JPH10123137A - Highly sensitive immunoassay method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunoassay method for determining an antigen substance accurately by allowing antigens or antibodies that are related to a reaction to exist as much as possible in an antigen antibody reaction system without causing agglutination inhibition due to the steric hindrance of an insoluble carrier and without conjuring up a non-specific agglutination. SOLUTION: In the immunoassay method, at least two types of different monoclonal antibodies for a specific antigen are carried by an insoluble carrier, are reacted with the antigen in a water-soluble medium, and at least two types of carries with different average particle diameters are used as the insoluble carrier when aggregating the combined substance of the insoluble carrier and the antigen selectively, and the insoluble caries carry each monoclonal antibody. A preferable insoluble carrier is a latex particle. At least two types of latex particles whose average particle diameter ranges from 0.05 to 0.500μm are preferably combined so that the ratio of the average particle diameter of other particle species to that of the minimum particle seed ranges from 3 to 6.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a high-sensitivity method in which a monoclonal antibody against a specific antigen is supported on an insoluble carrier, reacted with the antigen in an aqueous solvent, and agglutination reaction between the insoluble carrier and the antigen is performed. It relates to an immunoassay.

[0002]

2. Description of the Related Art In recent years, hospitals and testing centers have been demanding labor saving, cost reduction, and processing of a large number of samples.
Various tests such as clinical tests have been automated and the measurement time has been shortened. As a method suitable for such automation, an agglutination method for qualitatively or quantitatively determining an antigenic substance by utilizing an agglutination reaction of insoluble carrier particles is known (Japanese Patent Publication No. Sho 58).
-11575).

In the agglutination method, when measuring an antigenic substance in a test sample, the test sample is reacted with an insoluble carrier carrying an antibody or a fragment thereof that specifically binds to the antigenic substance. The antigenic substance is detected or quantified by measuring the degree of aggregation of the insoluble carrier. In this case, as the antibody,
Usually, a polyclonal antibody or a monoclonal antibody is appropriately selected. Since a monoclonal antibody reacts only with a specific epitope on an antigen molecule, even if the antigenic substance is a polyvalent antigen, the antigenic substance is not always polyvalent with respect to the specific epitope. Therefore, when a monoclonal antibody is used as the above antibody, the antigen is more likely to be a monovalent antigen with respect to the epitope corresponding to the specific monoclonal antibody. When the antigen substance to be measured is a monovalent antigen, aggregation usually occurs even when one kind of monoclonal antibody supported on an insoluble carrier reacts or binds with the antigenic substance in the test sample. Absent. For this reason, polyclonal antibodies are generally used frequently from the viewpoint of easy aggregation.

However, when a polyclonal antibody and a monoclonal antibody are compared, the monoclonal antibody is by far the best in terms of antigen specificity.
In addition, since polyclonal antibodies differ depending on the individual differences between animals and the time of blood sampling, it is difficult to always obtain the same quality, and the amount and activity of the antibody during purification to a specific antibody are large. In contrast, monoclonal antibodies are suitable for mass-producing antibodies of constant quality.

In view of the above, two or three different monoclonal antibodies against a specific antigen are supported on an insoluble carrier, reacted with the antigen, and the degree of aggregation of the insoluble carrier is measured. There has been proposed a method for detecting or quantifying the antigenic substance (Japanese Patent Publication No. 3-4 / 1991).
0341).

In recent years, in immunodiagnosis, trace substances contained in urine, serum, plasma, etc., for example, α-
It is desired that quantification of fetoprotein (AFP), carteinoembryoic antigeneene (CEA) and the like be performed down to a concentration as low as 1 ng / ml. In order to measure such a substance, a method has been proposed that enables measurement of antigens or antibodies in a wide range of concentrations from low to high by mixing two or more types of latex particles having different average particle sizes. (See JP-A-63-65369).

