JPH0452557A - Composite carrier for immunological measurement and immunological measuring method using the carrier - Google Patents

Abstract

PURPOSE:To improve the reaction efficiency of a measuring carrier by a method wherein a composite carrier having particles of a secondary solid state carrier bonded to the surface of a primary solid state carrier is used as a solid state carrier for fixation of ligand, thereby bonding much more ligands without saturation accompanied. CONSTITUTION:An anti IgG antibody 13 is fixated to the surface of a primary solid state carrier 11 and particles of a secondary solid state carrier 12. The antibody 13 is bonded with an antigen 14. Accordingly, the particles 12 are bonded to the carrier 11. Those of the antibodies 13 fixed to the surface of the particles 12 without being used for bonding to the carrier 11 and therefore in the free state are employed for immune reaction with an antigen present in a measuring sample. The antigen 14 is the same as an antigen to be measured by this composite carrier. In this manner, much more antibodies 13 can be fixated without saturating or lowering the reactivity. Therefore, if this composite carrier is used, the reaction efficiency can be improved in the immunological measurement.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a composite carrier for immunoassays.

The present invention also relates to an immunoassay method using this composite carrier.

[Conventional technology]

Enzyme immunoassay (EIA) is a method for measuring antigens, antibodies, hormones, etc.
ssay) and fluorescence immunoassay are used. These immunoassay methods are broadly classified into liquid phase methods and solid phase methods, but the solid phase method is used for boat fishing.

The solid-phase immunoassay method includes a step that captures the target substance to be measured on the surface of a solid-phase carrier through an immune reaction. For this purpose, an immunoassay carrier is known in which a ligand having an affinity for the substance to be measured is immobilized in advance on a carrier such as glass beads (US Pat. No. 3,652,761). The ligand used is an antigen or antibody that specifically reacts with the substance to be measured. As a method for immobilizing the ligand on the carrier surface, there is a method of physically adsorbing the antibody 2 etc. on the surface of the carrier 1 as shown in FIG. 7(A), and a method as shown in FIG. 7(B).
), a method is used in which antibody 2 or the like is covalently bonded to carrier surface 1 using coupling agent 3.

By reacting such an immunoassay carrier with a sample containing a substance to be measured, the substance to be measured is captured by the ligand on the surface of the carrier. Next, the analyte bound to the carrier (Bound) and the unbound analyte (Bound) are separated.
Free) (hereinafter referred to as B/P separation). Next, after reacting with a second ligand that has an affinity for the substance to be measured and is labeled with an enzyme or a fluorescent substance,
Perform B/P separation. When the second ligand is labeled with an enzyme, the amount of the analyte can be determined by adding a substrate for the enzyme to cause a coloring reaction depending on the amount of the enzyme, and colorimetrically quantifying the produced dye. can be measured indirectly. When the second ligand is labeled with a fluorescent substance, the amount of the substance to be measured can be indirectly measured by irradiating it with excitation light and measuring the generated fluorescence.

The sensitivity of the above-mentioned immunoassay depends on the probability of reaction between the ligand immobilized on the surface of the carrier and the substance to be measured, and this reaction probability is proportional to the concentration of both. Therefore, in order to improve measurement sensitivity, more ligands must be immobilized on the carrier surface.

For example, as explained in FIG. 7(A), a diameter of 6.3
When anti-1gG antibody 2 was physically adsorbed onto the surface of H polystyrene beads as the solid phase carrier 1 at various densities, the reactivity of the obtained immunoassay carrier was shown in Figure 8. It changed like this.

That is, the reactivity increases in proportion to the amount of antibody adsorbed per unit area of the polystyrene beads 1.

However, as is clear from the results shown in FIG. 8, the reactivity is saturated when the amount of antibody adsorption reaches a certain value, and beyond this, the reactivity decreases. The reason is shown in Figure 9 (A
), when the distance between adjacent anti-1gG antibodies 2 becomes shorter than a certain value a (width of anti-1gG antibodies 2), antigens 4 cannot be brought together by mutual electrical buffering. Because it will disappear. In this state, anti-1gG antibody 2 is no longer able to bind antigen 4 in a 1:1 ratio, resulting in decreased reactivity. Anti-1gG antibody 2 and antigen 4 = 1
In order to combine with 1, as shown in Figure 9 (B),
Experiments have revealed that the distance between adjacent anti-1gG antibodies 2 must be at least twice a.

