurement method of the present invention is to react the antibody or antigen with the biological sample component on a solid phase carrier on which the antibody or antigen that immunologically reacts with the biological sample component is immobilized. Then, the reacted biological sample component is reacted with a reagent containing a labeled antibody or antigen that immunologically reacts with the component, and then an immunity containing an alkyl glycoside surfactant or a steroid surfactant The solid phase carrier is washed with a solid phase washing solution for biological measurement, and the labeling substance on the solid phase carrier is measured. The immunological reaction may be a one-step reaction or a two-step reaction. As a method for measuring the labeling substance, a known method may be used. For example, when the labeling substance is an enzyme, after reacting with the substrate, the absorbance or the intensity of reflected light may be measured. When the labeling substance is a radioisotope, the radiation dose may be counted with a counter.

In another embodiment of the present invention, an alkylglycoside interface is prepared by reacting an antibody or antigen with a biological sample component on a solid support on which an antibody or antigen that immunologically reacts with the biological sample component is immobilized. The solid phase carrier is washed with a solid phase washing solution for immunological measurement containing an active agent or a steroid-based surfactant, and then reacted biological sample components and labeled antibodies that react immunologically with the components or The reagent containing the antigen is reacted, the solid support is washed with a solid phase washing solution for immunoassay containing an alkyl glycoside surfactant or a steroid surfactant, and the labeling substance on the solid support is measured. To do.

Furthermore, another embodiment of the present invention is the reaction of a biological sample component and an antibody or antigen that immunologically reacts with the component on a solid phase carrier, and then alkylglycoside surfactant or steroidal surfactant. And a step of washing with a solid phase washing solution for immunological measurement containing an agent. These immunological reactions may be any method using a solid phase carrier, and are not limited to the detection method by the sandwich method.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 Preparation of a solid phase washing solution
To phosphate buffered saline (pH 7.2) containing 0.05 (w / v)% polyoxysorbitan monolaurate (trade name: Tween20; manufactured by Nacalai Tesque), n-octyl-β-D-thioglucopyranoside ( Manufactured by Dojindo Laboratories), 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonic acid (trade name: CHAPS; manufactured by Dojindo Laboratories), and hexabromide bromide as a surfactant for comparison Decyltrimethylammonium (manufactured by Nacalai Tesque) was added to prepare the following 6 types of solid phase washing solutions.
(a) 0.05 (w / v)% n-octyl-β-D-thioglucopyranoside-added cleaning solution
(b) 0.2 (w / v)% n-octyl-β-D-thioglucopyranoside-added cleaning solution
(c) 0.05 (w / v)% CHAPS-added cleaning solution
(d) 0.2 (w / v)% CHAPS-added cleaning solution
(e) 0.05 (w / v)% hexadecyltrimethylammonium bromide added cleaning solution
(f) 0.2 (w / v)% Hexadecyltrimethylammonium bromide added cleaning solution

Example 2 Comparison of specific signals
(1) Measuring reagent As an immunological measuring reagent, it has a porous membrane (glass fiber filter) on which an anti-CEA (carcinoembryonic antigen) antibody, which is an immunological reaction layer, is immobilized on the upper part, and an immunoreactive part on the lower part. An immunological measurement reagent (trade name, Immunoflora / CEA, manufactured by Toyobo Co., Ltd.), a blocking liquid (including casein, etc.) comprising a reaction member having a layer that absorbs the liquid after reaction and a reagent containing a peroxidase-labeled anti-CEA antibody A diluent (including a buffering agent) and a color developing solution (including 10-N-methylcarbamoyl-3,7-dimethylamino-10H-phenothiazine (MCDP) and the like) were used. The solid phase washing solution was prepared by adding the n-octyl-β-D-thioglucopyranoside washing solution prepared in Example 1, (a), (b), (c), (d), (e) (comparative example), ( f) (Comparative Example) was used, and 0.05 (w / v)% Tween20-containing phosphate buffered saline (pH 7.2) was used as a control.
(2) Test sample CEA standard solution (manufactured by Toyobo) was used.
(3) CEA-specific signal measurement The upper part has a porous membrane (glass fiber filter) to which an anti-CEA (carcinoembryonic antigen) antibody, which is an immunological reaction layer, is immobilized. To the reaction member having a layer that absorbs lysate, the blocking solution, the test sample diluted with the diluent, the labeled antibody, the washing solution, and the coloring solution are dropped in this order in order, and the degree of coloration on the porous membrane is set to a wavelength of 670 nm. The CEA-specific signal of the CEA standard solution was calculated by measuring the reflected light when the light was applied. The measurement results are shown in Table 1. The value of the signal is the amount of change per unit time (ΔK / S) of the K / S value derived by the Kubelka-Munk equation (K / S = (1−reflectance) ^ 2 / (2 * reflectance)) It shows with.

