JP2002014098A - Immunoassay and kit used therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunoassay, by which immunoassay can be performed accurately from a low concentration region to a high concentration region, without or by performing dilution by the number of times which is smaller than that performed in the conventional method, even when the concentration of an antigen to be inspected in a specimen is high. SOLUTION: In this immunoassay, a labeled antibody bonding to solid phase is measured through an antibody specifically bonded to the solid phase, an antigen to be inspected, and a labeled antibody, by reacting the antigen to be inspected having a plurality of epitopes, the labeled antibody which has been labeled or will be later labeled and causes an antigen-antibody reaction with the antigen, and an antibody which causes an antigen-antibody reaction with the antigen competitively with the labeled antibody and is specifically bonding to solid phase in a liquid phase, and simultaneously with or after the reaction, reacting the antibody with a solid phase specifically bonding to with the antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an immunoassay method and a kit therefor.

[0002]

2. Description of the Related Art When a mouse monoclonal antibody is produced by a high-density culture method, 5 mg / m
1 to 0.5 mg / ml mouse IgG.
During the production of the monoclonal antibody, the concentration of the antibody in the culture supernatant is measured by ELISA in order to check the amount of antibody in the culture supernatant.
Since the concentration is about g / ml to about 100 ng / ml, conventionally, the culture supernatant was diluted about 10,000 to 50,000 times to measure the antibody concentration. According to this method, at least two steps of dilution are required, which is complicated, and it is difficult to accurately measure the antibody concentration due to a dilution error.

As a method for solving this problem, a method is known in which a free antibody that reacts with an antigen to be tested with an antigen-antibody is added to a reaction solution in order to lower the sensitivity of the entire measurement system. However, when this method is applied to the ELISA method, if the detection signal at the upper limit of measurement (coloring in the example) is moderately suppressed, the measurement sensitivity will be reduced more than necessary and accurate measurement in the low concentration region will not be possible. However, the intended measurement system could not be set up.

[0004]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a low concentration region without dilution, or at least with a smaller number of dilutions than in the conventional method, even when the concentration of the test antigen in the sample is high. It is an object of the present invention to provide an immunoassay method capable of accurately performing an immunoassay from a high concentration region to a high concentration region.

[0005]

Means for Solving the Problems As a result of earnest studies, the inventors of the present invention have found that an antibody that reacts with a test antigen through an antigen-antibody reaction,
Furthermore, by using an antibody that specifically binds to the solid phase and competing it with the labeled antibody in the liquid phase, the measurement sensitivity is appropriately suppressed, even when the test antigen is contained at a high concentration. The present inventors have found that a test antigen can be accurately immunoassayed in a wide concentration range, and have completed the present invention.

That is, the present invention provides a test antigen having a plurality of epitopes, a labeled antibody that is subjected to antigen-antibody reaction with the test antigen, and that is labeled or subsequently labeled, The test antigen and an antibody that reacts with the antigen-antibody, and further reacted in a liquid phase with a solid-phase specific binding antibody that specifically binds to a solid phase, simultaneously or after the reaction,
The solid phase specific binding antibody is reacted with a solid phase that specifically binds thereto, and the solid phase specific binding antibody, the test antigen and the labeled antibody bound to the solid phase via the labeled antibody. An immunoassay method comprising measuring a label is provided. Further, the present invention provides a test antigen having a plurality of epitopes,
A labeled antibody that undergoes an antigen-antibody reaction with the test antigen, a labeled antibody that is labeled or subsequently labeled, and an antibody that reacts with the test antigen with an antigen-antibody, and further solid-phase specific binding that specifically binds to a solid phase Antibody and a third free antibody that reacts with the test antigen in an antigen-antibody reaction in a liquid phase. Simultaneously with or after the reaction, the solid-phase specific binding antibody specifically binds thereto. Reacting with a solid phase to be subjected to, the immunoassay method comprising measuring the label bound to the solid phase through the solid phase specific binding antibody, the test antigen and the labeled antibody, An immunoassay method is provided wherein at least two of a labeled antibody, the solid phase specific binding antibody, and the third free antibody competitively react with the test antigen. Furthermore, the present invention provides an antigen-antibody reaction with a test antigen having a plurality of epitopes, a labeled antibody labeled or subsequently labeled, and an antigen-antibody reaction with the test antigen in competition with the labeled antibody. An immunoassay kit comprising an antibody, which further comprises a solid phase specific binding antibody that specifically binds to a solid phase, and a solid phase that specifically binds to the solid phase specific binding antibody. Further, the present invention relates to a labeled antibody which is subjected to an antigen-antibody reaction with a test antigen having a plurality of epitopes, and which is labeled or subsequently labeled, and an antibody which reacts with the test antigen in an antigen-antibody manner, further comprising a solid phase. Immunoassay comprising a solid-phase specific binding antibody that specifically binds to a test antigen, a third free antibody that reacts with the test antigen by an antigen-antibody, and a solid phase that specifically binds to the solid-phase specific binding antibody A kit for immunoassay, wherein at least two of the labeled antibody, the solid-phase specific binding antibody and the third free antibody competitively react with the test antigen. provide.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The immunoassay method of the present invention comprises a first immunoassay method using two kinds of antibodies, a “labeled antibody” and a “solid phase specific binding antibody”, which will be described in detail later. A second immunoassay method using three types of antibodies, “labeled antibody”, “solid phase specific binding antibody”, and “third free antibody” is included. First, the first immunoassay will be described.