[0007]

However, in the method in which all the monoclonal antibodies are supported on an insoluble carrier having the same average particle size, steric hindrance caused by the insoluble carrier causes a problem between the antibody and the antigenic substance to be measured. May inhibit the antigen-antibody reaction and subsequent agglutination. That is, when the antigenic substance simultaneously binds to one type of monoclonal antibody and at least another type of monoclonal antibody, aggregation of the insoluble carrier occurs, but all the monoclonal antibodies are supported on the insoluble carrier. When the epitopes of the antigens recognized by the two types of monoclonal antibodies are located at sites that are structurally close to each other, steric hindrance between the insoluble carriers causes
The simultaneous binding of two or three different monoclonal antibodies to one antigenic substance may be inhibited. Also, when the affinity constant between the antigen and the antigen is greatly different between the two or three types of monoclonal antibodies, the monoclonal antibody having the higher affinity constant preferentially binds to the antigen, as described above. May interfere with binding to the monoclonal antibody and subsequent aggregation. For these reasons, there is a possibility that antigenic substances cannot be accurately quantified by a method in which a monoclonal antibody is supported on an insoluble carrier having the same average particle size.

As described above, Japanese Patent Application Laid-Open No. 63-65369 discloses a method for detecting a low concentration of an antigenic substance.
Although a method of mixing two or more types of latex particles having different average particle sizes is disclosed, the antibody used in this method is a polyclonal antibody, and the invention described in this publication discloses the reaction in an antigen-antibody reaction system. It is intended that as many antigens or antibodies as possible are present as much as possible.
Therefore, the reaction amount at low concentration increases, but at the same time, there is a disadvantage that non-specific agglutination derived from substances other than the background and the antigenic substance is large.

In view of the circumstances as described above, an object of the present invention is to prevent an insoluble carrier from being inhibited by aggregation due to steric hindrance, not causing nonspecific aggregation, and causing an antigen or antibody involved in a reaction in an antigen-antibody reaction system. The present invention is to provide a highly sensitive immunoassay method in which antigens can be accurately quantified as much as possible.

[0010]

Means for Solving the Problems A highly sensitive immunoassay according to the present invention comprises the steps of: loading two or more different monoclonal antibodies against a specific antigen on an insoluble carrier, reacting the antigen with the antigen in an aqueous solvent; In selectively aggregating the conjugates, two particles having different average particle sizes are used as insoluble carriers.
This method is characterized by using at least one kind of carrier and carrying each monoclonal antibody on each of these insoluble carriers.

The antigenic substance suitable for detection according to the present invention is not particularly limited as long as it is a physiologically active substance contained in a biological sample and a monoclonal antibody corresponding to the antigenic substance can be prepared or obtained. However, a monovalent antigen which is undetectable with one type of monoclonal antibody is particularly desirable. Further, as a test item for detecting an antigenic substance, an item that is important in a clinical test and is considered to be insufficient in detection sensitivity by the conventional agglutination method is particularly useful.

For example, 1 ng / ml of CEA, CA19-9 or the like, which is measured in screening for cancer screening
Items that require detection sensitivity up to this point.

The monoclonal antibody used in the present invention may be composed of two or more different antibodies against a specific antigen (for example, two or three different antibodies).
(Preferably two). Monoclonal antibodies can be produced and obtained using the cell line, by appropriately selecting a technique known per se in the cell fusion technical field, and combining them to form a monoclonal antibody-producing fusion cell line. ("Monoclonal antibodies, hybridomas and ELISA", Tatsuo Iwasaki et al., Kodansha).

According to one embodiment of cell fusion, a certain complete antigen is used and is transferred to a suitable animal, such as a mouse, rat, rabbit, goat, sheep, horse, cow, etc., together with an adjuvant, for example. After administration using a technique such as subcutaneous injection to immunize the animal, the immunized animal, for example, antibody-producing cells against the antigen of the immunized mouse, such as spleen cells, thymocytes, lymph node cells and / or peripheral blood cells The cells are collected, and the cells are fused with a suitable cell line having self-proliferation but substantially no antibody-producing ability, for example, a mouse myeloma (myeloma) cell line by a method known per se. To process.

[0015] The combination of myeloma cells and antibody-producing cells to obtain a monoclonal antibody is based on the type of animal from which each cell is derived, as long as the cells can fuse and proliferate to produce the antibody. There is no limitation, and any combination may be used.

