JP3174402B2 - Competitive immunoassay and assay kit - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay and an immunoassay reagent. More specifically, the present invention relates to an immunoassay method and an immunoassay kit by a heterogeneous competition method.

[0002]

2. Description of the Related Art Conventionally, in a heterogeneous competitive immunoassay, an antibody or an antigen against a substance to be measured immobilized on a water-insoluble carrier is labeled with an enzyme, a fluorescent substance, a luminescent substance, or a radioisotope. Competition reaction between the test substance or the derivative of the test substance and the test substance in the sample causes an immune complex to be formed on the water-insoluble carrier, and the amount of the labeling substance in the immune complex is measured. Has been implemented.

As a method improved from the conventional method, Japanese Patent Laid-Open No.
No. 23251 discloses that a first receptor capable of binding to an antigen, a labeled third receptor and a second receptor capable of binding to the first receptor present in a liquid layer are used as an immobilized antibody. There is a description of an immunoassay method and a measurement reagent kit characterized by providing a solid-phased antibody corresponding to every measurement item.

[Problems to be solved by the present invention]

[0004] However, Japanese Patent Application Laid-Open No. 59-2325 describes the above.
No. 1 is a method limited to the sandwich method,
Haptens such as thyroid hormones such as T3 and T4; steroid hormones such as cortisol and estradiol; drugs such as digoxin and digitoxin cannot be applied to all items measured immunochemically in the laboratory. It cannot be measured by the above method.

In this method, a method using an antibody specific to the Fc portion of the first receptor as the second receptor is exemplified. In this case, the second receptor and the first receptor are respectively It is necessary to use antibodies obtained from different animal species.

In recent years, in the field of immunoassay, monoclonal antibodies have been widely used due to their high specificity and the ability to always obtain antibodies with uniform performance. Normally, monoclonal antibodies are very commonly derived from mice.
In the method as described in Japanese Patent Application Laid-Open No. H10-260, when a monoclonal antibody is used as the first receptor, it is usually difficult to use a monoclonal antibody obtained from a mouse as the second receptor. An antibody produced by an animal other than a mouse, that is, a polyclonal antibody had to be used as the receptor.

However, polyclonal antibodies have insufficient titer and specificity, and when polyclonal antibodies are used as reagents for immunoassay, problems such as reduced measurement sensitivity and reproducibility have occurred.

[0008]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have reached the present invention. That is, the present invention relates to a substance to be measured (A) in a sample,
The first monoclonal antibody (A) against (A), the labeled substance to be measured or a derivative of the substance to be measured (C) and immobilized on a water-insoluble carrier, and attached to the Fc portion of the first monoclonal antibody React specifically and
The amount of (A) in the sample is measured by measuring the amount of the labeling substance in the immune complex formed by reacting the second monoclonal antibody (D) obtained from the same animal species as (B). The present invention relates to an immunoassay method and an immunoassay kit comprising the above (B), (C) and (D).

In the present invention, it has become possible to use a monoclonal antibody specific to the Fc portion of the monoclonal antibody against the substance to be measured as the solid-phased antibody. Problems did not occur, and a stable performance immunoassay kit could be supplied at all times.

[0010] In the present invention, monoclonal antibodies obtained from mice, rats, etc. may be mentioned, but it is preferable to prepare a monoclonal antibody having low nonspecific adsorption of antibodies and having a strong affinity for antigenic substances. It is obtained from a mouse that can. Monoclonal antibodies can be obtained by the method of Keller et al. (Nature, Vol. 256, 495).
, 1975).

In the present invention, the substances to be measured (A) include thyroid hormones such as T3 and T4;
Steroid hormones such as estradiol and progesterone; and hapten antigens represented by drugs such as digoxin, digitoxin, theophylline, and diphenylhydantoin. In addition, AFP, CEA, C
Tumor markers such as A19-9; TSH, LH, FS
H, hormones such as insulin; proteins such as CRP and β2-microglobulin; infectious substances such as hepatitis antigens;
Substances that are usually measured by the sandwich method, such as antibodies such as hepatitis antibodies and IgE, can also be measured by the method of the present invention.

