JP2523171B2 - Immunoassay method and reagent kit used therefor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a non-specific reaction in an immunological method for measuring the amount of a substance by utilizing an antigen-antibody reaction in a solution without lowering the specific reaction. TECHNICAL FIELD The present invention relates to an immunoassay method for reducing and a reagent kit used therefor.

BACKGROUND ART Immunoassay methods using antibodies are widely used due to their high specificity and high sensitivity, and are expected to grow even further with the discovery of monoclonal antibodies in 1975.

Regarding the detection means of this immunoassay, radioactive substances (I ¹²⁵ ) have been used in the past, but in recent years, oxygen, fluorescent substances, etc. have come to be used, and the special substances of users for the use of radioactive substances have come to be used. The lack of training may also be one of the factors that have spurred the development of this immunoassay.

Now, in order for this immunoassay to be highly sensitive, it is an essential condition that the specific reaction derived only from the antigen-antibody reaction is high and the other nonspecific reaction is as low as possible.

Suppressing this non-specific reaction is an important technical point for completing the highly sensitive immunoassay. Therefore, various attempts have been made conventionally. The most widely used method is to add an additive for suppressing nonspecific reaction to the immune reaction system, and the method is roughly divided into two. The first method uses a non-proteinaceous substance such as a surfactant, and the second method
This method uses a body fluid or protein solution. As an example of the first method, in JP-A-57-182169, a polyanion soluble in a reaction medium in which an immune reaction is performed is used, and in JP-A-58-187862,
Non-specific adsorption removal is devised by using a nonionic surfactant. On the other hand, as a second method, JP-A-59-25184 is characterized by using 0.1% or more of hydrophobic protein in the presence of salts, and JP-A-61-65162 is used. By using mouse ascites, etc., we are seeing a reduction in non-specific adsorption.

However, each of the above-mentioned conventional methods has disadvantages and cannot be a definitive non-specific adsorption removal method at present.

That is, the first method often interferes with a specific reaction, resulting in a low-sensitivity measurement system. As a second method, the use of a hydrophobic protein is mentioned, but according to the study by the present inventors, the hydrophobicity had almost no effect on the nonspecific adsorption / removal.

In addition, when ascites of mouse is used as the second method, there is a problem in reproducibility of the ascites component, and the specific reaction is reduced by this component, which is contrary to the original purpose of the immunoassay. .

On the other hand, the reaction of carrying out the immune reaction on a membrane such as nitrocellulose instead of the above solution also helped with the expectation that high sensitivity can be achieved by concentrating the antigen on the membrane. It came to be used. Brian et al. J. Biochem. Biophys. Methods 12 271
~ 279 (1986) reported that a dramatic reduction in non-specific reaction was observed by blocking the membrane with a 10% suspension of skim milk.

As a reaction blocking agent on such a solid phase, Ahma
d et al. also use skim milk and they use J.Clin.Micr
obiol. 23 , 3,563-567 (1986), anti-pseudorabies virus antibody in patient serum was measured by blocking nitrocellulose membrane immobilized with pseudorabies virus antigen using 5% skim milk solution. .

However, when 5% to 10% skim milk suspension is used as a blocking agent, as described in the report of Brian et al. 2 because the method is sensitive
One of the two essential conditions (high specific reaction derived from the antigen-antibody reaction) is lacking, and high-sensitivity measurement cannot be achieved.

The reason why the specific reaction is disturbed is that skim milk is insoluble in water at such a concentration and blocking is performed by using the suspension, and therefore, in terms of microscopic view, a large insoluble matter in skim milk covers the antigen. , The antibodies do not come close to each other, and as a result, the antigen-antibody reaction is largely inhibited.

The conventional use of skim milk as a blocking agent mostly utilizes the above-mentioned solid-phase immune reaction, not the solution-state immune reaction.

Conventionally, there is no report that skim milk has been used as an additive for an immune reaction in a solution state. One of them is that Kuroki et al. Used a 2% skim milk suspension in Pediarr. Res. 19 .1017 (1985) to measure lung-surfactant apoproteins in amniotic fluid and used 2% as an additive. I use skimmed milk. However, use in such a method has two major problems. The problems are that, firstly, skim milk is a suspension and thus suppresses a specific immune reaction for the above-mentioned reason, and secondly, the concentration required to suppress a non-specific reaction. Using a 2% skim milk suspension, which resulted in the following, caused a precipitate after 2-3 weeks of storage in the refrigerator, which precipitation was not possible no matter how re-dissolution was attempted and the inhibition of non-specific reactions. The effect also disappears. That is, unless it is used immediately after preparation, it cannot play the role of suppressing non-specific reaction, and it must be said that the reagent is completely inadequate.

DISCLOSURE OF THE INVENTION Accordingly, the present invention eliminates the above-mentioned problems of the prior art, and when carrying out an immunoassay utilizing an antigen-antibody reaction in a solution, without specifically reducing the specific reaction, nonspecific It is an object of the present invention to provide an immunoassay method capable of measuring an immunoassay reaction with high sensitivity by suppressing a biological reaction and a reagent kit used therefor.

Other objects and advantages of the present invention will be apparent from the following description.

According to the present invention, when performing an immunoassay utilizing an antigen-antibody reaction in a solution, a protein having an average molecular weight of 16 to 50 thousand and an isoelectric point of 1.0 to 5.0 or a mixture containing the same in the immune reaction solution. There is provided an immunoassay method in which a solution containing the above is present as an antigen-antibody reaction regulator, and the final concentration of the antigen-antibody reaction regulator in the immune reaction solution is adjusted to 0.02 to 0.9% by weight.