The myeloma cells used are not particularly limited, and cell bodies of many animals such as mice, rats, rabbits and humans can be used. Preferred cell lines are drug resistant and are such that unfused myeloma cells do not survive in the selective medium, while only fused cells survive. A commonly used cell line is an 8-azaguanidine-resistant cell line, which is deficient in hypoxanthine / guanine / phosphoribosyltransferase and cannot grow on hypoxanthine / aminpterin / thymidine (HAT) medium. ing. Further, it is preferable that the cell line to be used is "non-secretory". For example, P ₃ / X derived from mouse myeloma MOPC-21 strain
_{63-Ag8U 1 (P 3 U} 1), P 3 / X63-Ag8
・ 6.5-3, P ₃ / NSI-1-Ag4-1, Sp2
/ O-Ag14, Rat Myeloma 210 ・ RCY3 ・
Ag1, 2, 3, etc. are preferably used.

The cell fusion treatment is carried out, for example, usually in a medium such as Eagle's minimal basic (MEM) medium or RPMI-1640 medium, from 1 × 10 ^{8 to} 5 × 1 spleen cells of the immunized mouse.
0 ⁸ and, the mouse myeloma cell line 1 × 10 ⁷ ~5 ×
10 ⁷ can be mixed and performed. As the fusion promoter, polyethylene glycol (PEG) having an average molecular weight of 1,000 to 6,000 is preferable, and other fusion promoters, for example, polyvinyl alcohol, virus and the like can also be used. The working concentration of PEG is preferably about 30-50%.

The fused cells obtained from the fused cell-containing system obtained as described above are subjected to selection, antibody activity screening, and cloning using known methods to form immunized mice. Thus, a fused cell line having the ability to produce a monoclonal antibody against the complete antigen and the ability to self-proliferate can be obtained.

The above-mentioned process for selecting fused cells is carried out, for example, in the step of
% Fetal bovine serum-containing RPMI-1640 medium cells having been subjected to the cell fusion, etc. were appropriately diluted, aliquoted into ¹⁰⁵ to about ¹⁰⁶ cells / well in 96-well microplate min, selective medium (e.g., HAT to each well Medium), and the culture is continued in a 5% CO ₂ incubator (37 ° C.) while replacing the selective medium. If an 8-azaguanine-resistant strain is used as the myeloma cell, the unfused myeloma cell is killed in the HAT medium, and the antibody-producing cell is a normal cell and cannot be grown for a long time in an in vitro (in vitro) culture. Therefore, cells that grow from about 10 to 14 days after culture are all fusion cells.

The antibody activity screening and cloning of the fusion cell strain obtained as described above can be carried out, for example, by a conventional method as follows.

A part of the culture supernatant in the well in which the fused cells have grown is collected, incubated with a fixed amount of a labeled antigen, and measured for its ability to bind to the labeled antigen, thereby determining the well secreting the desired antibody. Can be searched.
That is, after reacting the culture supernatant with an antigen labeled with a radioisotope or an enzyme such as ¹²⁵ I or ¹³¹ I, the antigen-antibody conjugate is separated from each reaction solution, and the amount of the label is measured, whereby the target amount is determined. The presence and binding ability of the antibody can be searched.

Since there is a possibility that two or more types of fused cells may grow in each well in which the desired antibody activity is observed, cloning is performed by limiting dilution or colony formation with soft agar, and monoclonal antibodies are cloned. A production fusion cell line can be obtained.

In order to obtain a monoclonal antibody against the complete antigen used to form the immunized animal by using the monoclonal antibody-producing cell line obtained as described above, the monoclonal antibody-producing cell line is used, for example, Using a method known per se, such as a method of culturing the cells in a suitable medium and collecting the monoclonal antibody from the medium, and a method of transplanting the cell line into an animal of the same strain as the myeloma cell-derived animal and collecting the monoclonal antibody in ascites fluid. Can be.

According to the former method, for example, a monoclonal antibody-producing fusion cell line is cultured in a culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, and the culture supernatant is subjected to ammonium sulfate fractionation or antigen extraction. The target monoclonal antibody can be collected by purification by affinity chromatography or the like such as Sepharose 4B to which is bound.