In the present invention, it is necessary to use a whole antibody containing an Fc portion as the first monoclonal antibody (B).

Further, (B) includes IgG, IgM,
Antibodies of each class of IgA, IgD and IgE can be used. Of these, Ig is preferred.
G, IgM and IgA classes.

In the present invention, an enzyme, a fluorescent substance, a luminescent substance or a radioisotope is suitably used as a substance for labeling a substance to be measured or a derivative (C) of the substance to be measured.

As the labeling substance, any of the substances already known in immunoassays can be used. Enzymes include horseradish oxidase, alkaline phosphatase, glucose oxidase, β-D
Fluorescein and europium derivatives as fluorescent substances; acridinium ester and N-aminobutyl-N-ethylisoluminol (ABEI) as luminescent substances; ¹²⁵ I and ¹³¹ as radioisotopes I.

As the labeling method, any of the above types can be prepared by a conventionally known labeling method.
For example, as a method of labeling with an enzyme, the method of Ishikawa et al. (Analytical Letters, vol. 15, p. 147, 1
982); As a method for labeling a fluorescent substance, a method such as Hemira (Analytical Biochemistry, No. 1)
37, 335 (1984); Woodhead et al. (Clinical Chemistry, Vol. 29, p. 1480, 1983) as a method for labeling a luminescent substance;
Examples of a method for labeling a radioisotope include a method using a hunter and the like (Nature, 194, 495, 1962).

In the present invention, the second monoclonal antibody (D) is obtained from the same animal species as the first monoclonal antibody (B). (D) is F
The whole antibody including the c portion may be used, or the F (ab ′) ₂ , Fab fragment, or F (ab ′) ₂ fragment obtained by decomposing the whole antibody with an enzyme such as pepsin or papain may be used as a mercapto antibody. Fa obtained by reduction with a reducing agent such as ethylamine
It may be a b 'fragment.

Further, (D) shows that when (B) is IgG,
An IgG-derived Fc portion; if (B) is IgM,
An Fc portion derived from M; if (B) is IgA,
It can be obtained by immunizing the Fc portion corresponding to the class and subclass of (B), such as the derived Fc portion. Further, in order to obtain a more specific antibody, polyclonal mouse IgG, IgM, I
A more specific monoclonal antibody can be obtained by immunizing a monoclonal mouse IgG, IgM, or IgA-derived Fc portion than by immunizing a gA-derived Fc portion.

In the present invention, as the water-insoluble carrier for immobilizing (D), any known carrier such as glass beads, plastic beads, plastic test tubes, glass filters, and microparticles can be used.

The method for binding the above (D) to the water-insoluble carrier may be the same as the method described in JP-A-2-205774. That is, a method of chemically bonding glass and (D) (for example, US Pat. No. 4,280,992)
And the method of physically adsorbing an antibody to plastic (for example,
E. Engbal et al .; Biokim Biofizs Acta, Vol. 251, 427, 1971).

In the present invention, the immune complex is (B)
A sample containing (C), (D), and (A) can be formed on a water-insoluble carrier by reacting the sample with the following three methods [Method 1] to [Method 3].
That is, (Method 1) is a method in which (B), (C), (D) and (A) are simultaneously reacted; [Method 2] is that (B), (C) and (A) are reacted, and And (B) as [Method 3].
(D) and (A) are reacted, unreacted substances are removed by B / F separation, and then reacted with (C). can do.