According to the present invention, further, a solution containing a protein having an average molecular weight of 16 to 50,000 and an isoelectric point of 1.0 to 5.0 or a mixture containing the protein in a completely dissolved state is used as an antigen-antibody reaction modifier to perform an antigen-antibody reaction. There is provided a reagent kit for use in an immunoassay, which comprises an amount of a regulator in an immunoreaction solution in a final concentration of 0.02 to 0.9% by weight as a part of its components.

In the present invention, the “average molecular weight” of a protein means a molecular weight measured by an osmotic pressure method, and specifically, a polymer solution, a pure solvent, and a solvent molecule are freely permeable, but an eluted polymer is not permeable. The average molecular weight of a protein is measured by utilizing the fact that the osmotic pressure difference between the two liquids becomes a parameter of the molecular weight of the polymer when the membranes are contacted with each other.
It is a value measured at 4 ° C. using a 66 M urea solution. Also, "isoelectric point" means a value measured by a chromatofocusing method in which proteins are separated according to their isoelectric points,
Specifically, it refers to the value measured with a column (0.5 cmφ × 45 cm) packed with PBE94 (Pharmacia) gel with an eluent of 0.025 M imidazole hydrochloric acid (pH 7.4).

Brief Description of the Drawings Fig. 1 shows the retention rate of the dissolved state of skim milk aqueous solutions at various concentrations during storage, and Fig. 2 shows the skim milk concentration and its addition effect in the α ₂ PI-plasmin complex measurement system. Fig. 3 and Fig. 4 show α ₂ PI and protein C, respectively.
Fig. 5 shows a calibration curve comparing the effect of adding skim milk in the measurement system with that without adding skim milk. Fig. 5 shows 0.5% by weight of skim milk in the immune reaction solution.
Fig. 6 shows a calibration curve for the immunological measurement of human protein S by the present assay method (Example 5) in the case of adding sucrose, and Fig. 6 shows human protein S in the case where skim milk was not added to the immune reaction system. Fig. 7 shows a calibration curve for immunological measurement (comparative example 2), Fig. 7 shows the effect of skim milk addition concentration on the immune reaction in the human-placenta-derived acidic GST measurement system, and Fig. 8 shows chondrocalcin measurement. Fig. 9 shows the effect of skim milk addition concentration on the immune response in the system, Fig. 9 shows the effect of skim milk addition concentration on the immune reaction in the IC measurement system, and Fig. 10 shows the correlation between the non-specific reaction rate and the molecular weight of various proteins. Fig. 11 shows the correlation between the non-specific reaction rate and the isoelectric point of various proteins, Fig. 12 shows the effect of the addition concentration of orosomucoid on the immune reaction, and Fig. 13 shows the addition of pepsin. Shows the effect of the degree by immune reaction and Figure 14 shows the effect on the immune reaction by the addition concentration of the skimmed milk in the measurement system of a human pulmonary surfactant apoprotein.

Preferred embodiment for carrying out the invention In the present invention, when carrying out an immunoassay utilizing an antigen-antibody reaction in a solution, the immune reaction solution has a molecular weight of 16 to 50,000, preferably 20 to 46,000 and is isoelectric. Point is 1.0
-5.0, preferably 1.2-4.8 is a protein-
Used as an antibody reaction modifier.

Examples of such proteins in the present invention include casein,
Examples include pepsin, ovoglycoprotein, orosomucoid and the like. When a protein with a molecular weight of less than 16,000 is used, nonspecific adsorption tends to increase, and with a molecular weight of more than 50,000, a reduction in immune nonspecific reaction is insufficient and a reduction in specific immune reaction occurs. The protein used in the present invention has a molecular weight of 1.6
It is 10,000 to 50,000, preferably 2.0 to 46,000.

On the other hand, regarding the isoelectric point, when a protein having an isoelectric point of more than 5.0 is added, non-specific adsorption is increased, and when the isoelectric point is less than 1.0, a specific reaction is suppressed. Has an isoelectric point of 1.0 to 5.0, preferably
It is 1.2 to 4.8.

According to the present invention, a mixture containing the above-mentioned proteins can be used as an antigen-antibody reaction modifier. Such a mixture includes, for example, 10 to 60% by weight of the protein as a main component, preferably 20 to 50% by weight, sugar (e.g., lactose) 30 to 80% by weight, preferably 40 to 60% by weight, other fats (e.g., 0.5-2% by weight), ash (eg 5-12)
% By weight, water (for example, 2 to 8% by weight), and the like. A typical example of such a mixture is skim milk. Skim milk contains casein as a protein, but skim milk has better dispersibility in an immune reaction solution than NSA (Non-spec
It has the advantages of high ific binding effect and good storage stability at 4 ° C (precipitation does not easily occur). In addition,
The skim milk used in the present invention may be milk of any origin as long as it is defatted milk. The most typical is commercially available skim milk from Difco.