According to the latter method, for example,
After intraperitoneal administration of mineral oil such as pristane (2,6,10,14-tetramethylpentadecane) to syngeneic animals,
In vivo administration of fused cells by intraperitoneal administration
3.) Proliferate the fused cells in large quantities. As a result, the ascites fluid formed contains high concentrations of monoclonal antibodies. The target monoclonal antibody can be obtained from the ascites by ammonium sulfate fractionation and, if necessary, the affinity chromatography.

A monoclonal antibody which can be obtained as described above can be obtained as a commercial product and can be used in the method of the present invention.

As the insoluble carrier used in the present invention, a carrier of fine particles generally used in an immunochemical agglutination reaction and an agglutination inhibition reaction can be used.

As such an insoluble carrier, industrially,
Organic particles that can be mass-produced are preferred, but are not limited
It is not something to be done. Organic fines that can be mass-produced industrially
Examples of the particles include styrene, vinyl chloride, and acrylic.
Lonitrile, vinyl acetate, acrylate, methacrylate
Homopolymers and vinyl monomers such as lylic acid esters
And / or copolymer, styrene-butadiene copolymer
Body, methyl methacrylate-butadiene copolymer, etc.
Fine particles such as butadiene copolymer and functional groups
Carboxyl group, primary amino group, or carboxamide group
No group (-CONH _Two), Hydroxyl group, aldehyde group, etc.
And a reactive organic system wherein the substrate comprises the organic fine particles.
Fine particles and the like. Excellent antibody adsorption,
And the ability to maintain biological activity stably for a long period of time
And especially polystyrene latex particles are preferred.
No.

Other examples include biological particles such as animal red blood cells and bacterial cells, and non-biological particles such as bentonite, collodion, cholesterol crystals, silica, kaolin, and carbon powder.

The average particle size of the insoluble carrier used in the present invention is such that the aggregate on the antibody on the insoluble carrier and the agglutination produced by the antigen-antibody reaction of the antigen substance to be measured are visually or optically. Any size can be used as long as the size is large enough to be detected. In particular, the average particle size is 0.05 to
Two or more insoluble carriers (preferably latex particles) in the range of 0.500 μm are combined with other particle types with respect to the average particle size of the smallest particle type (the smallest average particle size of the two or more insoluble carriers). It is preferable to use them in combination such that the ratio of the average particle size of (one or two or more of the two or more insoluble carriers other than the minimum particle type) is in the range of 3 to 6.

When mixing two kinds of insoluble carriers, the volume ratio is appropriately selected in the range of 1:10 to 10: 1 according to the required detection lower limit of the substance to be measured. When mixing three or more insoluble carriers, the mixing ratio may be determined in the same manner as described above.

Various techniques for sensitizing the surface of the insoluble carrier with the monoclonal antibody are known and can be appropriately used in the present invention. For example, such a sensitizing method is a method of physically adsorbing a monoclonal antibody on the surface of an insoluble carrier, or a method of chemically binding or covalently bonding a monoclonal antibody to a surface of an insoluble carrier having a functional group by a known method. There is a method of sensitizing efficiently.

The reaction between an insoluble carrier carrying an antibody and an antigen is an antigen-antibody reaction and an accompanying agglutination reaction.
The reaction conditions are not particularly limited as long as the reaction can take place, but the reaction temperature is constant, particularly 25 ° C to 37 ° C.
It is preferable that the temperature is within the range described above. The reaction time is not particularly limited, but is preferably 5 seconds to 15 minutes.

The reaction solution may be any solution as long as it satisfies physiological conditions under which an antigen-antibody reaction can take place. Phosphate buffer, glycine buffer, Tris-HCl buffer, Good buffer and the like can be used. preferable. The pH of the reaction solution is
Preferably 5.5 to 8.5, particularly preferably 6.5 to 8.5.
8.0. The above reaction solution is prepared by adding bovine serum albumin and sucrose as stabilizers, water-soluble polysaccharides such as polyethylene glycol and dextran, which are expected to enhance sensitivity, sodium azide as a preservative, and chloride for adjusting salt concentration. An additive such as sodium may be appropriately dissolved.