A test antigen to be immunoassayed by the first immunoassay of the present invention has a plurality of epitopes. Here, “a plurality of epitopes” refers to a case where a plurality of epitopes of the same type exist, a case where a plurality of epitopes of different types exist, and a case where epitopes of different types including a plurality of one or two or more types of the same epitope are included. Any of the cases where there are a plurality of them may be used.

In the first immunoassay of the present invention, a labeled antibody that reacts with the test antigen by an antigen-antibody is used. Here, the “labeled antibody” may be a labeled antibody, or may be an antibody that is labeled in a later step.

[0010] The term "labeled antibody" used herein refers to an antibody obtained by labeling an antibody that reacts with an antigen to be tested with an antigen. As the label, an enzyme label, a fluorescent label, a radioactive label, and the like, which have been widely used, can be used, and a method of binding these labels to an antibody is also well known.

[0011] The "antibody to be labeled later" refers to the time of an immunoreaction between the test antigen and the solid phase specific binding antibody described below (hereinafter referred to as "antibody to be labeled").
"Primary reaction") is an antibody that is not labeled but is labeled after the immune complex has bound to the solid phase or during the primary reaction as described below. For example, as a “labeled antibody”, an unlabeled goat IgG antibody that reacts with the test antigen as an antigen-antibody is used, and after the immune complex has bound to the solid phase, the labeled anti-goat IgG antibody is reacted, In the case of measuring the label bound to the phase, the goat IgG antibody used in the primary reaction is labeled in a later step, and thus corresponds to the “labeled antibody” in the present invention. As described above, the method of using an antibody labeled with an antibody that reacts with an antigen and an antibody without labeling the antibody that reacts with the test antigen by an antigen-antibody is widely used in the field of immunoassay. is there. If this method is adopted, it is not necessary to label each antibody that reacts with the test antigen by an antigen-antibody. For example, in the above example, if an anti-goat IgG antibody is labeled, it can be used for measurement of various test antigens. There is an advantage. However,
There is also the disadvantage of using one more type of antibody and increasing the immune response by one step or one more antibody to participate in the primary reaction.

