JPH02152999A - Monoclonal antibody-originated substance for removal and suppression of non-specific reaction in immunoassay, its production and use - Google Patents

Abstract

NEW MATERIAL:A substance originated from a monoclonal antibody and having non-specific activity of the monoclonal antibody in spite of the complete loss of the specific antibody activity characteristic of the monoclonal antibody to be used in immunoassay. USE:Elimination or suppression of non-specific reaction in immunoassay using a monoclonal antibody. PREPARATION:The objective monoclonal antibody-originated substance for suppressing non-specific reaction can be prepared e.g. by fusing a spleen cell of Balb/c mouse immunized with an antigen (e.g. lowly differentiated human adenocarcinoma) with a mouse myeloma cell, screening and cloning the fused cell, diluting the prepared monoclonal antibody solution with 0.05M phosphate-buffered sodium chloride solution and heating the diluted solution at 60 deg.C for 120min. A determination object can be quantitatively determined in high sensitivity while suppressing non-specific reaction by an enzyme immunoassay by supporting immobilized monoclonal antibody on beads and adding and reacting the beads and the monoclonal antibody-originated substance to a specimen solution, thereby performing non-specific reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to immunoassay methods, and more particularly, to immunoassays using monoclonal antibodies, and non-specific reactions in the 11+1 standard method. This article relates to a method for removing or inhibiting the use of monoclonal antibodies, and a monoclonal antibody-derived substance used therein.

(Prior art) Measurement methods that utilize immune reactions have been applied to various clinical tests, but in recent years, the use of the high specificity of monoclonal antibodies has made it possible to conduct tests that are impossible with conventional polyclonal antibodies. Or where the country fl T-was,
It has become possible to develop specific or similar detection and theorems for microorganisms, and as a result, it has become possible to obtain useful information regarding more advanced testing and diagnosis than ever before.

For example, various monoclonal antibodies were produced by immunizing mice with cancer cells and using cell fusion techniques, and the nature of the antigenic determinants recognized by these antibodies was investigated.

As a result, it was revealed that these monoclonal antibodies often recognized sugar chains, and it was also discovered that these sugar chains changed as cells became cancerous, and that these monoclonal antibodies often recognized sugar chains. It has been found that if a monochrome antibody that can be obtained can be used as a powerful means for detecting cancerous changes in sugar chains.

Therefore, sugar chains detected using such monoclonal antibodies can be used as markers (tumor markers) for detecting cancerous cells and tumors.

In fact, various seven clonal antibodies that recognize sugar chains have been created and applied to the measurement of tumor markers.
Carbo, a sugar chain antigen discovered from 1S W-1116
-bydrate antigen 19-9 (CA
CA 19-9) was found to be useful as a tumor marker for pancreatic and biliary tract cancer, and it was reported that monoclonal antibody 1116-N519-9 against this antigen specifically recognized CA 19-9 (Koprowski, Il
,,et al,: Co1orectalCa
rCin0111a anti(]enS det
eCt(3d by hybridI!1aanti
bodies, Soma(ic Ce1l Ge
netics 5:9571979). In particular, if such antigens are present in body fluids such as serum and can be measured, this could be a very useful method for clinically diagnosing cancer. In fact, the above-mentioned CA 19-9 has been evaluated as a marker for leprosy incisions mainly in pancreatic and biliary tract cancers.

However, since such antigens exist only at a low temperature of 11 degrees in body fluids such as serum, their detection requires extremely sensitive measurement methods. Examples include radioimmunoassay ([IA)], enzyme immunoassay (EIA), and fluorescence immunoassay (FIA), and furthermore, application of chemiluminescence method, latex agglutination method, etc. are also considered. In these measurement methods,
The detection means are different, but they are all based on immunological reactions, and each one quantitatively reflects the immune reaction.
] and the previous set. Among these measurement methods, RIA is the most commonly used high-sensitivity measurement method at the actual clinical testing level, followed by EIA, but recently FIA has also become possible in some cases. . It is also possible to further categorize these measurement methods.

Among the above measurement methods, RIA is commercially available.