Therefore, in order to increase the reactivity above the saturation value mentioned above, the surface area of the polystyrene beads 1 must be increased. As a means for this purpose, it is also possible to subject the beads 1 to surface roughening treatment.

However, the effect of increasing the surface area by this method is slight, and the above object cannot be satisfied.

[Problem to be solved by the invention]

The present invention has been made in view of the above circumstances, and its object is to significantly increase the surface area of the solid support,
The objective is to bind more ligands without saturation and improve the reaction efficiency of immunoassay carriers.

[Means to solve the problem]

In order to achieve the above object, the carrier for immunological assay of the present invention, as a solid phase carrier for immobilizing a ligand,
A composite carrier was used in which multiple carriers were bound together through an antigen-antibody reaction.

That is, the composite carrier for immunoassay according to the present invention comprises a primary solid phase carrier having a first antibody immobilized on its surface, a secondary solid phase carrier particle having a first antibody immobilized on its surface, and both of these particles. a first antigen that specifically binds to the first antibody of the assembly and connects both the assembly; and a second antigen that is immobilized on the surface of the second carrier particle and reacts specifically with the second antigen to be measured. It is characterized by comprising two antibodies.

In the above composite carrier for immunoassay, the first antibody for linking the primary carrier and the secondary carrier may be the same as or different from the second antibody for specifically reacting with the measurement target. good.

Furthermore, the second antibody may be immobilized not only on the secondary solid phase carrier particles but also on the surface of the primary solid phase carrier.

Furthermore, although the above-mentioned composite carrier for immunoassay is for measuring an antigen, according to the present invention, a carrier for immunoassay that targets antibodies to be measured can be constructed in the same manner. It is. That is, in this case, the relationship between antigen and antibody may be reversed.

The immunoassay #j standard method according to the present invention is characterized by using the above-mentioned composite carrier for immunoassay, and is otherwise the same as the conventional method.

[For production]

The composite carrier particles for immunoassay according to the present invention use a composite carrier in which secondary solid phase carrier particles are connected to the surface of a primary solid phase carrier as a solid phase carrier for immobilizing a ligand. Therefore, the surface area of the carrier is significantly increased compared to conventional carriers. Therefore, more ligands can be immobilized without causing saturation of reactivity, and much better immunoreactivity than before can be obtained.

〔Example〕

Embodiments of the present invention will be described in detail below with reference to the drawings.

FIG. 1(A) is a diagram showing an example of a composite carrier for antigen measurement. In this example, the same antibody is used as the first and second antibody.

In the figure, 11 is a primary solid phase carrier, and 12 is a secondary solid phase carrier particle. As the primary solid phase carrier 11, for example, spherical particles such as glass beads and polystyrene beads, or well walls of a microplate can be used. Also,
As the secondary solid phase carrier particles 12, for example, glass beads,
Polystyrene beads, latex beads, etc. can be used.

Anti-1gG antibody 13 is immobilized on the surfaces of primary solid phase carrier 11 and secondary solid phase carrier particles 12 by physical adsorption or via a coupling agent. Anti-1 immobilized on the primary solid phase carrier 11 and the secondary solid phase carrier particles 12
Both of the gG antibodies 13 and 13 are bound to the antigen 14, so that the secondary solid phase carrier particles 12 are bonded to the primary solid phase carrier 1.
1. Among the anti-1gG antibodies 13 immobilized on the surface of the secondary solid phase carrier particles 12, those remaining in a free state without being used for coupling with the primary solid phase carrier 11 are contained in the measurement sample. used for immune reactions with antigens present in Incidentally, the antigen 14 is the same as the antigen to be measured using this composite carrier for immunoassay.

According to the above embodiment, more anti-1gG antibody 13 can be immobilized than before without saturation or reduction of reactivity. Therefore, if this composite carrier is used,
The reaction efficiency in immunological measurements can be improved.

