JPH04344465A - immunoassay - Google Patents

Abstract

PURPOSE:To prevent prozone phenomenon so as to enable stable and accurate measurement by reacting a complex with a second reagent while effecting physical action so that the degree of monodispersion of the complex is more than a specific value. CONSTITUTION:A first reagent, which is formed when a first material immunologically active to a material for measurement in a sample is bonded to the surface of solid particulates, is reacted with the sample in a liquid medium so as to form a complex 1 of the first reagent and the material for measurement and then a second reagent, which is formed when a second material immunologically active to the material for measurement and different from the first reagent is labeled in advance, is added to the complex 1 to generate a complex 2 and the amount by which the complex 2 is labeled is measured whereby the quantity of the material for measurement contained in the sample is determined. In this case, the complex 2 is reacted with the second reagent in such a manner that the degree X(%) of monodispersion of the complex 1, which is defined by the formula X=n/SX100 wherein n represents the number of complexes 1 which are monomers and S the total number of particulates, is more than 90. Prozone phenomenon is thereby prevented and stable and accurate measurement is made possible.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring immunologically active substances such as antigens and antibodies in a specimen using solid microparticles. More specifically, the present invention is a method for quantifying a analyte in a sample using two types of solid microparticles carrying an immunologically active substance and a labeled immunologically active substance. Regarding the method.

0002

[Prior Art] A dispersion (latex reagent) in which microparticles such as polystyrene carrying an immunologically active substance, such as an antibody, are dispersed in a liquid medium such as water is used to The latex agglutination immunoassay method (LAIA), which measures a analyte by observing the agglutination that occurs when a selectively reactive substance (analyte, e.g., antigen) in a specimen acts on it, was developed by J.
J.M. Singer et al.
) [Am. J. Med. ,21 888
(1956)], and various studies have been made since then. Among them, the method of visually determining the degree of aggregation is
Although quantitative measurement is difficult, it is widely used in practice because it is simple and results can be obtained in a short time. In addition, in recent years, quantitative analysis has become possible by optically capturing the degree of aggregation as a change in turbidity, and has become widely used.

0003

[Problems to be Solved by the Invention] However, in either method, whether the degree of aggregation is visually or optically detected, for example, in the case of a dispersion of solid particles carrying antibodies, the degree of aggregation cannot be determined by When the number of antigen molecules in a sample is greatly excessive compared to the number of antibody molecules in the sample, the excess antigen binds to antibodies and forms multimers, blocking the active site of the antibody involved in agglutination. The resulting antigen excess zone phenomenon (so-called prozone phenomenon) can be seen. In other words, when the amount of antigen in a specimen becomes greatly excessive, the concentration of the antigen and the state of agglutination after reaction do not have a 1:1 correspondence, and there are multiple concentrations corresponding to one state of agglutination after reaction with the specimen. will be seen. For this reason, for example, the sample may be diluted and measured at two or more concentrations, and the state of agglutination may be measured at multiple points after the reaction, and temporal changes may be measured over time using materials with known concentrations. Methods are being used to measure the concentration by comparing it with changes. However, since the above-mentioned method is complicated to operate, a more preferable method for avoiding the prozone phenomenon is desired. Furthermore, since the antigen concentration in a specimen at which the prozone phenomenon becomes apparent varies depending on the type of antigen, the type of antibody used, etc., it is difficult to set uniform measurement conditions.

[0004] In the above explanation, an example was shown in which an antigen in a specimen is measured using an antibody bound to a solid fine particle, but the same applies to the reverse mode, that is, an embodiment in which an antibody in a specimen is measured by an antigen on a solid fine particle. You can think about it. The following explanation will be based on the former, but the antibody and antigen can be interchanged, and such embodiments are also included within the technical scope of the present invention. However, in general, it is easier for antibodies to bind to solid microparticles than antigens, and from the viewpoint of specificity, it is easier to carry monovalent antibodies and use the antigen as the substance to be measured.

The present invention solves the problems of the prior art described above, and provides a method that can suppress the prozone phenomenon that tends to occur in measurement methods based on immune reactions and perform stable and accurate measurements. .

[0006]

[Means for Solving the Problems] The present inventors investigated the above-mentioned problems and found that the above-mentioned first reagent consisting of solid fine particles bound to an antibody (first substance) and a sample A first reagent produced by reacting with an antigen (substance to be measured), and then reacting with a second reagent labeled with an antibody (second substance) to the antigen in the specimen, which is different from the antibody. In a method for quantifying an analyte in a sample by measuring the labeled amount of a complex consisting of /analyte/analyte/second reagent, the first reagent is responsible for the reaction that generates the complex. By applying physical action to prevent the formation of multiple complexes (multimers) between fine particles mediated by the substance to be measured, or to keep the ratio of formed multimers below a certain level, prozone They discovered that the phenomenon can be reliably suppressed and completed the present invention.