To explain the solid-phase Nando Inch method, which is used in most measurement kits for EIA, etc., the Sand Inch R
When measuring antigens using the IA method, first, glass,
A monoclonal antibody that specifically reacts with the analyte is adsorbed and in phase with an inert carrier made of plastic or the like, and then the antigen in the sample is brought into contact with this to cause the antigen and antibody to bind through an immune reaction. . Furthermore, when this bound antigen is reacted with a radioisotope-conjugated antibody (radioisotope-labeled antibody), a sandwich-shaped antigen-antibody complex is formed bound to the solid phase with the antigen in between. . Thereafter, the amount of radioactivity of the radioisotope bound to the antigen-antibody complex is measured to determine the target amount of antigen. In EIA and FIA, an enzyme or fluorescent substance is used instead of a radioactive isotope as a substance to label antibodies, and the amount of activity or fluorescence intensity of the enzyme bound to the antigen-antibody complex is measured to determine the target. Determine the amount of antigen.

In any case, the common point is that a labeled antibody is used, and the measurement method takes advantage of the fact that the amount of antigen to be determined is positively reflected in the labeled substance.

In this way, each measurement method attempts to detect the target substance using immune reactions, but immune reactions generally involve non-specific reactions that do not depend on the original antigen-antibody reaction. are often observed, which often impairs the reliability of measurements. Conventionally, in order to remove and suppress such non-specific reactions and obtain correct measurement values, surfactants, gelatin, serum of various animals, ascites or immunoglobulin fractions, or additives used in the measurement system have been used. A method has been proposed in which a monoclonal antibody or the like is added which has a different reaction specificity from those used in the measurement and does not inhibit the reaction involved in the measurement.

(The problem that FF Ming is trying to solve) The above method of adding surfactants and various animal serums to remove and suppress non-specific reactions is a method that can be used in various immunoassay systems. However, this method is not always satisfactory in practical terms because it may not be sufficient to remove or suppress nonspecific reactions, or it may partially inhibit the target antigen-antibody reaction. However, even if the desired specific antigen-antibody reaction is suppressed to some extent, non-specific reactions will be completely eliminated or suppressed to a negligible extent, and as a result, sufficient sensitivity for measurement will still be maintained. If it is in a stable state, it can be used as a measurement system.

However, in measurement systems using monoclonal antibodies, simply adding the above substances cannot sufficiently remove or suppress non-specific reactions, making it impossible to obtain monoclonal antibodies with excellent properties. However, there was a major problem in that it was subject to major restrictions when it was actually used, and its utility value was lost.

Another method for eliminating this non-specific reaction is to pre-treat the sample to be measured. This is done by mixing the sample to be measured with an acidic buffer and heating it at 60 to 70°C, or treating it with perchloric acid to remove substances or sites that cause non-specific reactions. This is the method of trying to remove it. This method takes advantage of the fact that sugar chains, which are the main antigenic determinants, are resistant to heat and acid, but this method requires complicated procedures such as sample processing before immune reaction and centrifugation to remove denatured proteins. It is necessary to carry out the process. Particularly when measuring multiple samples, the pretreatment requires a lot of time, which is not preferable. Furthermore, depending on the specimen, the antigen-determining site that participates in the antigen-antibody reaction may also be partially inactivated or reduced in activity by the above-mentioned treatment, and as a result, the measured value may become small. Especially Ill
For markers such as the 1tA marker, for which a cutoff value is set, there is a possibility that the measured value will be lower than the cutoff value, resulting in a negative result. Therefore, it is difficult to say that methods for removing non-specific reactions such as heat treatment of specimens are satisfactory not only in terms of operation but also in terms of measured values.

(Means for Solving the Problems) Therefore, the present inventors investigated various methods for eliminating and suppressing such non-specific reactions without complicating the operation, and as a result, they obtained the following knowledge. Ta.

That is, certain non-specific reactions that could not be removed or suppressed by known methods are due to a site different from the original antigen-antibody reaction site of the monoclonal antibody used in the measurement system; Then, this monoclonal antibody is subjected to, for example, heat treatment, decomposition treatment, or a combination of both treatments I! By applying certain treatments, including the following, the original antibody activity is completely or substantially lost, and the activity of non-specific reaction (hereinafter referred to as "non-specific activity").
Furthermore, if a sample is pretreated with a monoclonal antibody treated in this way (hereinafter referred to as "monoclonal antibody-derived material") before a specific immune reaction, the above-mentioned non-active substances can be retained. The specific reaction is thereby absorbed, and the non-specific reaction in the measurement system can be completely or substantially eliminated or suppressed to a negligible extent.