[0022] The amount of the labeling substance in the immune complex can be measured by any known method. When the label is an enzyme, its enzymatic activity can be measured by a colorimetric method using a spectrophotometer (Engbal et al., J. Immunol, 109
Vol. 129, 1972); Fluorescence method using a fluorometer (Watanabe et al., Clinical Chemistry, 25, 80)
1979); Luminescence method using a luminometer (Solpe et al., Mesos in Enzymology, 133)
Vol. 331, 1986). When the label is a fluorescent substance, a method such as Hemira (Analytical Biochemistry, Vol. 137,
335, 1984) using a fluorimeter. When the label is a luminescent substance, a method such as Weeks (Clinical Chemistry, vol. 29,
1474, 1983) using a luminometer, and when the label is a radioisotope, a scintillation counter by a method such as Espinosa (Clinical Chemistry, 33, 1439, 1987). Can be measured. The amount of the labeling substance in the immune complex can be measured by any known method.

The present invention also relates to an immunoassay kit comprising the above-mentioned first and second types of monoclonal antibodies, particularly to a competitive immunoassay kit.

The immunoassay kit of the present invention may contain various adjuvants in addition to the above-mentioned two kinds of antibodies, which are essential components, in order to facilitate the use of the immunoassay kit. For example, various buffers or buffers used to avoid the influence of the pH of the sample on the immune reaction; various buffers or agents for efficiently proceeding the immune reaction and avoiding inhibition of various serum components to the immune reaction. A protein component or a sugar component; a surfactant used to prevent nonspecific adsorption in an immunoassay; a preservative for storage stability of the kit, and the like can be included in any combination.

[0025]

The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

EXAMPLE Measurement of T4

(1) Preparation of Monoclonal Antibody for Solid Phase A BALB / c mouse was immunized by intraperitoneally administering 100 μg of the Fc portion of the anti-AFP monoclonal antibody together with Freund's complete adjuvant. Anti-AFP again after 2 months
100 μg of the Fc portion of the monoclonal antibody was intravenously administered, and three days later, the spleen was removed to collect splenocytes. RP
After washing with MI 1640 medium, the total amount of spleen cells was reduced to 2 ×
10 ⁷ mouse myeloma cells (P3-NS1 / 1-
Ag4.1) and fused for 1 minute at 37 ° C. in 1 ml of RPMI 1640 medium containing 42.5% polyethylene glycol 1540 and 7.5% dimethyl sulfoxide. One minute later, the cell suspension was washed with RPMI 1640.
Diluted gradually with 5 ml of medium. After centrifuging and washing the cells, HAT medium (hypocysanthin, aminopterin, thymidine, RPMI containing 10% fetal bovine serum) was used.
(1640 medium) was added to a volume of 20 ml, 0.2 ml was dispensed into a 96-well microplate, and the cells were cultured for 2 weeks. The antibody activity of the culture supernatant in the grown wells was measured.

Next, the cells in the wells in which the activity was observed were repeatedly cloned by the limiting dilution method, and the anti-mouse Fc was cloned.
Cells producing monoclonal antibodies were obtained. The cells were cultured in a serum-free culture medium, and a culture supernatant was collected. The monoclonal antibody in this culture solution was purified and isolated using Affi-Gel Protein A MAPS kit (manufactured by Bio-Rad) and used in the following studies.

(2) Binding of Antibody to Solid Phase The anti-mouse Fc monoclonal antibody obtained above was dissolved in a carbonate buffer (0.02 M, pH 9.5) to a concentration of 100 μg / ml. The monoclonal antibody-containing carbonate buffer (500 μl each) was dispensed into a Nunc polystyrene test tube (12 × 75 mm), and reacted at 4 ° C. for 48 hours to immobilize the antibody on the inner wall of the test tube. After the reaction was completed, the liquid in the test tube was removed using an aspirator. The test tube is washed three times with 1 ml of physiological saline, and 1 ml of a phosphate buffer (0.02 M, pH 7.2) containing 1% bovine serum albumin (hereinafter abbreviated as BSA) is added. saved.