The protein solution (or skim milk solution) in the present invention is prepared as follows. That is, a protein or a mixture thereof (for example, skim milk) having an appropriate concentration is added to phosphate buffered saline and stirred for about 1 hour.
Then, it is dissolved by applying ultrasonic waves and used as a 0.45 μmillipore passage solution, for example. When immunoassays were carried out using protein solutions (or skim milk solutions) of various concentrations, when protein solutions (or skim milk solutions) of less than 0.02% by weight were used, non-specificity was obtained even though the antigen was 0. The lower limit of the protein concentration is 0.02% by weight because the biological reaction was significantly increased. Further, when the concentration exceeds 0.9% by weight, the specific immune reaction is decreased, and the storage stability in the refrigerator tends to decrease, so the upper limit of the protein concentration is 0.9% by weight. Considering the above two facts, the protein concentration that satisfies the stability of the reagent and effectively reduces the non-specific reaction is appropriately 0.02 to 0.9% by weight, preferably 0.05 to 0.7% by weight. Is.

The reagent kit used in the immunoassay according to the present invention is a known reagent kit, for example, a reagent kit used in the immunoassay by the sandwich method using two antibodies.
Consists of enzyme-labeled antibody, (b) antibody-immobilized beads, (c) assay buffer, (d) substrate solution, (e) color former, (f) color stop solution, (g) standard, (h) wash solution, etc. It is preferable that any one of these contains a solution of the protein. Among them, (a) the enzyme-labeled antibody or (c) the assay buffer, and particularly (c) the assay buffer, is preferable. The protein solution is kept in a freeze-dried state, and when it is reconstituted by reconstitution with water etc. at the time of use,
You may make it a predetermined density range.

According to the present invention, a protein satisfying the conditions of the present invention or a mixture containing the protein (typically skimmed milk) is added to an immunoassay system so that the specific reaction is hardly reduced, and the reaction is sufficiently nonspecific. It was confirmed that it has the effect of lowering the reaction, and a highly sensitive immunoassay became possible.

A conventional aqueous solution of skim milk capable of exhibiting a blocking action has a problem that even after a short standing time, skimmed milk aggregates and precipitates during the standing and cannot be redissolved. And in that state, the blocking effect of skim milk was completely lost. However, when the skim milk of the present invention is used as the antigen-antibody reaction modifier, that is, the skim milk solution of 0.02 wt% to 0.9 wt% used in the present invention is
Even if it was stored in a refrigerator in the form of an aqueous solution for a year, no aggregates were generated, and it could be used for an immunoassay, and its storage stability could be satisfied as a measuring reagent.

Moreover, when an immunoreaction was carried out using the skim milk solution prepared in this way, it was possible to sufficiently reduce the nonspecific reaction even with a dilute skim milk solution, which had not been considered to have a blocking effect in the past, of 0.02% by weight. Moreover, it was confirmed that the skimmed milk concentration in this range did not significantly reduce the specific immunoreactivity, which enabled a highly sensitive immunoassay.

The reason why the assay method of the present invention hardly reduces specific immunoreactivity is considered as follows.

That is, it is considered that conventionally, the particles of the skimmed milk, which is water-soluble, surrounds the antigen and the antibody, so that the antibody cannot come close to each other, and as a result, the specific immune reaction is lowered. However, by making skim milk into a dilute aqueous solution and using ultrasonic waves or the like, skim milk in a completely dissolved state can be prepared. Even if an immune reaction is carried out using this skim milk solution, it is considered that skim milk does not inhibit the antigen-antibody reaction because the skim milk is in a completely dissolved state. Moreover, it was confirmed that this concentration sufficiently inhibits the non-specific reaction.

By using the method of the present invention, various antigens can be measured. Examples of such an antigen include α ₂
Plasmin inhibitor (α ₂ PI), α ₂ PI-plasmin complex, coagulation and fibrinolysis system factors such as protein C and protein S, lung-surfactant apoprotein and AFP, CEA
Tumor markers and the like.

The antibodies used when measuring various antigens as described above include polyclonal antibodies against these antigens, monoclonal antibodies, and their fragments such as F (ab ′) ₂ , Fab ′, Fab, Facb and the like. Alternatively, it can be used as a fixed antibody. Of these, when a monoclonal antibody is used as the antibody, highly specific measurement is possible, and when Fab 'is used, higher sensitivity measurement is possible, which is preferable.

Such polyclonal antibodies, monoclonal antibodies, and fragments thereof can be prepared by known methods, for example, ed.
Page, Tokyo Kagaku Dojin, 1986; G.Kohler and Milstein, Nature (Lo
ndon), 256 , 495-497 (1975)); and A. Nisonov et al. Purham's method (A. Nisonoff et al., Arc
h.Biochem.Biophys., 89 , 23 (1960); P.Parham, J.Immuno
l., 131 , 2895 (1983).

Among such monoclonal antibodies, the anti-human protein S monoclonal antibody is, for example, Japanese Patent Application No. 61-296766 (filed Dec. 15, 1986:
The invention is described in detail in the patent application specification (Japanese Patent Laid-Open No. 63-148994) entitled "Monoclonal antibody, hybridoma, method for producing monoclonal antibody and method for separating human protein S" (a) Human・ Protein S
Monoclonal antibody against human protein S that specifically recognizes and binds to the complex of human and the c4b binding protein of human complement system regulatory factor, and (b) selectively recognizes free human protein S and A monoclonal antibody against human protein S, which does not recognize the complex between S and the c4b binding protein of the human complement system regulatory factor, can be mentioned.

Examples of the antigen-antibody reaction include conventionally known immunological measurement methods such as a so-called sandwich method or a two-antibody method using two kinds of antibodies that recognize different antigen determining sites of an antigen. However, the sandwich method is particularly preferable.

Examples Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the technical scope of the present invention is not limited to these Examples. In the following examples, "%" is based on weight unless otherwise specified.