That is, the present invention provides a first substance that is immunologically active against a substance to be measured in a specimen by solid fine particles (hereinafter referred to as
They are called fine particles. ) is reacted with the sample in a liquid medium to form a complex 1 of the first reagent and the analyte, and then immunologically reacted with the analyte against the analyte. A second reagent pre-labeled with an active second substance different from the first substance is added and reacted with the first reagent/analyte complex 1 to form the first reagent/analyte. A method for quantifying an analyte in a sample by forming a complex 2 consisting of an analyte/a second reagent and then measuring the amount of labeling of the complex 2. This is an immunoassay method characterized in that the reaction of the reagents in step 2 is carried out while exerting a physical action so that the monodispersity x (%) of complex 1 defined below becomes 90 or more.

[0008]

##EQU00002## (In the formula, n indicates the number of complexes 1 that are monomers, and S indicates the total number of particles.) The present invention will be described in detail below. First, in the present invention, the specimens targeted are blood,
Sample fluids include biological samples such as lymph fluid, ascites, and urine, tissue culture fluids, cell culture fluids, and tissue extracts. Measured substances include immunoglobulins, hormones, plasma proteins, bacteria, protozoa, and drugs. and their antibodies, etc., and monovalent or multivalent antigens or antibodies that can be measured based on immune reactions can be targeted.

FIG. 1 is a schematic representation of the reaction according to the present invention and the reaction according to the prior art which produced a multimer. However, although this figure shows an embodiment in which antibodies are used as the first and second substances and antigens are used as the analyte, as described above, in the present invention, they can be considered interchangeably. The present invention is not limited to this figure in any way.

As shown in FIG. 1, in the prior art,
When the first reagent and the sample are reacted, the substance to be measured captures multiple first substances, so in this state it is no longer monodisperse, and the labeled second substance is bound to it. Even if an attempt is made to measure the substance to be measured, accurate measurement is impossible because the reactivity with the second substance changes depending on the state of formation of the multimer. On the other hand, in the present invention, the reaction between the first reagent and the sample (i.e., the reaction between the first substance and the analyte) is performed while physically acting on the reaction solution (stirring in this figure). is not produced, or even if it is produced, it dissociates again, and the number of multimers is controlled below a certain level, so the reactivity with the second substance remains constant. This is because the bonding force between the particle surface and the first substance is stronger than the bonding force between the first substance and the substance to be measured, and the latter bond is broken without breaking the former bond by the action of a predetermined physical force. This is because it becomes possible to selectively cut off those involved in multimer formation.

That is, as will be described later, the bond between the fine particles and the first substance is, for example, a chemical bond via an amino group, a carboxyl group, an aldehyde group, etc., or a physical bond due to van der Waals force, electrostatic attraction, etc. On the other hand, the bond between the first substance and the analyte is based on an immune reaction, such as a hydrophobic bond or a hydrogen bond, and the latter bond is generally weaker than the former and is a suitable bond. It can be selectively cut by processing. Here, when the complex 1 of the first substance and the substance to be measured is in an aggregated state (multimer) rather than monodisperse (monomer), the action of appropriate physical force is the destruction of the aggregate, the monomer This occurs in the following order: destruction of the body, and separation of the first substance from the surface of the fine particles. Therefore, by applying a certain physical force, it is possible to selectively generate monomers.

[0012] Each element related to the measuring method of the present invention will be explained in detail below. The fine particles that are one of the components constituting the first reagent used in the present invention include biologically derived fine particles, inorganic fine particles, and organic fine particles. Examples of the microparticles derived from the living organisms include bacteria such as Staphylococcus and Streptococcus treated with red blood cell dispersion. Examples of the inorganic fine particles include silica, alumina, bentonite, and the like. Examples of the organic fine particles include homopolymers and/or copolymers of vinyl monomers such as styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylic esters, and methacrylic esters, and styrene-butadiene. Copolymer, methyl methacrylate
Examples include fine particles of butadiene-based copolymers such as butadiene copolymers. The binding of immunologically active substances to such fine particles can be done physically and/or chemically, as described below, but it is preferable that the surface of the fine particles is hydrophobic for physical binding, and styrene Particularly preferred are single polymer particles, vinyl polymer particles containing styrene as a main component, or styrene-butadiene copolymers containing styrene as a main component. The particle diameter of the above-mentioned fine particles is preferably 0.1 μm to 10 μm, regardless of whether they are biologically derived, inorganic, or organic fine particles.
．． Particularly preferred is 5 μm to 5 μm. Particle size is 0.1μ
If it is less than m, optical measurement becomes difficult, and if it is more than 10 μm, the stability of the dispersion becomes poor.