The method for measuring the degree of aggregation of the insoluble carrier is not particularly limited. For example, when agglutination is measured qualitatively or semi-quantitatively, it is possible to visually determine the degree of aggregation of the conjugate from comparison with the degree of turbidity of a known sample. When the aggregation is quantitatively measured, for example, it is desirable to measure the aggregation optically from the viewpoint of simplicity and accuracy.

As an optical measurement method of aggregation, a known method can be used. More specifically, for example, a so-called turbidity method (the formation of aggregates is regarded as an increase in turbidity),
Measurement method based on particle size distribution (the formation of agglomerates is regarded as a change in particle size distribution or average particle size), integrated sphere turbidity method (change in forward scattered light due to the formation of agglomerates is measured using an integrating sphere, and transmitted light Various methods are available, such as comparing the ratio with the intensity.

For each of these assays, a rate test (rate assay; at least two measurements were taken at different time points, and the extent of aggregation based on the increment (ie, the rate of increase) of the measurements between these time points) Ask for)
And an end point test (end point assay; one measurement is taken at some point (usually the end point of the reaction) and the degree of aggregation is determined based on this measurement). From the viewpoint of simplicity and quickness of measurement, it is desirable to conduct a speed test using a turbidimetric method.

[0038]

Embodiments of the present invention will be described below.

The reagents and materials used in the examples and comparative examples are as follows.

1) Reagents and Materials The following reagents and materials were prepared or prepared.

Anti-human CEA monoclonal antibody: anti-CE
A monoclonal antibody (Clone No. CEA4-G67,
IgG-1, DAKO, and Clone No. CEA4
-G11, IgG-1, manufactured by DAKO).

Latex: average particle size 0.087 μm,
0.120 μm, 0.210 μm 0.400 μm,
Five types of latex each containing 0.464 μm polystyrene particles (all having a solid content of 10% (W / V), manufactured by Sekisui Chemical Co., Ltd.) were used.

Latex dilution buffer: 50 mM Na
₂ HPO ₄ and 50 mM NaH ₂ PO ₄ at pH 7.50
And the resulting mixture was used as a latex dilution buffer.

Antibody dilution buffer: The above latex dilution buffer was also used as an antibody dilution buffer.

Blocking buffer: 100 mM Na
₂ HPO ₄ and 100 mM NaH ₂ PO ₄ at pH 7.4
0, and the resulting mixture was added to bovine serum albumin (Bovine serum albumin, Fraction V, Reagent
Grade, manufactured by Miles Corp.) to 1% (W / V), and NaN ₃ (reagent grade, manufactured by Nacalai Tesque)
Was added to a concentration of 0.1% (W / V) and used as a blocking buffer.

CEA standard products: 0, 5, CEA standard products (manufactured by Dynabot, attached to CEA / rear beads kit)
20, 100 and 500 ng / ml were used as they were.

Diluent for sample dilution (R1 solution): polyethylene glycol 6,000 (average molecular weight 7,500, Wako Pure Chemical Industries) 3% (W / V) in blocking buffer
The diluted solution for sample dilution (R1
Liquid).

Example 1 (1) Preparation of Reagent for CEA Measurement To 1 volume of polystyrene latex (solid content 10% (W / V)) having an average particle size of 0.087 μm, 9 volumes of a latex dilution buffer were added. The latex was diluted to a 1.0% latex solution. Anti-CEA antibody (Clone No. CEA4-G
No. 67) was diluted with an antibody diluting buffer so that the protein concentration became 66.7 μg / ml to obtain a sensitizing antibody solution. Add 600 μl of 1.0% (W / V) latex solution to 25
While stirring with a magnetic stirrer in an incubator at ° C, 1200 µl of the antibody solution was quickly added thereto, followed by stirring at 25 ° C for 1 hour. Thereafter, 3.0 ml of a blocking buffer was added, and the mixture was continuously stirred at 25 ° C. for 2 hours. Then, at 15 ° C and 15,000 rpm, 1
Centrifuged for 5 minutes. 4.0 ml of a blocking buffer was added to the obtained precipitate, and the precipitate was washed similarly by centrifugation. The washing operation was performed three times. 1.8 ml of a blocking buffer was added to the precipitate, and the mixture was stirred well, and then subjected to a dispersion treatment with an ultrasonic crusher. To this, 1.8 ml of a blocking buffer was further added to obtain a CEA measuring reagent [1] having a solid content of 0.17% (W / V). The reagent for CEA measurement [1] thus prepared was stored at 4 ° C.