The present invention is based on the above findings. That is, if the sample to be measured is brought into contact with the above-mentioned monoclonal antibody-derived substance before a specific immune reaction, the monoclonal antibody with only the non-specific reactant or its reaction site remaining in the sample and the non-specific activity can be removed. The derived product ′ζJ reacts. As a result, the non-specific reactive substance or its reactive site in the sample reacted non-specifically with the 1!7I substance derived from the monoclonal antibody, and therefore no longer caused a non-specific reaction with the Tomoclonal antibody. 1q is absent, in other words, the non-specific reaction is absorbed, so from then on, the influence of the non-specific reaction can be eliminated, and moreover, this monochrome antibody-derived substance is not the original specific one. Since the antibody activity has been lost, accurate measurements can be made without any effect on this original, specific antigen-antibody reaction.

Therefore, the present invention provides monoclonal antibodies for use in immunoassays that have completely or substantially lost their inherent specific antibody activity, but have substantially retained their nonspecific activity. The purpose is to provide monoclonal antibody-derived substances.

Furthermore, the present invention provides a method for producing the monoclonal antibody-derived substance and a method for removing and suppressing non-specific reactions using the same. This is what we provide.

Here, "substantial" means to the extent that there is no problem when judged from the viewpoint of measurement sensitivity, reliability, etc.

By the way, when measuring antigens in a sample using the sandwich method, especially the two-step sandwich method, which is the most commonly used method for measurement, it is necessary to Before the first immune reaction in which the immobilized antibody reacts with the antigen in the specimen, a special method is used to perform an absorption reaction, such as mixing the specimen with the monoclonal antibody-derived substance in advance and incubating it in the dark for a certain period of time. No further operations are required.

By simply adding the monoclonal antibody-derived substance to the first step reaction buffer, the monoclonal antibody-derived substance is allowed to coexist with the in-phase antibody and the sample in the same immune reaction. It is possible to obtain the desired effect without the need for any operations, especially without any complications or problems such as squirting. This is because, compared to the solid-liquid reaction between the in-phase antibody and the specimen, the non-specific reaction between the monoclonal antibody-derived substance and the specimen existing in the same immune reaction buffer is liquid-liquid. This is because the rate of progress is extremely fast since it is a liquid-intermediate reaction, and an absorption reaction by the monoclonal antibody-derived substance can substantially occur before a specific immune reaction occurs.

Furthermore, it is also possible to further improve the reliability of the measurement by allowing a monoclonal antibody-derived substance to coexist not only in the first immune reaction but also in the second immune reaction.

Furthermore, in the one-step sandwich method, the monoclonal antibody-derived substance is mixed with the sample prior to the immune reaction and an absorption reaction is performed for a certain period of time, or the monoclonal antibody-derived substance is simply added to the reaction buffer. Just by allowing them to coexist during the same immune reaction, the manipulation
Accurate values can be easily obtained by excluding the effects of non-specific reactions without substantially increasing the

Therefore, the most unexpected and essential feature of the present invention is that monoclonal antibodies that have both original specific antibody activity and non-specific reaction activity,
It is important to note that even if certain treatments are performed to eliminate the original antibody activity, the non-specific activity will not be lost. If absorbed in advance, non-specific reactions in immunoassays can be removed and suppressed, allowing accurate measurement values to be obtained. Briefly speaking, the feature of the present invention is that a part of the properties of the antibody protein is extracted and used to solve a problem.

Next, a method for producing the monoclonal antibody-derived substance of the present invention for non-specific reaction absorption will be described.

The monoclonal antibodies used as starting materials are the same as those used in each reaction step. That is, it is the same monoclonal antibody as the antibody that is adsorbed and immobilized on the homogeneous carrier and/or the antibody that is labeled with a radioisotope, enzyme, fluorescent substance, or the like. These monoclonal antibodies have the property of performing non-specific reactions that may or may not be recognized depending on the individual specimen and that vary in degree in the reaction pool based on the original specific antibody activity. Moreover, among these reactive activities, the original antibody activity is deactivated by treating the monoclonal antibody under certain conditions, whereas the non-specific activity is retained without being deactivated. have characteristics. Utilizing this property, monoclonal antibody-derived substances for non-specific reaction absorption are manufactured.