Example 1: Solubility of skim milk and dissolution state retention test Skim milk aqueous solution (2.0, 1.5, 1.0, 0.8, 0.7,
0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.02, 0.01%) was stirred at room temperature with a stirrer bar, and then ultrasonic waves were applied for 10 minutes to dissolve them. Add 0.01% thimerosal to each of these solutions, store it in the refrigerator for 12 months, compare the protein at the start of storage with that of the product stored for 12 months, and use the Bradford protein measurement kit to dissolve at each concentration. The degree of state retention was examined. FIG. 1 shows the dissolution state retention rate (%) at each concentration. In FIG. Was used. As shown in Fig. 1, there is no skimmed milk.
It can be seen that if the content is 8% or less, the solution can be sufficiently and stably stored.

Example _2: α 2 PI- with specific reactivity and nonspecific reactivity of the skim milk concentration dependence skim milk solution study Example 1 of the immunoassay of plasmin complexes, alpha ₂ PI- plasmin complexes 0 , 50,100,200ng / ml immunoassay, non-specific reaction (Ag = 0) and specific reaction (Ag = 200ng / ml)
ml) was compared with the system without skim milk.

The results are shown in Fig. 2, and it was found that when skim milk was smaller than 0.02%, non-specific reaction to immunity increased. It was also confirmed that when the skim milk content exceeds 0.8%, the specific reaction begins to be inhibited, and that the skim milk concentration of 0.02 to 0.8 is optimal for highly sensitive immune reaction in the solution.

Example 3: α ₂ - plasmin inhibitor (alpha ₂ PI) polystyrene balls with a fixed monoclonal antibody against immunoassay human alpha ₂ plasmin-Inhibuta (alpha ₂ PI) (diameter 6 mm), and the monoclonal antibodies A peroxidase-labeled anti-human α ₂ PI-monoclonal antibody having different reaction sites for α ₂ PI was used in 0.01 M phosphate buffered saline (pH 7.4) containing 0.5% BSA and 0.1% skim milk. , Human α ₂ PI concentration 0,200,400,8
After reacting each level of 00 ng / ml for 60 minutes at a temperature of 37 ° C., the ball and the reaction solution were separated, and the pole was washed well with physiological saline. Next, this is reacted in an aqueous solution containing a color forming system of tetramethylbenzidine-H ₂ O ₂ , and then a reaction terminator is added to stop the oxygen reaction,
The absorption intensity at a wavelength of 0 nm was measured, and the concentration and the absorption intensity were plotted to obtain a calibration curve.

This was compared with a calibration curve prepared by reacting in exactly the same manner as above except that 0.1% skim milk was not present. As shown in FIG. It was confirmed that the human α ₂ PI concentration of 0 ng / ml had a lower absorption intensity and less non-specific reaction, and was better than the system without addition.

Example 4: Immunoassay method for protein C Polystyrene pole (6 mm in diameter) to which an anti-human protein C-monoclonal antibody against the Gla region of human protein C is immobilized, and an anti-human protein C-monoclonal having a different reaction site from this monoclonal antibody 0.5% BSA, 0.1% with peroxidase-labeled antibody
In 0.05M Tris-buffered saline (pH7.4) containing skim milk, 5 mM calcium chloride, and 1 U / ml heparin, at 37 ° C, protein C concentrations of 0, 12.5, 25, and 50 ng / ml were obtained. After reacting for 30 minutes, the ball and the reaction solution were separated, and then the pole was thoroughly washed. Next, this was developed with a color-forming system of tetramethylbenzidine-H ₂ O ₂ , and then a reaction terminator was added to stop the oxygen reaction, and 650n
The absorption intensity at the wavelength of m was measured, and the concentration and the absorption intensity were plotted to obtain a calibration curve. When this was compared with a calibration curve which was reacted in exactly the same manner as above except that 0.1% skim milk was not present, as shown in FIG.
It was confirmed that the assay system with the addition of skim milk had a lower absorption intensity at a protein C concentration of 0 ng / ml and less non-specific reaction than the system without the addition of skim milk.

Reference Example 1: Production and purification of anti-human protein S (PS) monoclonal antibody Purified human PS was immunized four times at 14-day intervals to two female Balb / C mice (4 weeks old). The first immunization was dissolved in PBS. 50 μg of human PS was mixed with an equivalent amount of Freund's adjuvant and the emulsion was intraperitoneally administered (0.5 mg / he
ad) In the second and third times, 50 μg of human PS was also used in Freund's incomplete adjuvant (Freund's imcomplete).
adjuvant) and administered intraperitoneally. For the final immunization, 30 μg of human PS was additionally administered as a PBS solution through the tail vein of mice. Pancreatic cells of the immunized mouse were used for cell fusion 3 days after the final immunization.

Pancreatic cells of immunized mouse and myeloma cells of syngeneic mouse (P3U1) are mixed at a ratio of about 2: 1 to about 15: 1, and 50% polyethylene glycol 1540 (Wako Pure Chemical Industries, Ltd.) is used as a fusion accelerator. As a result, cell fusion was performed according to the method of Kohler and Milstein. After fusion, the cells were suspended in 10% FCS-RPMI-1640 medium at a cell concentration of 1 × 10 ⁶ cells / ml and 96wel.
100μ per well in ls microplate (Coster)
1 was dispensed.

Fused cells are CO ₂ incubator (5% CO ₂ , 37 ℃)
The cells were cultured in a medium, the medium was exchanged with a medium containing hypoxanthine, aminopterin and thymidine (HAT medium), the medium was grown in HAT medium, and a hybridoma composed of pancreatic cells and myeloma cells was screened.