In the first immunoassay of the present invention, an antibody which reacts with the test antigen by an antigen-antibody, and which specifically binds to a solid phase, is used. Here, the “solid-phase specific binding antibody” may be any antibody capable of specifically binding to a solid phase, and is preferably a specific antibody obtained by binding a specific binding compound that specifically binds to a certain substance to an antibody. Binding compound binding antibodies can be mentioned. Here, preferred examples of the “specific binding compound” include biotin. In this case, avidin or a biotin-binding derivative thereof (for example, streptavidin or the like) is bound to the solid phase. That is, in a preferred example,
As the solid-phase specific binding antibody, a biotinylated antibody that reacts with the test antigen by an antigen-antibody is used, and as the solid phase, an avidin (or derivative thereof) -modified solid phase is used. In addition, biotinylation of an antibody and avidinization of a solid phase can be performed by a well-known method, and are described in detail in the following Examples.
However, the "specific binding compound" is not limited to biotin, and may be any compound having a substance that specifically binds. In this case, a solid phase on which a “specific binding-compatible compound” that specifically binds to the “specific binding compound” is used. Further, as the solid phase specific binding antibody, it is not always necessary to bind a “specific binding compound”, but any antibody having antigenicity is bound, and an antibody that specifically binds to this arbitrary compound is fixed. It is also possible to use a phased solid phase. Furthermore, a normal antibody may be used as the solid phase specific binding antibody, and a solid phase in which an antibody that specifically binds to the F _C region of the antibody may be used. However, in this case, it is necessary to prevent the labeled antibody from binding to the solid phase, and this can be achieved by using an antibody derived from a different animal as the solid phase specific binding antibody and the labeled antibody.

The above-mentioned labeled antibody and solid phase specific binding antibody are:
Reacts competitively with the test antigen. Here, the term “competitively reacts” with the test antigen means not only when the corresponding epitopes of both antibodies are the same, but also when one antibody binds to the test antigen and the other antibody reacts. The case where binding cannot be performed due to steric hindrance is also included. further,
If at least one of the labeled antibody and the solid-phase specific binding antibody is a polyclonal antibody against the test antigen, it can be said that both of them react competitively. Because, the polyclonal antibody contains various antibodies against various epitopes of the test antigen, even if the other antibody is a monoclonal antibody, some of the polyclonal antibodies compete with the monoclonal antibody This is because an antibody molecule is always included. Even when both the labeled antibody and the solid-phase specific binding antibody are polyclonal antibodies to the test antigen, they naturally “competitively react” to the test antigen. Preferably, at least one of the labeled antibody and the solid phase specific binding antibody is a polyclonal antibody.
Because by doing so, it "competitively reacts"
This is because the requirement is always satisfied. However, if the test antigen has a plurality of the same epitopes, or has a plurality of combinations of epitopes that differ in the epitope but cannot simultaneously bind to both antibodies due to steric hindrance, a monoclonal antibody that specifically reacts with each of these epitopes is required. It is also possible to use the above labeled antibody and solid phase specific binding antibody.

In the first immunoassay of the present invention,
As will be described in detail later, the test antigen is measured by sandwiching the solid phase specific binding antibody and the labeled antibody. Therefore, the test antigen is composed of at least one molecule of the solid phase specific binding antibody and at least one molecule of the solid phase specific binding antibody. It is necessary to have a plurality of epitopes that can simultaneously bind to the labeled antibody.

In the first immunoassay according to the present invention, the antigen to be tested, the labeled antibody and the solid phase specific binding antibody are subjected to an antigen-antibody reaction in a liquid phase. In this case, by reducing the number of moles of one of the labeled antibody and the solid-phase specific binding antibody with respect to the other, the proportion of the test antigen to which the smaller number of antibodies cannot bind can be increased. Overall sensitivity can be reduced. The test antigen that could not bind to either the labeled antibody or the solid phase specific binding antibody is not detected in a later step, so even if the labeled antibody or the solid phase specific binding antibody is reduced, Sensitivity suppression is achieved. The addition ratio of the labeled antibody and the solid phase specific binding antibody can be appropriately set based on routine preliminary experiments depending on how much the sensitivity is to be suppressed. This involves preparing standard samples with different antigen concentrations, and performing immunoassay using antibody systems with variously changed concentration ratios of the labeled antibody and the solid-phase specific binding antibody to achieve the desired suppression of sensitivity. This can be easily performed by selecting a concentration ratio.

The amount of a compound (an avidin or the like, hereinafter sometimes referred to as a "trapping compound") that is immobilized on a solid phase and traps a solid phase-specific binding antibody is not particularly limited. It is preferable to increase as much as possible so as to correspond to the concentration. This can be achieved by bringing a sufficient concentration of the trapping compound solution into sufficient contact with the solid phase and then thoroughly washing. For example, when using avidin or a derivative thereof as a trapping compound, 100 μg / ml as described in the following example.
Avidin solutions of moderate concentration can be contacted with the solid phase.