Further, as the insoluble carrier liquid, an average particle diameter of 0.1 is used.
464 μm polystyrene latex (solid content 10%
(W / V)) as a sensitive antibody solution
An antibody (Clone No. CEA4-G11, IgG-1) diluted with an antibody diluting buffer so that the protein concentration becomes 12.5 μg / ml is used.
The same operation as in the case of the measurement reagent [1] was performed to obtain a CEA measurement reagent [2] having a solid content of 0.17% (W / V).
Stored at 4 ° C.

(2) Measurement of CEA Amount The CEA amount was measured using an automatic analyzer for biochemistry Model 7150 (manufactured by Hitachi, Ltd.). Equal amounts of the reagents [1] and [2] for CEA measurement having a solid content of 0.17% (W / V) obtained in the above (1) are mixed in equal amounts, and the obtained mixture is mixed with the reagent (solution R2) (solid 0.17% (W / V)).
The measurement conditions are as follows.

Sample volume 20 μl Diluent for sample dilution (R1 solution) 210 μl Reagent (R2 solution) 30 μl Measurement wavelength 570 nm Measurement temperature 37 ° C.

The difference between the absorbance approximately 80 seconds after the addition of the reagent (R2 solution) to the measurement system and the absorbance approximately 320 seconds later (ΔOD
570), and the difference in absorbance multiplied by 10,000 was defined as the change in absorbance. C of known concentration as a sample
The measurement was performed using the EA standard, and a calibration curve was created.

Comparative Example 1 (Measurement of CEA amount using latex agglutination immunoreagent having the same average particle size) CEA measurement reagent [1] (latex agglutination immunoreagent (solid content 0.17%) obtained in Example 1 Example 1 except that (W / V)) was used as a reagent (R2 solution) as it was.
The same operation was performed.

Comparative Example 2 (Measurement of CEA amount using latex agglutination immunoreagent having the same average particle size) CEA measurement reagent [2] (latex agglutination immunoreagent (solid content 0.17%) obtained in Example 1 Example 1 except that (W / V)) was used as a reagent (R2 solution) as it was.
The same operation was performed.

Test Results Table 1 and FIG. 1 show the amounts of change in absorbance measured in Example 1 and Comparative Examples 1 and 2 and the calibration curves prepared.

[0056]

[Table 1]

As is clear from Table 1 and FIG. 1, the calibration curve prepared from the measured values of Example 1 by the method of the present invention showed good linearity from the low concentration range to the high concentration range of CEA. . On the other hand, the calibration curves of Comparative Examples 1 and 2 using the latex agglutination immunoreagent having the same average particle size show lower linearity and lower linearity in the CEA low concentration region and high concentration region than the method according to the present invention. Did not show.

This is because, in Example 1, a latex reagent comprising two types of monoclonal antigens supported on insoluble carriers having different average particle diameters was used. It is presumed that the steric hindrance between them was resolved, and the antigen-antibody reaction and the subsequent agglutination reaction were promoted.

From the above results, it was confirmed that the CEA quantification method according to the present invention was a quantification method superior in sensitivity and superior to the conventional latex agglutination immunoreagent.

Example 2 (1) Preparation of Reagent for CEA Measurement Two kinds of polystyrene latex having an average particle size of 0.120 μm and 0.400 μm were used as an insoluble carrier solution, and a CEA antibody Clone was used as a sensitive antibody solution. No. C
EA4-G67 diluted with an antibody dilution buffer so that the protein concentration becomes 48.3 μg / ml; and
The CEA antibody Clone No. CEA4-G11 was diluted with an antibody diluting buffer so that the protein concentration was 14.5 μg / ml.
The same operation as in the case of the measurement reagent [1] was performed to obtain CEA measurement reagents [3] and [4] having a solid content of 0.17% (W / V), and stored at 4 ° C.