That is, in order to obtain the monoclonal antibody-derived substance of the present invention, the same antibody as that used in the measurement system is subjected to certain treatments, including treatments such as heating or decomposition, alone or in combination, to improve the properties of the antibody. It is prepared so that the original antibody activity is completely or substantially eliminated, while non-specific activity is substantially retained. Certain other treatments that can be used in the present invention include ultrasonic treatment, organic solvent treatment, acid/alkali treatment, and the like.

The monoclonal antibody to be treated does not need to be particularly purified, and a sufficient effect can be obtained even if the ascites of a mouse or the like containing the monoclonal antibody is used as it is. Therefore, Ltsuku [? - It is simplest and economically advantageous to directly subject the ascites fluid containing Nor antibody or its diluted form b to a certain treatment. For example, is it non-specific due to heat treatment? When attempting to obtain a substance derived from antibodies for four-way reaction absorption, use ascites containing monoclonal antibodies as an undiluted solution, bt, < is an appropriate pH-+
For example, the solution is diluted with a slow pH solution adjusted to pH 7.0 to 75, and heated for a certain period of time. The heat treatment conditions can be determined by the appropriate authorities of the Ministry of the Arts, but - the dilution ratio of ascites in the shell is from undiluted solution to 1 (10) times, preferably 5 to 20 times, and the heating temperature is 40 ° C. or higher, preferably is 50-75°C.

The heating time also varies depending on the heat resistance of the antibody activity or the temperature;
For example, at 60°C, the temperature is 15~15°C.
Suitably, the heating time is 240 minutes, preferably 60 to 150 minutes, and at 70°C, 2 to 60 minutes, preferably 5 to 20 minutes. However, if the heating temperature or heating time is set higher than necessary, not only the antibody activity but also non-specific reactions will be impaired, and the antibody will be completely inactivated, so care must be taken. The monoclonal antibody-derived substance thus obtained has a structure whose amino acid sequence is identical to that of the seven-crop antibody, which is the starting material. This 1. You can use it immediately by adding it to the reaction Li box solution to the desired concentration, for example, or if you want to store it, you can put it in the refrigerator at 2 to 8 degrees Celsius, or −
It may be frozen at 80°C or freeze-dried by a generally known method.

As a method for producing the monoclonal antibody-derived substance of the present invention, a method of decomposing a monoclonal antibody with a proteolytic enzyme such as pepsin, trypsin, or papain to remove, for example, the Fab portion or the F(al+′)2 portion; Furthermore, it is also possible to perform both decomposition and heating treatments. For example, monoclonal antibody 50II purified by the known ammonium sulfate precipitation method and gel filtration method.
fj containing 4M urea and 0.1M l-1,1 salt vi[
l! Water (1) II 8.0) 5d1. :Add, 25℃
, 16 hours in 4: Juvecht, then 1.5 ■ TPC
K-Trypsin (Wort former ngton company%j
), add 0.05M calcium chloride and decompose at 25°C for 8 hours. The Fc portion can be obtained by fractionating this decomposition product by gel filtration according to a conventional method. However, from the point of view of simplicity, a method that does not deactivate specific antibody activity by heat treatment is most suitable.

The amount of 7-clonal antibody-derived substances required to eliminate and suppress non-specific reactions can be determined as appropriate based on the knowledge in the art. This amount varies depending on the measurement system and the type of specimen, but if it is added in advance to the reaction buffer, the amount used should be converted to the concentration as ascites stock solution.
- in the shell o, oos ~ 5% v/v, preferably 0.
It is preferable to use a concentration of 0.05 to 1%. The protein concentration of the monoclonal antibody-derived substance in the reaction buffer was 0. (10) 5 to 0.111?9/rd is suitable.

Eliminate or suppress non-specific reactions using the method of the present invention υ1
However, immunoassay methods such as R[A and ERA, which can reliably determine the minute substances in a specimen with <1 lllll, are generally known measurement methods and are widely used in clinical tests. It is. (For example, the immunoassay fl research group (planned):
Examples of immunoassay usage and laboratory reagents/treatments? ? ! Application to Development, Management Education Publishing, 1985).

(Effect of the invention) In an immunoassay method using a monochrome full antibody,
By using a monoclonal antibody-derived substance that absorbs and removes non-specific reactions in this measurement system, we can eliminate the influence of non-specific reactions and obtain highly reliable measurement values that accurately reflect the original antigen-antibody reaction. Obtainable.