The primary reaction may be carried out on a solid phase to which the solid phase specific binding antibody specifically binds, or after the reaction is carried out in another container, the reaction solution is added to the solid phase specific binding antibody. May be brought into contact with a solid phase to which is specifically bound. However, performing on a solid phase to which the solid phase specific binding antibody specifically binds is simpler because the operation of transferring the reaction solution can be omitted.

As a result of the above reaction, a solid phase specific binding antibody is bound to the solid phase by the trapping compound, a test antigen is bound to the solid phase specific binding antibody, and a labeled antibody is bound to the test antigen. You. That is, the immune complex in which the test antigen is sandwiched between the solid phase specific binding antibody and the labeled antibody is bound to the solid phase. Therefore, after washing the solid phase, the amount of the test antigen in the sample can be quantified by measuring the label bound to the solid phase. The method of measuring the label itself is well known. When an antibody to be labeled later is used as the labeled antibody, as described above, after the immune complex is bound to the solid phase, the labeled antibody that specifically reacts with the antibody is reacted. Let it. Alternatively, a labeled antibody that specifically reacts with an antibody to be labeled later can be included in the primary reaction system.

In the above-described method of the present invention, in the primary reaction, the labeled antibody and the solid-phase specific binding antibody react competitively in the liquid phase. Therefore, as described above, by making the number of moles of one of the antibodies smaller than the number of moles of the other antibody, the proportion of the test antigen that does not bind to the smaller number of antibodies increases. The test antigen that failed to bind to either one of the antibodies is not detected because it cannot bind to the solid phase or the label does not bind to the solid phase. Therefore, since the measurement sensitivity is reduced, it is not necessary to perform dilution even when the test antigen is contained at a high concentration, or at least the number of dilutions can be reduced as compared with the conventional case. Nevertheless, the test antigen can be accurately quantified over a wide concentration range, as specifically shown in the examples below.

In the second immunoassay method of the present invention, a third free antibody that reacts with the test antigen in an antigen-antibody manner is present in the liquid phase in which the primary reaction is performed. Here, "free antibody" means an antibody that is not bound to a solid phase, has no solid phase specific binding property, and does not have the above-mentioned label.
A normal antibody against the test antigen may be used. The third free antibody competes with at least one of the labeled antibody and the solid-phase specific binding antibody (if not competing, it is considered that the method uses only the two types of antibodies described above. it can). In the second immunoassay, since the third free antibody competes with at least one of the labeled antibody and the solid phase specific binding antibody, the labeled antibody and the solid phase specific binding antibody need not compete with each other. No (of course, they may be competing). In order to ensure that the third free antibody competes with at least one of the labeled antibody and the solid phase specific binding antibody, at least one of the labeled antibody, the solid phase specific binding antibody and the third free antibody is used. Is preferably a polyclonal antibody. When the third free antibody is a polyclonal antibody, both the labeled antibody and the solid phase specific binding antibody can be monoclonal antibodies. However, when the test antigen has a plurality of the same epitopes or a plurality of combinations of epitopes in which at least two of these three types of antibodies cannot simultaneously bind due to steric hindrance, the monoclonal antibody is used as the labeled antibody, the solid phase. Specific binding antibody and third
It is also possible to use a free antibody. In the second immunoassay, similarly to the first immunoassay, the test antigen is sandwiched between the solid-phase specific binding antibody and the labeled antibody for measurement, so that the test antigen has at least one molecule. It is necessary that the solid-phase specific binding antibody and at least one molecule of the labeled antibody have a plurality of epitopes that can bind simultaneously.