(3) Horseradish peroxidase-labeled anti-T
Preparation of 4 T4 (manufactured by Calbiochemistry) and horseradish peroxidase (manufactured by Toyobo) were subjected to a method such as Abrames (Immunochemistry, Vol. 6, p. 43, 1969).
To obtain enzyme-labeled T4.

(4) Preparation of Luminescent Reagent 0.18 g of Luminol (Tokyo Kasei), 0.10 g of p-iodophenol (Nacalai Tesque) and 30%
500 μl of a hydrogen peroxide solution was dissolved in 1 liter of a 0.1 M, pH 8.0 Tris / HCl buffer solution, and this was used as a stock solution.

(5) Measurement of T4 in Blood Preparation of Primary Antibody Solution Anti-T4 monoclonal antibody (Boehringer Mannheim) was added to 1% BSA-containing phosphate buffer (0.02 M, p
H7.2) and diluted to 100 μg / ml.

Preparation of Enzyme-Labeled T4 Solution The peroxidase-labeled T4 obtained in (3) was treated with 0.2% BS.
A, 50% with 0.04% ANS (ammonium 8-anilino-1-naphthalenesulfonate, manufactured by Nacalai Tesque) in a barbital buffer (0.02 M, pH 8.6).
Diluted to 00 medium.

Preparation of Standard Solution Standard Solution The standard solution was prepared by the method of Miyai et al. (Endocrinol Japan,
Vol. 27, p. 375, 1980), T4 (manufactured by Calbiochemistry) was added to T3 and T4 free serum obtained by treating human serum with activated carbon, and 2.0, 6.0, 12 0.02, 24.0 μg / dl.

Measurement operation method (a) Measurement by [Method 1] The immersion liquid in the antibody binding test tube prepared in (2) was removed using an aspirator. The test tube was washed once with 1 ml of physiological saline. Sample 20 μl of the standard solution or sample (human pool serum) into the washed test tube,
(5) First monoclonal antibody solution 25 prepared in
0 μl and 250 ml of enzyme-labeled T4 solution prepared in (5)
μl was added, mixed well, and incubated at 37 ° C. for 30 minutes. After completion of the reaction, the reaction solution was removed using an aspirator, and 1 ml of physiological saline was added, and the same operation was performed again. This operation was repeated three times to remove unreacted substances.

The test tube after washing was set in a sample holder of a luminescence reader BLR-201 type manufactured by Aloka, and 500 μl of the luminescent reagent prepared in (4) was added to perform a chemiluminescence reaction. The measured value was obtained as an integral value for 10 seconds.

(B) Measurement by [Method 2] 20 μl of the standard solution or sample (human pool serum) was sampled in a test tube different from that prepared in (2), and (5)
250 μl of the first monoclonal antibody solution prepared
1 and 250 μl of the enzyme-labeled T4 solution prepared in (5)
Was added, and the mixture was stirred well and incubated at 37 ° C. for 30 minutes.

The immersion liquid in the antibody binding test tube prepared in (2) was removed using an aspirator. 1 ml of physiological saline
The inside of the test tube was washed once using. 500 μl of the above reaction solution was added to the washed test tube, and further reacted at 37 ° C. for 30 minutes. After completion of the reaction, the reaction solution was removed using an aspirator, and 1 ml of physiological saline was added, and the same operation was performed again. This operation was repeated three times to remove unreacted substances.

The test tube after washing was set in a sample holder of a luminescence reader BLR-201 manufactured by Aloka, and a chemiluminescence reaction was performed in the same manner as in [Method 1]. The measured value was obtained as an integral value for 10 seconds.

(C) Measurement by [Method 3] The immersion liquid in the antibody binding test tube prepared in (2) was removed using an aspirator. The test tube was washed once with 1 ml of physiological saline. Sample 20 μl of the standard solution or sample (human pool serum) into the washed test tube,
Further, 250 μl of the first monoclonal antibody solution prepared in (5) was added, and the mixture was stirred well and incubated at 37 ° C. for 30 minutes. After completion of the reaction, the reaction solution was removed using an aspirator, and 1 ml of physiological saline was added, and the same operation was performed again. This operation was repeated three times to remove unreacted substances.