FIG. 1(B) is a diagram showing a modification of the embodiment of FIG. 1(A). In this modification, a free anti-1gG antibody 13 capable of reacting with the antigen in the sample to be measured is also provided on the surface of the primary solid phase carrier 11. Everything else is the same as the embodiment shown in FIG. 1(A). Thereby, immunoreactivity can be further improved.

FIG. 2(A) is a diagram showing another example of a composite carrier for antigen measurement. In this example, different antibodies are used as the first and second antibodies. That is, as the first antibody, anti-1gG antibody 13 was used as in the example of FIG. 1(A).
However, anti-HBs antibody 15 was used as the second antibody. Therefore, the secondary solid phase carrier 11 and the secondary solid phase carrier particles 12 are linked via the antigen 14. Further, this composite carrier for immunological measurement is used for measurement of HBs antigen.

The rest is the same as the embodiment shown in FIG. 1(A), and the same effects can be obtained.

FIG. 2(B) is a diagram showing a modification of the embodiment of FIG. 2(A). In this modification, a free anti-HBs antibody 15 capable of reacting with the HBs antigen in the sample to be measured is also provided on the surface of the primary solid phase carrier 1. Other than that,
Everything is the same as the embodiment shown in FIG. 2(A). Thereby, the immunoreactivity can be further improved than in the example shown in FIG. 2(A).

FIG. 3(A) is a diagram showing an example of a composite carrier for antibody measurement. In this example, the same antigen is used as the first and second antigen.

In the figure, 21 is a primary solid phase carrier, and 22 is a secondary solid phase carrier particle. As the primary solid support 21, for example, spherical particles such as glass beads and polystyrene beads, or well walls of a microplate can be used. Also,
As the secondary solid phase carrier particles 22, for example, glass beads,
Polystyrene beads, latex beads, etc. can be used.

An antigen 23 that specifically binds to the anti-1gG antibody is immobilized on the surfaces of the primary solid phase carrier 21 and the secondary solid phase carrier particles 22 by physical adsorption or via a coupling agent. The antigens 23 and 23 immobilized on the primary solid phase carrier 21 and the secondary solid phase carrier particles 22 are both bound to the anti-1gG antibody 24, so that the secondary solid phase carrier particles 22 is connected to. Among the antigens 23 immobilized on the surface of the secondary solid phase carrier particles 22, those that are not used for coupling with the primary solid phase carrier 11 and remain in a free state are present in the measurement sample. It is used for immunoreaction with anti-1gG antibody. Anti-IgG antibody 2
4 is the same antibody to be measured using this composite carrier for immunoassay.

According to the above embodiment, more antigens 23 can be immobilized than before without saturation or reduction of reactivity. Therefore, if this composite carrier is used, anti-1gG
The reaction efficiency in immunoassay of antibodies can be improved.

FIG. 3(B) is a diagram showing a modification of the embodiment of FIG. 3(A). In this modification, a free antigen 23 that can react with the anti-IgG antibody in the sample to be measured is also provided on the surface of the primary solid phase carrier 21. Everything else is the same as the embodiment shown in FIG. 3(A). Thereby, immunoreactivity can be further improved.

FIG. 4(A) is a diagram showing another example of a composite carrier for antibody measurement. In this example, different antigens are used as the first and second antigens. That is, as the first antibody, the same antigen 23 as in the example of FIG. 3(A) was used, but as the second antibody, HBs antigen 25 was used. Therefore,
The primary solid phase carrier 21 and the secondary solid phase carrier particles 22 are anti-1gG.
linked via antibody 24. Moreover, this composite carrier for immunological measurement is used for the measurement of anti-HBs antibodies. The rest is the same as the embodiment shown in FIG. 3(A), and the same effects can be obtained.

FIG. 4(B) is a diagram showing a modification of the embodiment of FIG. 4(A). In this modification, free HBs that can react with the anti-HBs antigen to be measured is also present on the surface of the primary solid phase carrier 1.
25 antigens are provided.

Everything else is the same as the embodiment shown in FIG. 4(A).

This allows the immunoreactivity to be further improved than in the example shown in FIG. 4(A).