In the case of using a labeled antibody, a solid phase specific binding antibody and a third free antibody, the number of moles of the labeled antibody and / or the solid phase specific binding antibody with respect to the total of the three is reduced. This increases the proportion of the test antigen that cannot bind to at least one of these two antibodies, thereby suppressing the measurement sensitivity. The addition ratio of the labeled antibody, the solid phase specific binding antibody and the third free antibody is
As in the case of the first immunoassay, it can be appropriately set based on a routine experiment depending on how much the sensitivity is suppressed. For example, standard samples with different antigen concentrations are prepared, while a labeled antibody, a solid phase specific binding antibody and a
The immunoassay can be easily performed by performing immunoassay using an antibody system in which the concentration ratio of the free antibody is variously changed, and selecting the concentration ratio that achieves the desired suppression of sensitivity. In addition,
By using the third free antibody, the amount of the solid phase specific binding antibody and / or the labeled antibody can be reduced, and the cost can be saved. Therefore, the ratio of the third free antibody can be increased. By conducting a routine preliminary experiment, the preliminary experiment can be simplified even with three kinds of antibodies used.

Even when the third free antibody is used, other conditions are the same as those when the above-mentioned two kinds of antibodies are used.

The antibody can be decomposed with a proteolytic enzyme such as pepsin or papain to obtain an antigen-binding fragment such as a Fab fragment or F (ab ') ₂ fragment, which maintains the antigen-binding property. It is widely known that it occurs. Since such an antigen-binding fragment can undergo an antigen-antibody reaction in the same manner as an antibody, it is clear that such an antigen-binding antibody fragment can be used in place of the "antibody" in the present invention, In the following examples, such an antigen-binding antibody fragment is used. Thus, as used herein, the term "antibody" also includes antigen-binding antibody fragments, unless otherwise specified or otherwise apparent in context. In particular, the term "antibody" in the claims also covers antigen-binding antibody fragments.

[0024]

The present invention will be described in more detail with reference to examples and comparative examples. However, the present invention is not limited to the following examples.

Reference Example 1 Preparation of Anti-Mouse IgG Antibody The first 2 mg of mouse IgG (Zymet) and the second and subsequent 1 mg were immunized subcutaneously in goats 11 times at weekly intervals with complete Freund's adjuvant to give anti-mouse IgG antiserum. Obtained. 400 ml of antiserum was salted out with ammonium sulfate, and mouse Ig
After affinity chromatography using G (γ, κ) and mouse IgA (α, κ) immobilized columns, gel filtration chromatography was performed to obtain anti-mouse IgG antibody 14.
0 mg was prepared.

Reference Example 2 Preparation of Peroxidase (POD) Labeled Antibody and Biotinylated Antibody 100 mg of the anti-mouse IgG antibody prepared in Reference Example 1 was digested with pepsin to obtain 37 mg of F (ab ') ₂ fragment. POD (Peroxidase, Boehringer) 50
mg of a cross-linking agent EMCC (Dojindo) and POD-labeled anti-mouse IgG antibody (labeled antibody) by maleimide method
Was prepared.

Similarly, a biotin-labeled anti-mouse IgG antibody (solid phase specific binding antibody) was prepared from biotin-BMCC (Pierce) and Fab '.

Reference Example 3 Preparation of Antibody-Immobilized Plate The anti-mouse IgG antibody prepared in Reference Example 1 was adjusted to 70 μg / ml with a phosphate buffer pH 7.5, and 100 μl was dispensed into a 96-well microplate at 37 ° C. It was left overnight to immobilize the anti-mouse IgG antibody. After washing the microplate with a phosphate buffer, 300 μl of a phosphate buffer containing 1% BSA was dispensed and allowed to stand at 37 ° C. overnight. The next day, the phosphate buffer containing 1% BSA was removed by suction to obtain an anti-mouse IgG antibody-immobilized plate.

Example 1 (1) Preparation of avidin-immobilized plate Neutravidin (Pierce) was adjusted to 100 μg / ml with a phosphate buffer pH 8.5, and 100 μl was dispensed into a 96-well microplate at 37 ° C. overnight. This was left to immobilize the anti-mouse IgG antibody. After washing the microplate with a phosphate buffer, 300 μl of a phosphate buffer containing 1% casein was dispensed and allowed to stand at 37 ° C. overnight. The next day, the phosphate buffer containing 1% casein was removed by suction to obtain an avidin-immobilized plate.