(2) Measurement of CEA Amount of CE having a solid content of 0.17% (W / V) obtained in (1) above.
Example 1 was repeated except that equal amounts of the reagents A for measurement [3] and [4] were mixed and the resulting mixture was used as the reagent (solution R2).
The same operation as described above was performed to measure the CEA amount.

Comparative Example 3 (Measurement of CEA amount using latex agglutination immunoreagent having the same average particle size) CEA measurement reagent [3] obtained in Example 2 (latex agglutination immunoreagent (solid content 0.17% Example 2 except that (W / V)) was used as a reagent (solution R2) as it was.
The same operation was performed.

Comparative Example 4 (Measurement of CEA amount using latex agglutination immunoreagent having the same average particle size) CEA measurement reagent [4] obtained in Example 2 (latex agglutination immunoreagent (solid content 0.17% Example 2 except that (W / V)) was used as a reagent (solution R2) as it was.
The same operation was performed.

Test Results Table 2 and FIG. 2 show the change in absorbance measured in Example 2 and Comparative Examples 3 and 4, and the prepared calibration curves.

[0065]

[Table 2]

As is clear from Table 2 and FIG. 2, the calibration curve prepared from the measured values of Example 2 according to the method of the present invention shows good results from the low concentration region to the high concentration region of CEA as in Example 1. It showed excellent linearity. On the other hand, the calibration curves of Comparative Examples 3 and 4 using the latex agglutination immunoreagent having the same average particle size show lower linearity in the CEA low concentration region and high CEA concentration region than in the method according to the present invention. Did not show.

Example 3 An equal amount of the CEA measurement reagent [1] obtained in Example 1 and the CEA measurement reagent [4] obtained in Example 2 were mixed.
The amount of CEA was measured by performing the same operation as in Example 1 except that the obtained mixture was used as a reagent (solution R2).

Example 4 An equal amount of the CEA measuring reagent [2] obtained in Example 1 and the CEA measuring reagent [3] obtained in Example 2 were mixed.
The amount of CEA was measured by performing the same operation as in Example 1 except that the obtained mixture was used as a reagent (solution R2).

Test Results Table 3 and FIG. 3 show the change in absorbance measured in Examples 3 and 4 and the prepared calibration curve.

[0070]

[Table 3]

As is clear from Table 3 and FIG. 3, the calibration curves prepared from the measured values of Examples 3 and 4 according to the method of the present invention are similar to those of Example 1 from the low concentration range of CEA to the high concentration range. It showed good linearity.

Example 5 (1) Preparation of Reagent for CEA Measurement A polystyrene latex having an average particle size of 0.210 μm was used as an insoluble carrier liquid, and
The EA antibody Clone No. CEA4-G67 was diluted with an antibody dilution buffer so that the protein concentration became 27.6 μg / ml, and in the other respects, the CEA measurement reagent [1] of Example 1 was used. The same operation as described above was performed to obtain a solid content of 0.1%.
17% (W / V) CEA measuring reagent [5] was obtained at 4 ° C.
Saved in.

(2) Measurement of CEA content CE of 0.17% (W / V) solid content obtained in (1) above
An A-reagent [5] and a CEA-reagent [2] obtained in Example 1 were mixed in equal amounts, and the resulting mixture was used as a reagent (R2 solution). The same operation as in Example 1 was performed to measure the amount of CEA.

Comparative Example 5 (Measurement of CEA Amount Using Latex Agglutination Immunoreagent with Same Average Particle Size) (1) Preparation of CEA Measurement Reagent A polystyrene latex having an average particle size of 0.464 μm was used as an insoluble carrier liquid. As a sensitizing antibody solution, C
Using the EA antibody Clone No. CEA4-G67 diluted with an antibody diluting buffer so that the protein concentration becomes 12.5 μg / ml, in other respects the same operation as in the case of the CEA measurement reagent [1] is used. 0.17% solids
(W / V) CEA reagent [6] was obtained and stored at 4 ° C.