Next, specific examples regarding the production of a composite carrier for immunoassay according to the present invention and immunoassay using the same will be described.

Example 1: Production of composite carrier particles for HBs antigen measurement (1) Preparation of first carrier particles 50 polystyrene beads (diameter 8.35 mm) manufactured by Sekisui Chemical Co., Ltd.
M NaCJ-0, 1M glycine-NaOH buffer (p
H-8,0) 1001 and reacted for 60 minutes at 4°C with stirring. After immobilizing the anti-HBs antibody in this manner, the reaction solution was discarded. followed by 0.15M NaC
j-0,1M glycine-NaOH buffer (pH-8,0
) 100ml was added and washed, and the same washing operation was performed two more times.

After discarding the washing solution, add 10% BSA-0.15M N
aCJ-0, 1M glycine-NaOH buffer (pH-7
, 5) was added thereto, and the mixture was reacted at 4° C. for 60 minutes with stirring. As in the case of the previous solid phase reaction, after discarding the reaction solution, add 0.05% Tween20-0.15M NaCj-
0.1M glycine-NaOH buffer (pH 48.0)
The operation of adding 100+al and washing was performed three times.

(2) Preparation of second carrier particles Latex beads N-200 manufactured by Sekisui Chemical Co., Ltd. (diameter 0
．． 2μIl) was added to a 0.1M glycine-NaOH aqueous solution (p
H-8.0) to prepare a 0.1% latex suspension. Anti-HBs antibody lI was added to this latex suspension.
Ig was dissolved and allowed to react at 4° C. for 60 minutes with stirring. After the reaction was completed, centrifugation was performed at 5000 rpm for 5 minutes to sediment the latex beads immobilized with antibodies, and the supernatant was removed by suction. Then 0.05%T
An operation of adding LOOml of ween20-0.15M NaCJ-0, 1M glycine-NaOH buffer (pH-8,0) and washing was performed three times.

After washing, 10% BSA-0, 15M NaCj-0,
1M glycine-NaOH buffer (pH=7.5) was added, and the mixture was reacted at 4°C for 60 minutes with stirring. After the reaction was completed, centrifugation was performed at 5000 rpm for 5 minutes to sediment the latex beads immobilized with antibodies, and the supernatant was removed by suction. Next, 0.05% Tween2
0-0.15M NaCj-0,1M glycine-NaOH
An operation of adding buffer solution (pH-8, 0) 1001 and washing was performed three times.

(3) 50 first carrier particles prepared in (1) and HBs at the manufacturer of composite carrier particles.
10% goat serum containing 50 μg of antigen - 1% BSA - 3%
Gelatin-0.05% Tween20-0.1M PB
S(pH-7,0) LOOml was reacted at 4°C for 30 minutes. After the reaction is complete, remove the reaction solution by suction and reduce to 0.
．． 05% Tween20-0.1M PBS (pH=7
．． 0) Added 100ml and washed.

This washing operation was repeated two more times.

Meanwhile, 10% goat serum - 1% BSA - 3% gelatin - 0
．． 05%Tveen20-0.1M PBS (pH-7
, 0) A 0.025% suspension of the second carrier particles prepared in (2) was prepared using LOOml.

Next, the first carrier particles treated above were added to this second carrier particle suspension and allowed to react.

After removing the reaction solution by suction, add 0.05% T-seen.
10 hl of 20-0.1M PBS (pH=7.0) was added for washing, and this washing operation was repeated two more times.

Example 2: Measurement of HBs antigen (1) HBs antigen positive human serum was mixed with 10% goat serum -
1% BS^-3% gelatin-0.05% Tveen20
-0, diluted with OIM PBS (pH-7,0). In this way, a large number of serum lysate samples with different dilution ratios were prepared.

(2) The composite carrier particles produced in Example 1 were placed in a test tube, and 300 μg of the serum solution sample prepared above was added thereto, followed by reaction at 37° C. for 60 minutes.

After removing the reaction solution by suction, add 0.05% Tween20-
Washing was carried out by adding 100+ nl of 0.01 MPBS (pH=7.0), and this washing operation was repeated two more times.