As is clear from Table 1, a cleaning solution to which n-octyl-β-D-thioglucopyranoside, which is one of alkyl glycoside surfactants, or CHAPS, which is one of steroid type surfactants, was used. In some cases, no signal decrease was observed, but when a washing solution to which hexadecyltrimethylammonium bromide was added was used, a clear signal decrease was observed.

Example 3 Measurement of serum CEA concentration
(1) Measurement reagent In the same manner as in Example 2, a CEA measurement reagent (Immunoflora / CEA), a block solution, a diluent, and a color former were used. As the washing solution, the n-octyl-β-D-thioglucopyranoside-added washing solutions (a) and (b) prepared in Example 1 were used, and phosphate buffered saline containing 0.05 (w / v)% Tween20 was used as a control. (pH 7.2) was used. For comparison, an immunoassay reagent (Immunoball CEA (manufactured by Toyobo)) using plastic beads as a solid phase carrier was similarly subjected to immunoassay.
(2) Test sample human serum was used as serum containing non-specific reactants.
(3) CEA concentration measurement In the same manner as in Example 2, the CEA concentration in serum was measured using the above-described measuring reagent using a porous membrane or plastic beads. The measurement results are shown in Table 2.

As is apparent from Table 2, when measured using a control solution (without addition of n-octyl-β-D-thioglucopyranoside), it was more porous than when measured with a plastic bead solid phase reagent. The measurement value using a porous membrane is high, but when using a cleaning solution to which n-octyl-β-D-thioglucopyranoside is added, the measurement reagent using a porous membrane is It is almost the same as the measurement value measured with the reagent. This indicates that the washing solution containing n-octyl-β-D-thioglucopyranoside suppresses the influence of non-specific reactive substances in the serum.

Example 4 Preparation of solid phase washing solution
To phosphate buffered saline (pH 7.2) containing 0.05 (w / v)% Tween20, add n-octyl-β-D-thioglucopyranoside and sodium deoxycholate (manufactured by Nacalai Tesque). A cleaning solution was prepared.
(g) 0.2 (w / v)% n-octyl-β-D-thioglucopyranoside-added cleaning solution
(h) 0.2 (w / v)% n-octyl-β-D-thioglucopyranoside and 0.2 (w / v)% sodium deoxycholate
(i) Wash solution with 0.3 (w / v)% n-octyl-β-D-thioglucopyranoside and 0.2 (w / v)% sodium deoxycholate

Example 5 Measurement of serum AFP concentration
(1) Measurement reagent Immunoflora AFP (manufactured by Toyobo Co., Ltd.), a block solution, a diluent, and a color former, which are immunoassay reagents using a porous membrane as a solid phase carrier, were used. To the washing solution, 0.05 (w / v)% Tween20-containing phosphate buffered saline (pH 7.2) was further added with n-octyl-β-D-thioglucopyranoside, or the compound and sodium deoxycholate. The cleaning solutions (g), (h) and (i) prepared in Example 4 were used. As a control, phosphate buffered saline (pH 7.2) containing 0.05 (w / v)% Tween20 without addition of n-octyl-β-D-thioglucopyranoside and sodium deoxycholate was used for further comparison. Immunoball AFP Neo (manufactured by Toyobo), which is an immunoassay reagent using plastic beads as a solid phase carrier, was used.
(2) Test sample human serum was used as serum containing non-specific reactants.
(3) Measurement of AFP concentration In the same manner as in Example 3, the AFP concentration in serum was measured using the above-described measurement reagent using a porous membrane or plastic beads. The measurement results are shown in Table 3.

As is apparent from Table 3, when measured using a washing solution of the control composition (no addition of n-octyl-β-D-thioglucopyranoside and sodium deoxycholate), it was measured with a bead solid phase reagent. In contrast, the measurement value of the reagent using a porous membrane is higher, but when using a washing solution containing n-octyl-β-D-thioglucopyranoside and sodium deoxycholate, It is almost the same as the measured value measured with the reagent. This indicates that the washing solution to which n-octyl-β-D-thioglucopyranoside and sodium deoxycholate were added suppressed the influence of non-specific reactants in the serum.