Examples will be described below, but the technical scope of the present invention is not limited to these examples.

Experimental example (1) Obtaining mouse ascites containing monoclonal antibodies After fusion of Ba1b/c mouse tile cells immunized with human poorly differentiated adenocarcinoma and mouse myeloma cells (P3-X63-8g8-01) according to a conventional method, By screening using immunohistological staining, hybridomas that produced antibodies that did not react with normal tissues but reacted with cancer tissues were selected. This hybridoma was intraperitoneally administered to Balb/c mice, allowed to proliferate, and mouse ascites containing the monoclonal antibody (IaM) was drained.

(2) Preparation of purified monoclonal antibodies Purified monoclonal antibodies were obtained from the monoclonal antibody-containing mouse ascites obtained in (1) by the known ammonium sulfate precipitation method and gel filtration method.

(3) Enzyme-linked immunosorbent assay (EIA) using purified monoclonal antibodies, that is, a two-step sandwich method using polystyrene beads, as shown in Table 1. First, 50 g of the sample was collected in a test tube, and bovine serum albumin ([3SA) 0 was added as the am solution for reaction.
．． Phosphoric acid*iir saline containing 5% (pH 7,4)
2 (10) m was added. Next, one polystyrene bead in which the monoclonal antibody was insolubilized was added by a conventional method, and the mixture was reacted at 37° C. for 2 hours (first immune reaction). Next, after washing the beads three times with physiological saline, 2 (10) p of the monoclonal antibody solution labeled with horseradish peroxidase (+-I RP >) was added, and the beads were incubated in room 4 (25°C
) for 2 hours (second immune reaction). After washing the beads again with physiological saline, transfer the beads to another test tube and add 30% hydrogen peroxide solution 0.2M and ABTS' (
Boehringer Mannheim) containing 0.45115F 0.1M citrate phosphate buffer (pH 4.0) 3
0) IJl was added and reacted at 37°C for 30 minutes (color reaction). 5% Shu! After stopping the reaction by adding 2 precipitates, the absorbance at a wavelength of 405 nm was determined. The results are shown in Table 1.

Table 1 Results of Experimental Examples Sample Healthy person serum Δ*1 Healthy person serum B81 Pancreatitis patient serum Pancreatic cancer patient serum Breast cancer patient serum Antigen solution "2 1% BSA-containing PBS*3 (77>) Measured absorbance 0,030 1. 232 0,035 1.068 0,614 Q,1381 0,023 Umbrella 1 No abnormalities found in various measurement items during regular health checkups Umbrella 2 Tissue obtained by transplanting human gastric cancer cells into nude mice and growing them The extracted solution was added to 0.05M PBS containing 1% 83A (I) 117.41Te1 (10
) 0.05M PBS containing 31×BSA (pH 7°4)
Among the above results, even though sample 2 was serum from a healthy person, an abnormally high value of absorbance was produced, which was determined to be due to a non-specific reaction.

Example 1 (1) Production of monoclonal antibody-derived substance for non-specific reaction absorption by heat treatment The monoclonal antibody-containing mouse ascites obtained in Experimental Example 1-(1) was mixed with 0.05M phosphorus RB*saline (pl+7).
．． 4) and heat-treated for 20 minutes at 60"C11 to obtain a monoclonal antibody-derived substance for non-specific reaction absorption.

(2) Measurement by enzyme immunoassay using a monoclonal antibody-derived substance Among the measurement operations shown in Experimental Example 1-(3), the reaction buffer of the first immune reaction was All experiments were performed in Experiment 1-(3
), and the same sample as in Experimental Example 1 was measured.

The results are shown in Table 2.

Table 2 Results of Example 1 Sample Measured absorbance 1 Healthy subject serum 0.0312 Healthy subject serum
0.0363 Pancreatitis patient side
O,0334 Pancreatic cancer patient serum
10535 Breast cancer patient serum 0
4226 Antigen solution 0.89
PBS containing 611% BSA (blank) 0.
024 Comparing this result with the results of Experimental Example, the high value of absorbance observed in Experimental Example in Sample 2 is different from that of Example 1.
This was not observed, and the absorbance decreased significantly. This shows that the high absorbance of Sample 2 in Example 1 was due to a non-specific reaction, and that the non-specific reaction was removed by the addition of the monoclonal antibody-derived substance. Also,
Sample 5 also had a non-specific reaction, although it was weaker than sample 2, and the absorbance decreased due to the addition of the monoclonal antibody-derived substance.