(2) Measurement of mouse IgG using an avidin-immobilized plate Using a phosphate buffer pH 7.5 containing 1% casein, the anti-mouse IgG antibody prepared in Reference Example 1 was used at a concentration of 71 μg / m 2.
1. A mixture was prepared so that the biotin-labeled anti-mouse IgG antibody prepared in Reference Example 2 was 7.1 μg / ml and the POD-labeled anti-mouse IgG antibody was 0.045 μg / ml. 100 μl of this mixed solution and a homemade mouse IgG standard solution (0, 0.625, 1.25, 2.5, 5, 10, 20, 20)
(μg / ml) was dispensed to each of the avidin-immobilized plates prepared in (1), and reacted at 37 ° C. for 1 hour. After washing the microplate with PBS containing 0.1% Tween (trade name) (hereinafter, “PBST” five times,
TMBZ (tetramethylbenzidine) / hydrogen peroxide solution was dispensed in 100 μl portions, and reacted at 25 ° C. for 30 minutes. After dispensing 100 μl of the reaction stop solution, absorbance was measured at 450 nm.

FIG. 1 shows the results. The calibration curve showed a quantitative response over the entire measurement range. Under these measurement conditions, a POD-labeled anti-mouse IgG antibody and a biotin-labeled anti-mouse IgG antibody form a complex via the mouse IgG to be measured, and the complex binds to the avidin-immobilized plate. In the formation, since free anti-mouse IgG antibody competes with the POD-labeled anti-mouse IgG antibody and the biotin-labeled anti-mouse IgG antibody in the same liquid phase, it is considered that the same suppression was applied in the entire reaction system.

Example 2 Measurement of Mouse IgG by Addition of Biotin-Labeled Antibody In the mixture of Example 1, mouse IgG was measured under the same conditions as in Example 1 except that a biotin-labeled anti-mouse IgG antibody was used instead of the anti-mouse IgG antibody. Was done. The results are shown in FIG. Even without using a free anti-mouse IgG antibody, the amount of POD-labeled anti-mouse IgG antibody bound to the avidin-immobilized plate is suppressed by the biotin-labeled anti-mouse IgG antibody. A quantitative response was obtained over the entire calibration curve while maintaining the measurement sensitivity.

Comparative Example 1 Measurement of Mouse IgG by Addition of Anti-Mouse IgG Antibody Mouse IgG was measured under the following conditions. Reference Example 3
The anti-mouse IgG immobilized plate prepared in
Anti-mouse IgG antibody solution (0.6 mg / m
l) and a homemade mouse IgG standard solution (0, 0.625,
1.25, 2.5, 5, 10, 20 μg / ml) to 50
The mixture was dispensed by μl and reacted at 37 ° C. for 1 hour. After washing the plate 5 times with PBS pH 7.5 containing 0.1% Tween (hereinafter referred to as PBST), 100 μl of the 3.2 μg / ml POD-bound anti-mouse IgG antibody prepared in Reference Example 2 was dispensed in 100 μl portions. It poured and reacted at 37 degreeC for 1 hour.
After washing the microplate five times with PBST, ABTS
(2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) / hydrogen peroxide solution was dispensed in 100 μl portions, and 25
The reaction was performed at 30 ° C. for 30 minutes. Add 100 μl of reaction stop solution,
The absorbance at a wavelength of 420 nm was measured.

FIG. 3 shows the results. The calibration curve showed low sensitivity in the low concentration range, and tended to become gentler in the higher concentration range. Under these conditions, there was a correlation between the change in the concentration of the antigen and the degree of color development, but they were not parallel, so it was determined that the antigen was not suitable for quantification.

As described above, in order to reduce the sensitivity of the entire measurement system, an attempt was made to add a free antibody to the first reaction solution. Under these conditions, a linear transition was observed during the set measurement range. No calibration curve was obtained. When an anti-mouse IgG antibody in an amount capable of measuring a low concentration region is added, a high concentration region cannot be measured, and when an anti-mouse IgG antibody sufficient to sufficiently suppress the high concentration region is added, sensitivity in the low concentration region becomes insufficient. Therefore, it is considered that a necessary measurement range cannot be secured by the method of adding a free anti-mouse IgG antibody.