To the test tube having been washed previously, add 500 μl of the enzyme-labeled T4 solution prepared in (5), and mix well.
Incubated at 7 ° C. for 30 minutes. After completion of the reaction, the reaction solution was removed using an aspirator, and 1 ml of physiological saline was added.
And the operation was repeated in the same manner. This operation was repeated three times to remove unreacted substances.

The test tube after the completion of the washing was set in a sample holder of a luminescence reader BLR-201 manufactured by Aloka Corporation, and a chemiluminescence reaction was carried out in the same manner as in [Method 1]. The measured value was obtained as an integral value for 10 seconds.

The amount of luminescence in each standard solution in [Method 1] to [Method 3] and the measured value of human pooled serum read from a calibration curve obtained by plotting the amount of luminescence on a graph (the same sample was n = 10 ) And the average of the measured values, standard deviation, C
V is shown in Table 1.

[0044]

[Table 1]

Comparative Example Measurement of T4

For comparison, an example of measurement using an anti-mouse Fc polyclonal antibody as a solid phase is shown below.

(1) Binding of Antibody to Solid Phase Anti-mouse Fc was added to carbonate buffer (0.02 M, pH 9.5).
100 µg / m of polyclonal antibody (Kappel)
1 was dissolved. The monoclonal antibody-containing carbonate buffer (500 μl each) was dispensed into a Nunc polystyrene test tube (12 × 75 mm), and reacted at 4 ° C. for 48 hours to immobilize the antibody on the inner wall of the test tube. After the reaction was completed, the liquid in the test tube was removed using an aspirator. Wash the inside of the test tube three times with 1 ml of physiological saline, and further add 1% BSA
1 ml of phosphate buffer (0.02M, pH 7.2) containing
And stored at 4 ° C. until use.

(2) Measurement of T4 in Blood Preparation of Reagents For the measurement in the comparative example, the reagents such as the luminescent reagent other than the immobilized antibody, the first monoclonal antibody solution, the enzyme-labeled T4 solution and the standard solution were prepared in the same manner as in the previous procedure. The reagents used in the studies in the examples were used as they were. However, the enzyme-labeled T4 was used at a dilution of 1000.

Measurement Procedure (a) Measurement by [Method 1] The immersion liquid in the antibody binding test tube prepared in (1) was removed using an aspirator. The test tube was washed once with 1 ml of physiological saline. Sample 20 μl of the standard solution or sample (human pool serum) into the washed test tube,
250 μl of the first monoclonal antibody solution prepared in
And 250 μl of enzyme-labeled T4 solution, add well, and mix well.
Incubated at 7 ° C. for 30 minutes. After completion of the reaction, the reaction solution was removed using an aspirator, and 1 ml of physiological saline was removed.
1 was added and the operation was repeated. This operation was repeated three times to remove unreacted substances.

The test tube after the completion of the washing was set in a sample holder of a luminescence reader BLR-201 manufactured by Aloka Co., Ltd., and 500 μl of the luminescent reagent prepared in the above was added to perform a chemiluminescence reaction. The measured value was obtained as an integral value for 10 seconds.

(B) Measurement by [Method 2] A standard solution or a sample (human pool serum) (20 μl) was sampled in a test tube separate from that prepared in (1), and 250 μl of the first monoclonal antibody solution prepared in Add 250 μl of enzyme-labeled T4 solution, mix well, and
For 30 minutes.

The immersion liquid in the antibody binding test tube prepared in (1) was removed using an aspirator. 1 ml of physiological saline
The inside of the test tube was washed once using. 500 μl of the above reaction solution was added to the washed test tube, and further reacted at 37 ° C. for 30 minutes. After completion of the reaction, the reaction solution was removed using an aspirator, and 1 ml of physiological saline was added, and the same operation was performed again. This operation was repeated three times to remove unreacted substances.