The immunologically active substance (first substance) to be bound to the surface of the microparticles used in the present invention and the immunologically active substance to be labeled (second substance) may each independently be IgG, Immunoglobulins such as IgM and IgE: Plasma proteins such as complement, CRP, ferritin, α1 microglobulin, β2 microglobulin, and their antibodies: α-fetoprotein, carcinoembryonic antigen (CE)
A), prostatic acid phosphatase (PAP), CA-
Tumor markers such as 19-9, CA-125 and their antibodies: luteinizing hormone (LH), follicle stimulating hormone (
Hormones such as FSH), human ciliated gonadotropin (hCG), estrogen, and insulin, and their antibodies: HBV-related antigens (HBs, HBe, HBc), H
Viral infection-related substances such as IV and ATL and their antibodies: Clostridium diphtheria, Clostridium botulinum, Mycoplasma,
Bacteria such as Treponema pallidum and their antibodies: Toxoplasma gondii, Trichomona, Thrishmania,
Protozoa such as trypanosomes and malaria parasites and their antibodies: antiepileptic drugs such as phenytoin and phenobarbital, cardiovascular drugs such as quinidine and digoxinin,
anti-asthmatic drugs such as theophylline, chloramphenicol,
Drugs such as antibiotics such as gentamicin, and their antibodies: Other enzymes, exotoxins, and their antibodies, etc. The type of specimen is a substance that specifically causes an antigen-antibody reaction with the analyte in the specimen. It is selected and used as appropriate.

[0014] The bonding between the fine particles and the first substance may be carried out by known physical and/or chemical bonding methods, such as those described in JP-A No. 53-52620, Japanese Patent Publication No. 53-12966, etc. Can be done.

In order to chemically bond the fine particles and the first substance, for example, amino groups, carboxyl groups,
Polyamide compounds, polyimide compounds, polyaldehyde compounds with oriented hydroxyl groups, oxirane groups, etc.
There are a method of chemically bonding with the first substance via a polyoxirane compound, and a method of orienting aldehyde groups, oxirane groups, etc. on the surface of the fine particles and chemically bonding with the first substance.

[0016] The binding reaction between the fine particles and the first substance is as follows:
It is preferable to conduct the reaction in water or a mixed solvent of water and an organic solvent compatible with water such as alcohols or ketones. In addition, the reaction system contains stabilization of fine particles as a monodisperse system,
For the purpose of preventing non-specific aggregation, it is preferable to add a buffer such as a phosphate buffer-physiological saline solution or a Tris-HCl buffer, an inert protein such as bovine serum albumin, a surfactant, or the like. The pH of the reaction solution (ie, dispersion medium) is usually 6 to 10, preferably 7 to 9. In addition, the concentration of fine particles in the reaction solution (dispersion medium) is usually 0.01 to 2.0 (
weight)%.

Next, a second reagent comprising a labeled immunologically active substance (second substance) used in the present invention can be prepared as follows.

First, the labeling of the second substance includes, for example, a method using a radioisotope (radioimmunoassay), a method using an enzyme (enzyme-labeled immunoassay), a method using a fluorescent reagent (fluorescent-labeled immunoassay), etc. A method using a fluorescent reagent is preferable from the viewpoint of handling safety and convenience. As the fluorescent reagent, conventionally known substances can be used, such as fluorescein isothiocyanate (FITC), rhodamine isothiocyanate, sulforhodamine 101 acid chloride, and the like. Labeling of the second substance with these fluorescent reagents can be performed by any known method. For example, for FITC-labeled antibodies, the antibody and FITC are combined at low temperature (4°C) in a pH 9 sodium carbonate buffer. After reacting for 12 hours, it is obtained by separating and purifying the product.

Next, the target substance in the sample is measured using the first and second reagents prepared as described above.