The reason why the measurement sensitivity in the low concentration range is reduced under these conditions is that the immunity to the immobilized antibody due to the formation of a complex between the free anti-mouse IgG antibody and the mouse IgG to be measured during the primary reaction. Inhibition of the reaction, inhibition of the immune reaction with the POD-labeled antibody by forming a complex between the immobilized antibody and the mouse IgG to be measured and a free anti-mouse IgG antibody, and the like can be considered.

Comparative Example 2 Measurement of Mouse IgG by Adding Biotin-Labeled Antibody The anti-mouse IgG antibody solution prepared in Reference Example 1 (0.44
mg / ml) and the biotin-labeled anti-mouse IgG antibody (0.44 mg / ml) prepared in Reference Example 2,
An anti-mouse IgG antibody solution (0.44 mg / ml) containing a 0.625% biotin label was prepared (this partition ratio was determined by preliminary experiments). A biotin-labeled mixed anti-mouse IgG antibody solution and a homemade mouse IgG standard solution (0, 0.625, 1.25, 2.5, 5, 10, 20, 20) were placed on the anti-mouse IgG antibody-immobilized plate prepared in Reference Example 3.
(μg / ml) was dispensed in 50 μl portions and reacted at 37 ° C. for 1 hour. After washing the plate 5 times with PBST, 0.34 mg
/ Ml of POD-labeled avidin (manufactured by the company) was dispensed in 100 μl portions and reacted at 37 ° C. for 1 hour. After washing the microplate five times with PBST, add ABTS / hydrogen peroxide solution for 1 hour.
The mixture was dispensed in an amount of 00 μl and reacted at 25 ° C for 30 minutes. 100 μl of the reaction stop solution was added, and the absorbance at a wavelength of 420 nm was measured.

FIG. 4 shows the results. The calibration curve showed the same shape as the result of Comparative Example 1. Since the measurement range was not expanded even when POD-labeled avidin was used, the cause was not the inhibition of the immune reaction to the labeled antibody due to the complex formation of the immobilized antibody, mouse IgG and free anti-mouse IgG antibody, but the free This was interpreted as inhibition of the immune response to the immobilized antibody due to the complex formation between the anti-mouse IgG and the mouse IgG.

Example 3 Using a phosphate buffer solution containing 1% casein (pH 7.5), the anti-mouse IgG antibody prepared in Reference Example 1 was 429 μg / m 2
1. A mixture was prepared so that the biotin-labeled anti-mouse IgG antibody prepared in Reference Example 2 was 7.1 μg / ml and the POD-labeled anti-mouse IgG antibody prepared by Nakane method was 7.5 μg / ml. 50 μl of this mixture and homemade mouse I
gG standard solution (0, 0.625, 1.25, 2.5, 5,
50 μl of (10, 20 μg / ml) were respectively dispensed to the avidin-immobilized plate prepared in (1) and reacted at 37 ° C. for 1 hour. After washing the microplate five times with PBS, 100 μl of ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid / hydrogen peroxide) was dispensed at 25 ° C. for 30 minutes. The reaction stop solution was 100
After dispensing each μl, the absorbance was measured at 420 nm.

FIG. 5 shows the results. The calibration curve showed a quantitative response over the entire measurement range.

[0041]

As described above, according to the present invention, even when the concentration of the test antigen in the sample is high, the dilution from the low concentration region to the high concentration can be carried out without dilution or at least with a smaller number of dilutions than in the conventional method. For the first time, an immunoassay method capable of accurately performing immunoassay up to an area has been provided.

[Brief description of the drawings]

FIG. 1 shows a calibration curve created in Example 1 of the present invention.

FIG. 2 shows a calibration curve created in Example 2 of the present invention.

FIG. 3 shows a calibration curve created in Comparative Example 1 of the present invention.

FIG. 4 shows a calibration curve created in Comparative Example 2 of the present invention.

FIG. 5 shows a calibration curve created in Example 3 of the present invention.