(3) After removing the cleaning solution by suction, 0.0023% 0-phenylenediamine-0.033% hydrogen peroxide-0.1M
Add 300 μg of carbonate buffer (p)I-5,0),
The enzyme-substrate reaction was performed at 37°C for 15 minutes.

(4) Next, the reaction was stopped by adding 100 μg of 2N sulfuric acid, and the absorbance at a wavelength of 492 nm was measured. By plotting this measured value against the dilution ratio of each sample, a calibration curve (Sl) shown in FIG. 5 was obtained.

For comparison, measurements similar to those described above were performed using a conventional immunoassay carrier. The calibration curve obtained by this (S2
) are also shown in Figure 5.

As is clear from comparing the two calibration curves (Sl, S2), according to the embodiment of the present invention, the measurement range can be widened and the measurement sensitivity can be improved at each concentration of HBs antigen. .

Example 3: Production of composite microplate for HBs antigen measurement (1) Preparation of first carrier (EIjS^ plate) Microplate (STRI PPLATE manufactured by NLINC)
4B8687), 100 μρ of 0.01 M PBS (pH-7,0) containing 1 μg of anti-HBs antibody.
and reacted at 37°C for 60 minutes. After immobilizing the anti-HBs antibody in this way, using a microplate washer, wash solution (0.01M
Washed three times with PBS (pH-7,0).

After discarding the washing solution, add 5% BSA-0,05M Tw
een20-0. OIM PBS (pH-7,0)
300 μg was added to each well and reacted at 37° C. for 60 minutes. After the reaction was completed, the plate was washed three times with a washing solution of 300 μp/well using a microplate washer. As a cleaning solution, 0.05% Tveen20-0. OIM
PBS (pH-7,0) was used.

(2) Preparation of second carrier particles Latex beads N-200 manufactured by Sekisui Chemical Co., Ltd. (diameter 0
．． 2μl), i River glycine-NaOH aqueous solution (p)
l-8,0), dissolved in 0. A 1% latex suspension was prepared. Anti-HBs antibody IB was dissolved in this latex suspension, and reacted at 4° C. for 60 minutes with stirring. After the reaction was completed, centrifugation was performed at 5000 rpm for 5 minutes to sediment the latex beads immobilized with antibodies, and the supernatant was removed by suction. Then 0.05%T
An operation of adding 100 ml of ween20-0.15M NaCj-0,1M Green-NaOH buffer (pH=8.0) and washing was performed three times.

After washing, 10% BSA-0, 15M NaCJ-0,
A 1M glycine-NaOH buffer (pH-7,5) was added, and the mixture was reacted at 4°C for 60 minutes with stirring. After the reaction was completed, centrifugation was performed at 5000 rpm for 5 minutes to sediment the latex beads immobilized with antibodies, and the supernatant was removed by suction. Then 0. Ω5% Tween2
D-0,15M NaCj-0,1M glycine-NaOH
An operation of adding buffer solution (pH-8, 0) 1001 and washing was performed three times.

(3) Preparation of composite microplate In each well of the ELISA plate prepared in (1), add 10% goat serum containing 3 ng of HBs antigen to 1% BSA.
-3% Gelatin-〇, 05% Tween20-0.1M
Add 100 μg of PBS (pH-7.0) and
The reaction was carried out at ℃ for 60 minutes. After the reaction was completed, the reaction solution was removed by suction.

Subsequently, using a microplate washer, 300
Washed six times with μg/well washing solution. As the cleaning solution, 0.05% Tween 20-0. DIM PBS(p
H-7,0) was used.

Meanwhile, 10% goat serum - 1% BSA - 3% gelatin - 0
．． 05% Tween20-0.1M PBS (pH-7
, 0) to prepare a 0.025% suspension of the second carrier particles prepared in (2).

Next, 100 μfI/well of this second carrier particle suspension was added and reacted at 37° C. for 60 minutes. After removing the reaction solution by suction, the plate was washed three times with 300 μg/well of washing solution using a microplate washer. Cleaning liquid Niha, 0.05% Tveen20-0.01MPBS
(pH-7, (1) was used.