The antibody in the culture supernatant of the hybridoma is ELIS using a microtiter plate coated with the antigen human PS.
It was detected by method A. As the second antibody, a rabbit anti-mouse IgG antibody labeled with alkaline phosphatase was used, and the binding property to the antigen PS was examined. Of the 494 wells in which the fused cells were seeded, formation of colonies was observed in 487 wells,
Among these, 94 wells were positive for antibody production that showed binding to the antigen PS.

Cloning by the limiting dilution method was repeated twice for 4 of these antibody production positive wells to obtain 6 clones. 90 clones were obtained.
Suspended in% FCS-10% DMSO and stored in liquid nitrogen.

The monoclonal antibody produced by each clone was grown in the abdominal cavity of the Balb / C mouse and purified from the ascites using a protein A-Sepharose 4B column.

Reference Example 2: Properties of purified monoclonal antibody IgG of each clone purified from mouse ascites was examined for class and human protein S-binding properties.

The class of mouse monoclonal antibody was determined by the Ouchterlony method using anti-mouse antiserum of each class specificity.

 The results are shown in Table 2 below.

Binding to human protein S is detected with goat anti-mouse IgG labeled with alkaline phosphatase by reacting human protein S immobilized on a microtiter plate with a monoclonal antibody diluted to an appropriate concentration. It was evaluated by

As a result, the binding strength of the 6 types of monoclonal antibodies to human protein S was 2B9F122B9C10> 3C3G8.
>3C4G4> 2B9G3 >> 2E12 C7 was found.

Reference Example 3: Reactivity to human C4bp and protein S complex 10 μg / ml of the purified 6 types of monoclonal antibodies
Coat microtiter plate at the concentration of 1
After Bocking with% BSA, human normal human plasma diluted to an appropriate concentration was added, and the C4bp-protein S complex in plasma was reacted with the monoclonal antibody. Next, add an alkaline phosphatase-labeled anti-C4bp antibody,
The binding properties of various types of monoclonal antibodies to the C4bp-protein S complex were detected and examined.

As a result, the monoclonal antibody 2E12C7 showed very weak binding to free protein S, but highly specific binding to C4bp-protein S complex. Binding strength to C4bp-protein S complex of 2E12C7 >> 2B9F102B
It was found that 9C12>3C3G8>3C4G4> 2B9G3.

As shown in Reference Examples 2 and 3, the complex of C4bp and protein S was not recognized and free human protein S was not recognized.
2B9F12 and 2B9C10 were obtained as monoclonal antibodies capable of specifically recognizing and binding to.

Example 5 (1) Preparation of antibody-immobilized beads Polystyrene beads (diameter 6 mm) were used as goat anti-human
Protein S antibody (polyclonal antibody; American Diag
nostica) 0.01 M pH 7.4 with a concentration of 20 μg / ml
1 in phosphate buffered saline (PBS) solution at a temperature of 4 ° C
After leaving it for 24 hours, it was washed with PBS, and then left for 1 day in a 0.5% bovine serum albumin (BSA) aqueous solution at a temperature of 4 ° C. for post-coating treatment to obtain antibody-immobilized beads.

(2) Preparation of horseradish peroxidase-labeled monoclonal antibody 1.0 ml of 1.0 mg / ml PBS solution of free monoclonal antibody (2B9F12) that specifically recognizes human protein S 1.0 ml
50 μl of a 10 mg / ml dimethylformamide solution of N- (m-maleimidobenzoic acid) -N-succinimide ester (MBS) was added to the mixture, and the mixture was reacted at a temperature of 25 ° C. for 30 minutes, and then Sephadex G-25. Using a column packed with.
Gel filtration was performed with 1 M phosphate buffer (pH 6.0) to separate maleimidated monoclonal antibody and unreacted MBS.

Meanwhile, horseradish peroxidase (HR
P) in 2.0 ml of 1.0 mg / ml PBS solution, N-succinimidyl-3- (2-pyridylthio) propionate (SPDP)
Of 10 mg / ml of ethanol was added and reacted at 25 ° C. for 30 minutes, and then purified by gel filtration with 0.01 M acetate buffer (pH 4.5) using a column packed with Sephadex G-25.
Fractions containing pyridyl disulfided HRP were collected and concentrated approximately 10 times under ice cooling in a collodion bag. Next, 1 ml of 0.1 M acetate buffer (pH 4.5) containing 0.85% NaCl and 0.1 M dithiothreitol was added thereto, and the mixture was added at 25 ° C.
After stirring for 30 minutes to reduce the pyridyl disulfide group introduced into the HRP molecule, gel filtration with 0.1M phosphate buffer (pH 6.0) using Sephadex G-25 column to contain thiolated HRP To obtain a fraction.

Next, the obtained maleimidated monoclonal antibody and thiolated HRP were mixed, concentrated to a protein concentration of 4 mg / ml under ice-cooling using a collodion bag, and allowed to stand at 4 ° C for 1 day, then Ultrogel AcA44 ( An HRP-labeled monoclonal antibody was obtained by gel filtration with PBS using a column packed with LKB (France).

(3) Measurement of human protein S 0, 50, 100, 200, 400 purified beads of goat anti-human protein S antibody immobilized on each 1 bead and purified human protein S
200 μl of 0.1% BSA and 0.1% skim milk-containing PBS solution (pH 7.4) containing each concentration of ng / ml and 0.1% BSA and 0.1% skim milk-containing PBS solution (pH 7.4) containing HRP-labeled monoclonal antibody 200 μl And were added to each test tube (n = 2) and incubated at a temperature of 37 ° C. for 1 hour.