First, the first reagent and the sample are mixed and stirred.
(Hereinafter, examples will be given in which the first and second substances are antibodies and the substance to be measured is an antigen). As a result, when the sample contains an antigen against the antibody (referred to as the first antibody) bound to the surface of the microparticles of the first reagent, the antibody in the first reagent and the antigen bind by an antigen-antibody reaction, A first reagent/antigen complex 1 is produced. If this reaction simultaneously produces a complex (multimer) of fine particles of the first reagent through the antigen, an agglutination reaction will also occur. In the present invention, by applying physical force during antigen-antibody reaction, the first reagent and the sample are uniformly mixed and reacted, and the resulting multimer is transferred between one particle of the first reagent and the antigen in the sample. It dissociates into complexes (monomers) and does not produce multimers during the first reaction. This dissociation from the multimer to the monomer utilizes the difference in the binding strength between the microparticle surface/first antibody and the first antibody/antigen. That is, multimers dissociate into monomers by the action of a physical force of a predetermined magnitude, but
The monomer is not destroyed any further and neither is the first antibody on the microparticle. Here, a physical force of a predetermined magnitude is a force of a magnitude sufficient to dissociate the multimer into monomers, but without destroying the monomers. Compared to a method of dissociating multimers by chemical action or a method of suppressing multimer formation by adjusting the dilution level, the physical dissociation method of the present invention can stably and easily realize monomerization.

Physical forces include, for example, stirring by a stirring tool, vibration of water molecules, etc. (cavitation) by ultrasonic waves, etc.
An example is the one by Etc. Stirring devices can be used to effectively apply physical force in an extremely convenient manner, and specific stirring means include stirring the target reaction solution at a predetermined rotation speed using various stirring blades or stirrers. Bye. Furthermore, the method using ultrasonic waves can reduce the amount of reaction liquid. The means for applying these physical forces can also be appropriately selected by taking advantage of their characteristics.

[0022] The physical action can be applied at our laboratory during the antigen-antibody reaction (analyte-first substance reaction), or before starting the labeled antibody (second substance) reaction. In any case, monomerization may be completed before the reaction of the labeled antibody. Preferably, a physical action is applied at the point where the antigen-antibody reaction occurs.

The magnitude of the physical effect imparted varies depending on the type of target substance to be measured, the first substance used, the type of fine particles, the supporting method, etc., and the amount of monomer can be determined as appropriate by the following measurements. All you have to do is measure it and set the appropriate conditions.

That is, the monomer can be confirmed using, for example, a flow cytometer.
The dissociation state of reagent/antigen) can be measured by the following method.

A portion of the product of the stirred first reaction is diluted and introduced into a flow cell. The degree of dilution is appropriately adjusted to a concentration that allows the masses of complex 1 to be delivered one by one. The composites 1 sent one by one are irradiated with light such as laser light or ultraviolet light, and the degree of aggregation (monodispersity) can be determined by measuring the resulting optical reaction, for example, scattered light. In order to avoid the prozone phenomenon, which is the objective of the present invention, the degree of monodispersity at this time has a great influence, but as a result of intensive research on the relationship between the degree of monodispersity and measurement accuracy, the present inventors found that
The ratio z (monodispersity) of the number n of single particle composite 1 to the total number S of particles of composite 1 is

[0026]

It has been found that the above object can be achieved by forming a monodisperse system that satisfies x≧90. When x is less than 90, the bias due to the multimer significantly influences the measured value, but when x is 90 or more, the influence can be suppressed within an acceptable range. Preferably it is 95 or more.

Next, after confirming that the monodispersity is within the above-mentioned range in the first reaction, proceed to the second reaction between the complex 1 and a second reagent consisting of a labeled antibody. By satisfying the monodispersity within the above-mentioned range, the reaction amount ratio between Complex 1 and the second reagent becomes substantially constant, that is, the reaction amount ratio with the second reagent is also substantially constant depending on the amount of antigen in the sample. Therefore, a complex 2 consisting of the first reagent/antigen/second reagent having a constant composition can be obtained depending on the amount of antigen in the sample.

The amount of antigen is measured by extracting the signal of the label caused by complex 2 contained in the reaction solution after adding the second reagent and completing the reaction. A corresponding constant measurement value will be obtained.

The label contained in complex 2 is measured, for example, by a method similar to that used for measuring the dissociation state of complex 1 described above. That is, the reaction solution containing the complex 2 obtained by the second reaction is diluted to a concentration such that a lump of the complex 2 is sent one by one, and then introduced into the flow cell. The complexes 2 sent one by one are irradiated with light such as laser light or ultraviolet light, and the resulting optical reactions, such as scattered light and fluorescence, are successively measured.

[0030] For the above measurement, for example, a clinical test 30 (11
) 1259, a flow cytometer with optical axes perpendicular to each other, a flow cytometer with the same optical axis, and the like are preferably used. In order to determine the amount of antigen in a sample using the above device, data on the amount of label in the monomer of complex 2 measured by the above method and a calibration curve showing the relationship between the amount of antigen and the amount of label prepared in advance are used. The amount of antigen in the specimen can be determined by comparing the data. As described above, according to the present invention, it is possible to perform highly sensitive and highly accurate measurements without the prozone phenomenon, regardless of the amount of the analyte in the sample.