Claims (13)

[Claims] 1. A test antigen having a plurality of epitopes,
A labeled antibody that undergoes an antigen-antibody reaction with the test antigen, a labeled antibody that is labeled or subsequently labeled, and an antibody that reacts with the test antigen in an antigen-antibody manner in a competitive manner with the labeled antibody, and further is specific to a solid phase. Reacting in a liquid phase with a solid phase specific binding antibody that specifically binds, and simultaneously or after the reaction, reacting the solid phase specific binding antibody with a solid phase specifically binding thereto, An immunoassay method comprising measuring the label bound to the solid phase via a solid phase specific binding antibody, the test antigen, and the labeled antibody. 2. The solid-phase specific binding antibody is a specific-binding compound-binding antibody in which a specific-binding compound that specifically binds to a certain substance is bound to an antibody that reacts with the test antigen by an antigen-antibody reaction. The method according to claim 1, wherein the solid phase is a substance to which a specific-binding corresponding compound that specifically binds to the specific-binding compound is bound. 3. The method according to claim 1, wherein the specific binding compound is biotin, and the specific binding corresponding compound is avidin or a biotin binding derivative thereof. 4. The method according to claim 1, wherein the test antigen, the labeled antibody, and the solid phase specific binding antibody are reacted on a solid phase that specifically binds to the solid phase specific binding antibody. The method according to any one of claims 1 to 4. 5. The method according to claim 1, wherein at least one of the solid phase specific binding antibody and the labeled antibody is a polyclonal antibody. 6. A test antigen having a plurality of epitopes,
A labeled antibody that undergoes an antigen-antibody reaction with the test antigen, a labeled antibody that is labeled or subsequently labeled, and an antibody that reacts with the test antigen with an antigen-antibody, and further solid-phase specific binding that specifically binds to a solid phase Antibody and a third free antibody that reacts with the test antigen in an antigen-antibody reaction in a liquid phase. Simultaneously with or after the reaction, the solid-phase specific binding antibody specifically binds thereto. Reacting with a solid phase to be subjected to, the immunoassay method comprising measuring the label bound to the solid phase through the solid phase specific binding antibody, the test antigen and the labeled antibody, An immunoassay method, wherein at least two of a labeled antibody, the solid phase specific binding antibody, and the third free antibody competitively react with the test antigen. 7. The method according to claim 6, wherein at least one of the solid-phase specific binding antibody, the labeled antibody, and the third free antibody is a polyclonal antibody. 8. The method according to claim 1, wherein the label is an enzyme label. 9. A labeled antibody that undergoes an antigen-antibody reaction with a test antigen having a plurality of epitopes, or a labeled antibody that is labeled later, and an antibody that reacts with the test antigen in an antigen-antibody competitively with the labeled antibody. An immunoassay kit further comprising a solid-phase specific binding antibody that specifically binds to a solid phase, and a solid phase that specifically binds to the solid-phase specific binding antibody. 10. A labeled antibody which is subjected to an antigen-antibody reaction with a test antigen having a plurality of epitopes, and which is labeled or subsequently labeled, and an antibody which reacts with the test antigen by an antigen-antibody,
Further, a solid phase specific binding antibody that specifically binds to a solid phase,
A third free antibody that reacts with the test antigen through an antigen-antibody reaction;
An immunoassay kit comprising the solid phase-specific binding antibody and a solid phase that specifically binds, wherein at least one of the labeled antibody, the solid phase-specific binding antibody, and the third free antibody is used. An immunoassay kit in which a subject competitively reacts with the test antigen. 11. The solid-phase specific binding antibody is a specific-binding compound-binding antibody in which a specific-binding compound that specifically binds to a certain substance is bound to an antibody that reacts with the test antigen through an antigen-antibody reaction. The kit according to claim 9, wherein the solid phase is a substance to which a specific-binding corresponding compound that specifically binds to the specific-binding compound is bound. 12. The kit according to claim 11, wherein the specific binding compound is biotin, and the specific binding corresponding compound is avidin or a biotin binding derivative thereof. 13. The kit according to claim 9, wherein the label is an enzyme label.