On the other hand, regarding the antigen solution, a difference in absorbance was observed depending on whether or not the monoclonal antibody-derived substance was added, indicating that the monoclonal antibody-derived substance did not affect the original specific immune reaction.

Comparative Example In Example 1-(2), except for using mouse serum, mouse ascites, rat serum, rabbit serum, and pig serum instead of the monoclonal antibody-derived substance, non-specific reactions were avoided under the same conditions and operations. Measurements were performed on the accepted samples 2, 5 and 7. As shown in the results shown in Table 3, it was not possible to sufficiently remove non-specific reactions in either Example 1 or 4.

Table 3 Results of Comparative Example Example 2 Measurements were made by changing the concentration of the monoclonal antibody-derived substance for non-specific reaction absorption in the first immune reaction in Example 1. However, using samples 2 and 5 in which non-specific reactions were observed, a monoclonal antibody-derived substance for non-specific absorption at the concentration shown in Table 4 was added to the reaction M buffer in the primary immune reaction. The same operation as in Example 1 was performed. As shown in the results shown in Table 4, the amount of the monoclonal antibody-derived substance added in Sample 2 is only 0.2 V/V% in terms of the concentration of the stock solution of mouse ascites containing the monoclonal antibody; No difference was observed in the effectiveness of eliminating or suppressing specific reactions. On the other hand, in Sample 5, a smaller amount of the monoclonal antibody-derived substance ω was required than in Sample 2, and non-specific reactions were eliminated at 0.05 v/V% in terms of the same mouse ascites stock solution.

Table 4 Results of Example 2 Example 3 Samples 2, 5 and 7 in which non-specific reactions were observed were measured by radioimmunoassay (RIA) using a monoclonal antibody labeled with a radioisotope using a conventional method. The operation is HRPI in the EIA method in Example 1-(2).
! The same procedure as in Example 1-(2) was carried out, except that a radioisotope-labeled antibody was used instead of the labeled antibody, and the radioactivity ID was determined instead of determining the enzyme activity amount. Specifically, 0.2 V/V% of the substance derived from the monoclonal antibody No. 11 in Example 1-(1) was added to the first immune reaction, and the reaction was measured by the 2-step German RIA method. For comparison, measurements were also conducted without using a monoclonal antibody-derived substance. The results are as shown in Figure 5 for 4 people, and as in Example 1, a large influence of non-specific reactions was observed unless a monoclonal antibody-derived substance was used.

Table 5 Results of Example 3

Claims (11)

[Claims] (1) Monoclonal antibodies used in immunoassays have completely or substantially lost their original specific antibody activity, but have substantially retained their non-specific activity. material. (2) The monoclonal antibody-derived substance according to claim 1, wherein the antigen recognized by the monoclonal antibody is a sugar chain antigen. (3) Claim 1 or 2, wherein the primary amino acid sequence structure of the monoclonal antibody is unchanged.
The monoclonal antibody-derived substance described in . (4) The monoclonal antibody-derived substance according to claim 1 or 2, wherein the Fab portion is substantially removed. (5) The method for producing a monoclonal antibody-derived substance according to claim 1, characterized in that the monoclonal antibody used in the immunoassay is subjected to a certain treatment. (6) The manufacturing method according to claim 5, wherein the certain treatment is a heat treatment, a decomposition treatment, or a combination thereof. (7) Claim 6, characterized in that the monoclonal antibody to be subjected to heat treatment is present in ascites or a diluted solution thereof.
Manufacturing method described. (8) A method for removing or suppressing a non-specific reaction using the monoclonal antibody-derived substance according to claim 1 in an immunoassay method, the method comprising: The above-mentioned removal/inhibition method is characterized in that a non-specific reaction is carried out in advance between the two. (9) The method according to claim 8, wherein the non-specific reaction is carried out by coexisting the immobilized antibody, the monoclonal antibody-derived substance, and the specimen in the same immune reaction. (10) The method according to claim 8 or 9, wherein the coexistence is achieved by adding the monoclonal antibody to a reaction buffer. (11) The method according to claim 8, wherein the immunoassay is a two-step sandwich method, and the non-specific reaction is performed before the first immune reaction.