The test tube after the washing was set in a sample holder of a luminescence reader BLR-201 manufactured by Aloka, and a chemiluminescence reaction was carried out in the same manner as in [Method 1]. The measured value was obtained as an integral value for 10 seconds.

(C) Measurement by [Method 3] The immersion liquid in the antibody binding test tube prepared in (1) was removed using an aspirator. The test tube was washed once with 1 ml of physiological saline. Sample 20 μl of the standard solution or sample (human pool serum) into the washed test tube,
Further, the first monoclonal antibody solution 25 prepared in
0 μl was added, mixed well, and incubated at 37 ° C. for 30 minutes. After completion of the reaction, the reaction solution was removed using an aspirator, and 1 ml of physiological saline was added, and the same operation was performed again. This operation was repeated three times to remove unreacted substances.

To the test tube after the completion of washing, 500 μl of the enzyme-labeled T4 solution prepared above was added, and the mixture was stirred well and incubated at 37 ° C. for 30 minutes. After completion of the reaction, the reaction solution was removed using an aspirator, and 1 ml of physiological saline was added, and the same operation was performed again. This operation was repeated three times to remove unreacted substances.

The test tube after the completion of the washing was set in a sample holder of a luminescence reader BLR-201 manufactured by Aloka, and a chemiluminescence reaction was carried out in the same manner as in [Method 1]. The measured value was obtained as an integral value for 10 seconds.

The amount of luminescence of each standard solution in [Method 1] to [Method 3] and the measured value of human pool serum read from a calibration curve obtained by plotting the amount of luminescence in a graph (the same sample was n = 10 ) And the average of the measured values, standard deviation, C
V is shown in Table 2. However, the results of the measurement using the polyclonal antibody were worse in both the sensitivity of the measurement calibration curve and the reproducibility of the measured values of the pooled sera than when the monoclonal antibody was used.

[0058]

[Table 2]

[0059]

According to the present invention, when a monoclonal antibody specific to the Fc portion of a mouse antibody is used as an immobilized antibody, problems such as deterioration in measurement sensitivity and reproducibility observed when a current polyclonal antibody is used. Thus, it has become possible to provide a measurement kit having uniform performance at all times without generation of odor.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-2-22659 (JP, A) Enzyme immunoassay method, 3rd edition, edited by Eiji Ishikawa, published by The Medical College, May 15, 1987, pp. 35-38 (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/543 G01N 33/577

Claims (3)

(57) [Claims] 1. A test substance (A) in a sample, a first monoclonal antibody (B) against (A), an enzyme, a fluorescent substance, a luminescent substance, or a target substance or a derivative thereof labeled with a radioisotope ( C) and only specifically binds to the Fc portion of the immobilized on a water-insoluble carrier and (B)
The amount of (A) in the sample is determined by measuring the amount of the labeling substance in the immune complex formed by reacting the second monoclonal antibody (D) obtained from the same animal species as in (B). An immunoassay method comprising: 2. The immunoassay according to claim 1, wherein the immune complex is formed by a method selected from the following [method 1] to [method 3]. [Method 1]: Method of simultaneously reacting (B), (C), (D) and (A) [Method 2]: Reaction of (B), (C) and (A) and then (D) Method of reacting [Method 3]: reacting (B), (D) and (A),
Method of reacting with (C) after removing unreacted substances by B / F separation 3. The method according to claim 1, wherein the first monoclonal antibody (B) against the target substance (A), the target substance or its derivative (C) labeled with an enzyme, a fluorescent substance, a luminescent substance or a radioisotope, and a water-insoluble carrier. Immobilized and
(B) specifically binds vital to the Fc portion of (B) and the same dynamic
A competitive immunoassay kit comprising the second monoclonal antibody (D) obtained from the species .