[0031]

[Examples] The present invention will be explained below with reference to Examples. Production of the first reagent: Anti-human CRP goat serum (manufactured by Bio Makor) was
Purify the IgG fraction by column chromatography using rotein-ASepharose (manufactured by Pharmacia), and add 10 mg to 0.1M phosphate buffer at pH 5.5.
It was diluted to a concentration of /ml. Particle size 0.71nm
1-cyclohexyl-3-[2-morpholinyl-(4) as a condensing agent was added to 10 ml of a 10% water suspension of carboxylated polystyrene (G0701 manufactured by Nippon Gosei Rubber Co., Ltd.).
-ethyl]carbodiimide 1% aqueous solution of metho-p-toluenesulfonate (hereinafter referred to as carbodiimide Ts) 25
ml and then 20 ml of the above-mentioned IgG fraction antibody were added and stirred at room temperature for 3 hours to obtain a sensitized latex. After centrifugal washing of the sensitized latex, 1% bovine serum albumin 3
% sucrose, pH 7.2 phosphate buffer-physiological saline (hereinafter referred to as PBS) was added, and the fine particle concentration was 0.
A 2% by weight dispersion of CRP antibody-sensitized fine particles (first reagent) was obtained. First reagent and CRP serum reaction: Standard CRP serum (manufactured by Kyowa Yuka) was diluted with Tris HCl buffer to a concentration of 50 μg/ml.
It was designated as P sample. While stirring 1 ml of the first reagent with a stirrer, 1 ml of the CRP specimen was added and reacted, a portion of the reaction solution was taken and the monodispersity x was determined to be 96 using a flow cytometer. Measurement of second reagent reaction and fluorescence intensity: While stirring 1 ml of the above reaction solution with monodispersity x of 96 using a stirrer, add FITC-labeled anti-human CRP sheep TgG (manufactured by Binding Site) to 0.1 M at pH 5.5. After diluting with phosphate buffer to a concentration of 5 μg/ml, add 1 ml and incubate at room temperature for 60 minutes.
The reaction was stirred with a stirrer for a minute. After washing the reaction solution by centrifugation, add PBS and stir with a stirrer to disperse it until the particle concentration is 0.
．． 1% by weight CRP antibody sensitized microparticles/CRP/FITC
A dispersion of labeled anti-human CRP sheep TgG complex was obtained. A portion of the reaction solution was taken, the fluorescence intensity of the complex was determined using a flow cytometer, and the CRP concentration was determined using a previously prepared calibration curve, and the value was obtained with good reproducibility.

[0032]

Effects of the Invention As explained above, in immunoassay methods using latex reagents, the first
When performing an antigen-antibody reaction between a substance and a substance to be measured, by applying a physical action to suppress multimer formation, measurement accuracy and sensitivity can be improved, and stable measurement can be performed. Become. Furthermore, according to the present invention, it is possible to always easily carry out a constant measurement depending on the amount of the substance to be measured in the sample, and the first substance is a monovalent or multivalent antibody or an antigen, and the substance to be measured is added to the first substance. Desired measurements can be made by selecting according to the conditions. In particular, the simplicity is further improved by causing the physical action during the reaction by stirring and by using the fluorescent labeling substance as the second substance.

[Brief explanation of the drawing]

FIG. 1 is a schematic reaction diagram for explaining the measurement principle of the present invention.

Claims (4)

[Claims] Claim 1: A first reagent comprising a first substance immunologically active against a substance to be measured in a sample bound to the surface of a solid fine particle is reacted with a sample in a liquid medium, A complex 1 of the reagent and the analyte is formed, and then a second substance different from the first substance is immunologically active toward the analyte.
A second reagent prepared by pre-labeling a substance is added to the first reagent.
By reacting with the reagent / analyte complex 1 of 1 to form a complex 2 consisting of the first reagent / analyte / second reagent, and then measuring the labeled amount of the complex 2. ,
In a method for quantifying an analyte in a sample, the reaction between the complex 1 and a second reagent is performed as a complex 1 defined below.
An immunoassay method characterized in that it is carried out while exerting a physical action so that the degree of monodispersity x (%) becomes 90 or more. [Formula 1] (In the formula, n is the number of complex 1 that is a monomer, and S represents the total number of particles.) 2. The measuring method according to claim 1, wherein the physical action is an action by stirring the liquid medium. 3. The measurement method according to claim 1, wherein the substance to be measured is an antigen, and the first and second substances are antibodies against the antigen. 4. The measurement method according to claim 1, wherein the label of the second reagent is a fluorescent label.