(2) Measurement of CEA content CE of 0.17% (W / V) solid content obtained in (1) above
An A-reagent [6] and an CEA-reagent [2] obtained in Example 1 were mixed in equal amounts, and the resulting mixture was used as a reagent (R2 solution), except that the mixture was used as a reagent (R2 solution). The same operation as in Example 1 was performed to measure the amount of CEA.

Test Results Table 4 and FIG. 3 show the amounts of change in absorbance measured in Example 5 and Comparative Example 5 and the prepared calibration curves.

[0077]

[Table 4]

As is clear from Table 4 and FIG. 3, the calibration curve prepared from the measured values of Example 5 according to the method of the present invention shows good results from the low concentration region to the high concentration region of CEA as in Example 1. It showed excellent linearity. On the other hand, the calibration curve of Comparative Example 5 using the latex agglutination immunoreagent having the same average particle size is as follows:
In the low concentration region and the high concentration region of CEA, the reactivity was lower than that of the method according to the present invention, so that good linearity was not exhibited.

[0079]

Since the immunoassay according to the present invention is constituted as described above, there is no inhibition of aggregation due to steric hindrance of the insoluble carrier as seen with the conventional latex agglutination immunoreagent, and non-specific agglutination can be achieved. The antigen or antibody involved in the reaction can be present in the antigen-antibody reaction system as much as possible without causing any elicitation. As a result, the antigenic substance can be accurately quantified, and high detection sensitivity can be exhibited even in a low concentration range. Particularly, when the epitopes of the antigens recognized by two or more types of monoclonal antibodies are structurally close to each other, or when the molecular weight of the antigenic substance is small and an insoluble carrier having the same average particle diameter does not easily cause agglutination, The effect is exhibited. Therefore, the measurement method according to the present invention enables accurate quantification of an antigenic substance in a test sample, and is useful for finding a disease, grasping a disease state, and determining a treatment method.

[Brief description of the drawings]

FIG. 1 shows the correlation between the amount of CEA in a CEA standard sample of known concentration used in Example 1 and the amount of change in absorbance according to the CEA measuring method of the present invention, and the known concentration of CEA used in Comparative Examples 1 and 2. 4 is a graph showing the correlation between the amount of CEA in a standard product and the amount of change in absorbance by a latex agglutination immunoreagent according to a conventional method.

FIG. 2 shows the correlation between the amount of CEA in a CEA standard sample of known concentration used in Example 2 and the amount of change in absorbance according to the CEA measurement method of the present invention, and the CEA of known concentration used in Comparative Examples 3 and 4. 4 is a graph showing the correlation between the amount of CEA in a standard product and the amount of change in absorbance by a latex agglutination immunoreagent according to a conventional method.

FIG. 3 shows the correlation between the amount of CEA in a CEA standard sample of known concentration used in Examples 3, 4 and 5, and the amount of change in absorbance according to the CEA measurement method of the present invention, and the known concentration used in Comparative Example 5. 4 is a graph showing the correlation between the amount of CEA in a CEA standard sample and the amount of change in absorbance by a latex agglutination immunoreagent according to a conventional method.

Claims (3)

[Claims] 1. An insoluble carrier in which two or more different monoclonal antibodies against a specific antigen are supported on an insoluble carrier and reacted with the antigen in an aqueous solvent to selectively aggregate a conjugate of the insoluble carrier and the antigen. A high-sensitivity immunoassay method comprising using two or more carriers having different average particle diameters and carrying each monoclonal antibody on each of these insoluble carriers. 2. The method according to claim 1, wherein the insoluble carrier is latex particles. 3. An insoluble carrier having an average particle size in the range of 0.05 to 0.500 μm, wherein the ratio of the average particle size of the other particle type to the average particle size of the minimum particle type is 3 to 6; 3. The measuring method according to claim 1, wherein the measuring method is used in combination so as to fall within the range.