Next, after the solution in the test tube was removed by suction, the tube was washed twice with PBS, and then 0.1 M phosphate-citrate buffer solution (pH 4.4) containing 0.02% tetramethylbenzidine hydrochloride and 0.005% hydrogen peroxide was added. 0) 400 μl each was added to each test tube and incubated at 37 ° C for 30 minutes.
1 ml of an aqueous solution containing 0.1% NaF and 2% acetic acid was added to each test tube to stop the enzymatic reaction.

Then, the absorption intensity of this solution at a wavelength of 650 nm was measured using a spectrophotometer, and this was plotted with the concentration of human protein S to obtain a calibration curve for measuring concentration of human protein S, which has concentration dependence. Obtained (see FIG. 5).

For measuring the concentration of human protein S in plasma samples,
Normal mixed human plasma containing 0.1% BSA and 0.1% skim milk P
200 μl of solution diluted 50 times with BS solution (pH 7.4), 200 μl of antibody-immobilized beads and HRP-labeled monoclonal antibody solution
Then, the sample was added to a test tube, and an immunoreaction and a color reaction were carried out in the same manner as in the method of preparing a calibration curve, and then the absorptivity was measured with a spectrophotometer. The value of human protein S was calculated by converting this value into the plasma concentration using a calibration curve.
The plasma concentration was 10.4 μg / ml.

Comparative Example 1 One goat anti-human protein S antibody-immobilized bead prepared in the same manner as in Example 5 and purified human
200 μl of 0.5% BSA-containing PBS solution (pH 7.4) containing protein S at concentrations of 0, 50, 100, 200, 400 ng / ml and HRP-labeled mouse anti-human protein S-monoclonal prepared in the same manner as in Example 5 0.5% B containing antibody
200 μl of SA-containing PBS solution (pH 7.4) and each test tube (n =
2) and incubated for 1 hour at a temperature of 37 ° C.

Next, after removing the solution in each test tube by suction, wash each with PBS twice and then add tetramethylbenzidine hydrochloride 0.02
% And 0.005% hydrogen peroxide in 0.1 M phosphate-citrate buffer (pH 4.0) 400 μl at a time, added to each tube 37
After incubating at a temperature of ° C for 30 minutes, 1 ml of an aqueous solution containing 0.1% NaF and 2% acetic acid as a reaction terminator was added to each test tube to stop the enzymatic reaction.

Then, the absorption intensity of this solution at a wavelength of 650 nm was measured using a spectrophotometer, and this was plotted against the concentration of human protein S to prepare a calibration curve (No. 6).
See figure).

This calibration curve has a baseline absorption intensity due to non-specific reactions.
The sensitivity was very high at 0.28 and good sensitivity was not obtained, which was insufficient as a measurement system.

Comparative Example 2 One goat anti-human protein S antibody-immobilized bead prepared in the same manner as in Example 5 and purified human
PBS solution containing 0.5% BSA and 2% skim milk containing protein S at concentrations of 0, 50, 100, 200 and 400 ng / ml (pH 7.
4) 200 μl, prepared in the same manner as in Example 5
PBS containing 0.5% BSA and 2% skim milk containing HRP-labeled mouse anti-human protein S-monoclonal antibody
200 μl of the solution (pH 7.4) was added to each test tube (n = 2) and incubated at a temperature of 37 ° C. for 1 hour.

Next, after carrying out a color reaction and a reaction termination in the same manner as in Example 5, the absorbance of this solution at a wavelength of 650 nm was measured using a spectrophotometer, and this was plotted with the human protein S concentration. Thus, a good calibration curve having the same concentration dependence as in Example 5 was obtained.

However, an immune reaction buffer consisting of 0.5% BSA and 2% skim milk-containing PBS solution (pH 7.4) and a 0.5% BSA containing 2% skim milk-containing PBS solution (pH 7.4) containing HRP-labeled monoclonal antibody were sterilized. Fill the vial with
When stored in the refrigerator for 1 month, skim milk in these solutions formed aggregates.

When this aggregate was separated and subjected to immunological measurement, a concentration-dependent calibration curve could no longer be obtained, and measurement was impossible.

Example 6: Skim milk addition test in human / placenta-derived acid glutathione S-transferase assay system Human / placenta-derived acid was measured with an immunoassay buffer (1% bovine serum albumin 0.01 M phosphate 0.85% NaCl buffer pH 7.2). Glutathione S-transferase (hereinafter referred to as human / placenta-derived acidic GST) solutions of 0, 25 and 50 µg / ml were prepared, and 100 µl of the solution was added to the above-mentioned buffer to give final concentrations of 0,0.05,0.1,0.2.
100 μl of skim milk-containing solution adjusted to
And the same buffer 200 containing an anti-human placenta-derived GST monoclonal antibody labeled with horseradish peroxidase
μl was placed in a test tube and mixed well. To each test tube, one bead on which the rabbit anti-human placenta-derived acidic GST polyclonal antibody was immobilized was placed in each test tube and reacted at a temperature of 37 ° C. for 2 hours. Next, after removing the solution in the test tube by suction,
After washing 3 times with 2 ml of physiological saline, 0.1M phosphate-citrate buffer solution (pH 4.3) containing 0.03% of 3,3 ′, 5,5′tetramethylbenzidine hydrochloride and 2.5 mM of H ₂ O ₂ (pH 4. 0) was added to each test tube in an amount of 0.4 ml and reacted at a temperature of 37 ° C for 30 minutes. Then, 1 ml of a 1N sulfuric acid aqueous solution was added as a reaction terminator to stop the oxygen reaction.