Example 6 Correlation with Bead Solid Phase Reagent when Using Washing Solution of Control Composition
(1) Measurement reagent As in Example 2, an immunoassay reagent (Immunoflora CEA, manufactured by Toyobo Co., Ltd.) using a porous membrane as a solid support, a block solution, a diluent, and a color former were used. As the washing solution, the control composition of the washing solution prepared in Example 4, 0.05 (w / v)% Tween20-containing phosphate buffered saline (pH 7.2) was used. For comparison, Immunoball CEA, an immunoassay reagent using plastic beads as a solid phase carrier, was used.
(2) Test sample 69 human sera were used as samples.
(3) Measurement of CEA concentration In the same manner as in Example 2, the CEA concentration in serum was measured using the above-described measurement reagent using a porous membrane or plastic beads. The measurement results are shown in FIG.

Example 7 Correlation with a bead solid phase reagent when using the cleaning solution of the present invention
(1) Measurement reagent As in Example 2, an immunoassay reagent (Immunoflora CEA, manufactured by Toyobo Co., Ltd.) using a porous membrane as a solid phase carrier, a block solution, a diluent, and a color former were used. The cleaning liquid (i) prepared in Example 4 was used as the cleaning liquid. For comparison, an immunoassay reagent (Immunoball CEA, manufactured by Toyobo Co., Ltd.) using plastic beads as a solid phase carrier was used.
(2) Test sample 53 human sera were used as samples.
(3) CEA concentration measurement The CEA concentration in serum was measured using the above-described measurement reagent. The measurement results are shown in FIG.

As is apparent from FIGS. 1 and 2, by applying the cleaning solution of the present invention to a reagent (Immunoflora / CEA) using a porous membrane as a solid phase carrier, the measured value of CEA can be obtained by using beads as a solid phase carrier. It shows the same value as the reagent used as (Immunoball / CEA). From this, it can be seen that the use of the cleaning liquid of the present invention suppresses the influence of non-specific reactive substances peculiar to the porous membrane and shows an accurate measurement value.

It is a figure which shows the measurement result of the CEA density | concentration in serum. It is a figure which shows the measurement result of the CEA density | concentration in serum.

Claims (8)

A washing liquid that is one of the components of an enzyme immunoassay reagent that uses a porous membrane as a solid phase carrier, and that contains an alkylglycoside surfactant or a steroid surfactant. The solid phase washing solution for immunological measurement according to claim 1, further comprising a polyoxyethylene nonionic surfactant. A solid-phase carrier comprising a porous membrane on which an antibody or antigen that immunologically reacts with a biological sample component is immobilized, a reagent containing an enzyme-labeled antibody or antigen that immunologically reacts with a biological sample component, and an alkyl A reagent for enzyme immunoassay comprising a solid phase washing solution for enzyme immunoassay containing a glycoside surfactant or a steroid surfactant. The reagent for immunological measurement according to claim 3, wherein the solid phase washing solution for enzyme immunoassay further contains a polyoxyethylene nonionic surfactant. On a solid phase carrier comprising a porous membrane on which an antibody or antigen that immunologically reacts with a biological sample component is immobilized, the antibody or antigen is reacted with the biological sample component, and then the reacted biological sample component; Reaction with a reagent containing an enzyme-labeled antibody or antigen that immunologically reacts with the component, and then with a solid phase washing solution for enzyme immunoassay containing an alkyl glycoside surfactant or a steroid surfactant An immunological measurement method comprising washing a solid phase carrier and measuring a labeling substance on the solid phase carrier. An alkylglycoside surfactant or steroid is prepared by reacting the antibody or antigen with the biological sample component on a solid phase carrier comprising a porous membrane on which an antibody or antigen that immunologically reacts with the biological sample component is immobilized. A solid phase carrier is washed with a solid phase washing solution for enzyme immunoassay containing a surfactant, and then contains a reacted biological sample component and an enzyme-labeled antibody or antigen that immunologically reacts with the component. The reagent is reacted, the solid phase carrier is washed with a solid phase washing solution for enzyme immunoassay containing an alkyl glycoside surfactant or a steroid surfactant, and the labeling substance on the solid phase carrier is measured. Immunological assay. Enzyme immunization containing a biological sample component and an antibody or antigen that immunologically reacts with the component on a solid phase carrier comprising a porous membrane, and then containing an alkyl glycoside surfactant or a steroid surfactant An immunological measurement method comprising a step of washing with a solid phase washing solution for measurement. The immunoassay method according to any one of claims 5 to 7, wherein the solid phase washing solution for enzyme immunoassay further contains a polyoxyethylene nonionic surfactant.

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