Example 4: Measurement of HBs antigen (1) HBs antigen positive human serum was mixed with 10% goat serum -
1% BSA-3% gelati:/-0,05% Tween2
0-0.01M Pt3S(J)H-7,0). In this way, a large number of serum lysate samples with different dilution ratios were prepared.

(2) Into each well of the composite microplate carrier prepared in Example 3, 100 samples of the serum lysate prepared above were added.
After adding μρ/well, the reaction was carried out at 37° C. for 60 minutes. After the reaction was completed, the plate was washed six times with a washing solution of 300 μp/well using a microplate washer. The cleaning solution includes 0.05% Tween20-0.01M PB
100 ml of S (pH=7.0) was used.

(3) After removing the cleaning solution by suction, 0.0028% 0-phinylene diamine-0.033% hydrogen peroxide-0.1M
Add carbonate buffer (pH = 5.0) 100μI),
The enzyme-substrate reaction was carried out for 15 minutes at 37°C.

(4) Next, the reaction was stopped by adding 100 μp of 2N sulfuric acid, and the absorbance at a wavelength of 492r+m was measured using an ELISA RECORDER. By plotting these measured values against the dilution ratio of each sample, a calibration curve (Sl) shown in FIG. 5 was obtained.

For comparison, measurements similar to those described above were performed using a conventional ELISA microplate. The calibration curve (S2) thus obtained is also shown in FIG.

As is clear from comparing the two calibration curves (SL, S2), according to the embodiment of the present invention, the measurement range can be widened and the measurement sensitivity can be improved at each concentration of HBs antigen. can.

〔Effect of the invention〕

As detailed above, according to the present invention, more ligands can be bound without saturation or decrease in reactivity,
Remarkable effects such as improving the reaction efficiency of the carrier for immunological measurements and increasing measurement accuracy can be obtained.

[Brief explanation of drawings]

FIG. 1(A) to FIG. 4(B) are diagrams showing examples of the composite carrier for immunoassay according to the present invention, and FIGS. 5 and 6 are diagrams showing examples of the composite carrier for immunoassay according to the present invention, respectively. Figures 7(A) and 7(B) are diagrams showing the general structure of immunoassay carriers, and Figures 8 and 9 (A) (B) are diagrams showing the calibration curve prepared in the example of the method. ) is a diagram showing problems with conventional immunoassay carriers. 11.21... Primary solid phase carrier, 12.22... Secondary solid phase carrier particles, 13.24... Anti-1gG antibody, 14°23... Antigen, 15... Anti-HBs antibody, 25...
HBs antigen

Claims (7)

[Claims] (1) A primary solid phase carrier with a first antibody immobilized on its surface, a secondary solid phase carrier particle with a first antibody immobilized on its surface, and a particle that specifically binds to the first antibody on both of these carriers. and a second antibody that is immobilized on the surface of the second carrier particle and reacts specifically with the second antigen to be measured. Composite carrier for quantitative measurements. (2) The composite carrier for immunoassay according to claim 1, wherein the first antibody and the second antibody are the same, and the first antigen and the second antigen are the same. (3) The composite carrier for immunoassay according to claim 1 or 2, wherein the second antibody is also immobilized on the surface of the primary solid phase carrier. (4) A primary solid phase carrier with a first antigen immobilized on its surface, a secondary solid phase carrier particle with a first antigen immobilized on its surface, and a particle that specifically binds to the first antigen of both carriers. and a second antigen that is immobilized on the surface of the second carrier particle and reacts specifically with the second antibody to be measured. Composite carrier for quantitative measurements. (5) The composite carrier for immunoassay according to claim 1, wherein the first antigen and the second antigen are the same, and the first antibody and the second antibody are the same. (6) The composite carrier for immunoassay according to claim 1 or 2, wherein the second antigen is also immobilized on the surface of the primary solid phase carrier. (7) The antigen or antibody is bound to the composite carrier by reacting the composite carrier for immunological assay according to any one of claims 1 to 6 with a sample solution containing the antigen or antibody to be measured. An immunological measurement method characterized by comprising a step of capturing the target.