Next, this solution was measured for absorption intensity at 450 nm using purified water as a control using a spectrophotometer, and the obtained results are shown in FIG.

Example 7: Skim milk addition test in chondrocalcin assay system Buffer for immunoassay (0.01% containing 1% bovine serum albumin
Bovine chondrocalcin was diluted with phosphoric acid and 0.85% NaCl buffer pH = 7.2 and thereafter abbreviated as 1% BSA-PBS to 0.2 ng /
Make a ml solution, and add 50 μl of it to the same buffer as above to give a final concentration of 0.0.
00625,0.0125,0.025,0.05,0.1,0.2,0.4 and 0.8% of skimmed milk-containing solution adjusted to 50 μl and mixed well, and then added to a microplate on which rabbit anti-bovine chondrocalcin polyclonal antibody was immobilized. 100 μl
Was charged and reacted at 37 ° C. for 2 hours (primary reaction). After washing with PBS-0.05% Tween-20, Fa of peroxidase-labeled anti-bovine chondrocalcin polyclonal antibody was used.
b ′ was diluted with an immunoassay buffer, 100 μl was added to each well, and the mixture was reacted at 37 ° C. for 1 hour (secondary reaction) PBS-0.05% Twe.
After washing with en-20, substrate solution for peroxidase (2.5 mM H ₂
100 μl of O ₂ , 0.0225% 3,3 ′, 5,5′-tetramethylbenzidine) was added, color was developed for 0.5 hours at 37 ° C., 25 μl of 1N sulfuric acid solution was added to stop the reaction, and the plate was read at 450 nm. The absorption intensity was measured at. The result is No. 8
As shown in the figure. As is clear from this figure, non-specific adsorption can be prevented at a skim milk concentration of 0.00625% or more.

Example 8: Skim milk addition test in IgA-IC (IgA type immune complex) measuring system Immunoassay buffer (0.5% bovine serum albumin, 0.01 M phosphate, 0.85% NaCl buffer pH 7.2, 0.5% BSA thereafter) at abbreviated as -PBS), standard substance (IgA-C ₃₎ 1,25 and 50 [mu] g / m
l was diluted 100-fold and 0.2 ml was collected in a test tube. next
0.2 ml of a skim milk-containing solution adjusted to a final concentration of 0, 0.05, 0.1, 0.2% with 0.5% BSA-PBS was added and mixed well. Rabbit anti-human C ₃ Facb-immobilized beads were put therein one by one and reacted at 37 ° C. for 1 hour (primary reaction). Then, the solution in the test tube was removed by suction, followed by washing 3 times with 2 μl of physiological saline (hereinafter referred to as saline), and then a stock solution of horseradish peroxidase labeled goat anti-human IgA antibody (manufactured by Kappel). The skim milk 0,0.05,0.1
And 0.5% BSA-PBS solution containing 0.2% each 10,000
A double dilution was added to each of the tubes having the same skim milk content in the primary reaction in an amount of 0.4 ml and reacted at 37 ° C. for 1 hour (secondary reaction). Then, the solution in the test tube was removed by suction, washed with 2 ml of saline three times, and then 2,2'-azinobis- (3-ethyl-6-benzthiazolinesulfonic acid) diammonium salt (ABTS) 0.05%, 0.1M phosphoric acid-citrate buffer (pH 4.5) containing 0.4mM of hydrogen peroxide was added to each test tube at 0.4ml and reacted at 37 ° C for 30 minutes, then 1ml of 0.1M oxalic acid aqueous solution as a reaction terminator. Was added to stop the enzymatic reaction.

The solution is then spectrophotometer at 420 nm.
The absorption intensity at the reagent blank (0.
5% BSA-PBS was used as a control, and the results are shown in FIG.

Example 9: Effect of addition of various proteins on non-specific reaction (N) / specific reaction (S) ratio in immune reaction Polystyrene balls (diameter 6 mm) immobilized with polyclonal antibody against human plasminogen and anti-human α ₂ PI-monoclonal antibody with peroxidase-labeled product, urease, gelatin, bovine serum albumin, egg albumin, pepsin, α-casein, β-casein, casein, skim milk, PFC (partial FC of IgG),
NZ Case (pepsin degradation product of casein), gelatin hydrolyzate enzymatic acid (Hydrolysat
e Enzymatic-Acid), orosomucoid, ovoglycoprotein in 0.01M phosphate buffered saline (pH7.4) containing 0.25% of various proteins, each of human α ₂ PI-plasmin complex concentration 0,100 ng / ml About the level, 37 ℃
After the reaction was performed at the temperature of 60 minutes, the ball and the reaction solution were separated, and the ball was thoroughly washed with physiological saline. Next, this was reacted in an aqueous solution containing a color forming system of tetramethylbenzidine-H ₂ O ₂ , and then a reaction terminator was added to stop the oxygen reaction, and the absorption intensity at a wavelength of 650 nm was measured.

The non-specific reaction rate was calculated using the following formula, and the correlation with the molecular weight or the isoelectric point is shown in FIG. 10 and FIG. 11, respectively.

From Fig. 10, the molecular weight is 16,000-50,000, and from Fig. 11, the isoelectric point is 1.
It is clear that the addition of proteins in the range 0-5.0 leads to a significant reduction in non-specific reactions. From Fig. 11, the isoelectric point
It can be seen that the addition of 1.0 to 5.0 protein has a good effect of reducing non-specific reactions.

Example 10: Effect of added concentration of orosomucoid and pepsin on immune response Using polystyrene balls on which a polyclonal antibody against human plasminogen was immobilized and peroxidase-labeled anti-human α ₂ PI monoclonal antibody, various types of orosomucoid and pepsin were used. Concentration of human α ₂ PI-plasmin complex in 0.1 M phosphate buffer (pH 7.2) containing 0, 50, 100 ng / ml of human α ₂ PI at 6 ° C at a temperature of 37 ° C.
After reacting for 0 minutes, the ball and the reaction solution were separated, and the ball was thoroughly washed with physiological saline. Next, this was reacted in an aqueous solution containing a tetramethylbenzidine-H ₂ O ₂ color-developing system, then a reaction terminator was added to stop the enzymatic reaction, and the absorption intensity at a wavelength of 650 nm was measured.

The results are shown in Fig. 12 (orosomucoid) and Fig. 13 (pepsin), respectively. From FIGS. 12 and 13, it is clear that at low protein concentrations, non-specific reactions significantly increase, and at high protein concentrations, specific reactions decrease.

Example 11: Skim milk addition test in lung-surfactant-apoprotein assay system Human lung surface apoprotein (apoprotein) was tested using an immunoassay buffer (1% bovine serum albumin 0.01M phosphate 0.85% NaCl buffer pH 7.2). (LSP), 0,50 ng / ml solution was prepared and
1 and same as above, but with final concentration of 0,0.1,0.2,0.3,0.4,0.5,0.
100 μm of skim milk-containing solution adjusted to 6,0.7%
l, and horseradish peroxidase-labeled anti-human
200 μl of the same buffer containing LSP monoclonal antibody was placed in a test tube and mixed well. One bead on which the anti-LSP monoclonal antibody was immobilized was placed in each test tube and reacted at a temperature of 45 ° C for 30 minutes. Next, the solution in the test tube was removed by suction, and then washed with 2 ml of physiological saline three times, and then 3,3 ', 5,5' tetramethylbenzidine hydrochloride 0.02
%, 0.1 M phosphate-citrate buffer (pH 4.0) containing H ₂ O ₂ 2.5 mM, 0.4 ml, was added to each test tube at the temperature of 45 ° C.
After reacting for 15 minutes, 1 ml of a 1N sulfuric acid aqueous solution was added as a reaction terminator to stop the oxygen reaction.

Next, the absorption intensity (OD) at 450 nm of this solution was measured using a spectrophotometer with purified water as a control, and the obtained results are shown in FIG.

Continuation of front page (56) Reference JP-A-61-275654 (JP, A) Journal of Clinical Microbiology, 25 (3) P. 509-515, (1987) Padiatric Research, 19 (10), P.P. 1017-1020, (1985) Journal of Biochemical & Biophysical Methods, 12, P. 271-279 (1986)

Claims (10)

(57) [Claims] 1. When performing an immunoassay utilizing an antigen-antibody reaction in a solution, the immune reaction solution has an average molecular weight of
A solution containing a protein having a isoelectric point of 1.0 to 5.0 and having an isoelectric point of 1.0 to 5.0 or a mixture containing the protein in a completely dissolved state is allowed to exist as an antigen-antibody reaction modifier, and the final solution of the antigen-antibody reaction modifier in the immune reaction solution. An immunoassay method comprising adjusting the concentration to 0.02 to 0.9% by weight. 2. The method according to claim 1, wherein the antigen-antibody reaction regulator is at least one protein selected from the group of casein and orosomucoid. 3. The method according to claim 1, wherein the antigen-antibody reaction regulator contains 10 to 60% by weight of protein and 30 to 80% by weight of sugar as main components. 4. The method according to claim 3, wherein the antigen-antibody reaction regulator is skim milk. 5. The subject to be measured is α ₂ plasmin inhibitor, α ₂ plasmin inhibitor-plasmin complex, protein C, protein S, human placenta-derived acid glutathione S-transferase, chondrocalcin, IgA type immune complex, The method according to claim 1, which is lung-surfactant apoprotein, AFP or CEA. 6. An average molecular weight of 16-50,000 and an isoelectric point of 1.0-
The final concentration in the immunoreaction solution of the antigen-antibody reaction modifier is 0.02 ~ as a solution containing the protein of 5.0 or a mixture containing it in a completely dissolved state as an antigen-antibody reaction modifier.
A reagent kit used for immunoassay, which contains 0.9% by weight as a part of its components. 7. The reagent kit according to claim 1, wherein the antigen-antibody reaction regulator is at least one protein selected from the group of casein and orosomucoid. 8. The reagent kit according to claim 1, wherein the antigen-antibody reaction regulator contains 10 to 60% by weight of protein and 30 to 80% by weight of sugar as main components. 9. The reagent kit according to claim 8, wherein the antigen-antibody reaction regulator is skim milk. 10. The subject to be measured is α ₂ plasmin inhibitor, α ₂ plasmin inhibitor-plasmin complex, protein C, protein S, human placenta-derived acid glutathione S-transferase, chondrocalcin, IgA type immune complex, The reagent kit according to claim 6, which is lung-surfactant apoprotein, AFP or CEA.