JPH0192659A - Anti-fr-900506 material antibody and high-sensitivity enzyme immunoassay method - Google Patents

Abstract

PURPOSE:To exactly measure the trace concn. of an FR-900506 material by constituting an antibody for recognizing the FR-900506 material of a polyclonal antibody and monoclonal antibody at the time of detecting said material. CONSTITUTION:The antibody to recognize the antigenic determinant in the FR-900506 material is constituted of the polyclonal antibody and the monoclonal antibody. The polyclonal antibody is obtd. by conjugating the FR-900506 which is an immunogen with a carrier such as bovine serum albumin (hereafter abbreviated as 'BSA'), etc., in order to enhance the immunogenicity and isolating and refining said material by salting out, dialysis, etc., from the resultant antiserum. The monoclonal antibody is obtd. by fusing the spleen cells and bone marrow cells of an animal immunized with the BSA-FR-900506 material conjugate, selecting the hybrodoma which produces the monoclonal antibody specific to the FR-900506 material and propagating this hybridoma in the abdominal cavity of an animal and obtaining the anti-FR-900506 material monoclonal antibody from the ascites.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to a novel antibody, a highly sensitive enzyme immunoassay, and a test kit for carrying out the assay.

More specifically, the present invention describes an antibody that recognizes an antigenic determinant present in the FR-900506 substance, a highly sensitive enzyme-linked immunosorbent assay (direct method) for the FR-900506 substance that utilizes an immobilized antibody, Highly sensitive enzyme-linked immunosorbent assay (indirect method) for low-molecular-weight substances, which consists of a first antibody that recognizes the substance and an immobilized second antibody that recognizes the first antibody, and FR by the direct or indirect method. -Relating to a test kit for detecting substance 900506.

't' and Ming is about to do it, FR-9
00506 substance is Streptomyces
genus, especially Streptomyces nkubaensis (5tra
tom ass tsukubaansis ) N
o.

9993 (Feikoken Jokyo No. 927) is a compound having pharmacological activities such as immunosuppressive activity and antibacterial activity, and is known to have the following structural formula (Japanese Unexamined Patent Publication No. 61-1
4FH8L).

Since the FR-900506 substance has very strong immunosuppressive activity even in minute amounts, in order to effectively and sustainably suppress the rejection reaction that occurs during transplantation, such as organ transplantation, it is necessary to A technique for easily and highly sensitively monitoring blood concentrations is required, and for this purpose, it is considered extremely important to establish a technique for accurately measuring trace concentrations.

However, the technology to easily and sensitively measure trace concentrations of the FR-900506 substance has not yet been developed, and the antibodies that recognize the substance, which are important in detecting the FR-900506 substance, have not yet been developed. I was in a situation.

Conventionally, gas chromatography, high performance liquid chromatography, radioimmunoassay, enzyme immunoassay, and the like have been used to measure trace amounts of low molecular weight substances contained in biological samples.

However, these methods (1) have insufficient sensitivity to measure trace concentrations of pharmacologically active substances, (2) are complicated to operate, (3) require large equipment, and (4)
have to use dangerous radioisotopes,
There were problems such as.

As a result of intensive research, the inventors of the present invention discovered FR-9005.
We succeeded in obtaining an antibody that recognizes the 06 substance, then immobilized this antibody, and competitively reacted it with a sample containing the FR-900506 substance and an enzyme-labeled FR-900506 substance, thereby producing trace amounts of the FR-900506 substance. R-9005
It has been found that the 06 substance can be detected easily and with high sensitivity.

Furthermore, as a result of intensive research, the inventors used a first antibody that recognizes the test substance and an immobilized second antibody that recognizes the first antibody, and added a sample containing the test substance and an enzyme. By competitively reacting the analyte labeled with
We have discovered a measurement method that can easily detect extremely small amounts of test substances with higher sensitivity. Furthermore, the present invention was completed by creating a test kit that is convenient for carrying out the above measurements.

An overview of the present invention can be divided into the following four inventions.

(I) An antibody that recognizes the antigenic determinant in the FR-900506 substance.

An antibody that recognizes an antigenic determinant present in the (I[)FR-900506 substance is immobilized, and FR-9
Sample containing 00506 substance and FR labeled with enzyme
- A highly sensitive enzyme immunoassay method (direct method) for the FR-900506 substance, which comprises detecting the FR-900506 substance labeled with an enzyme bound to an immobilized antibody by competitively reacting the FR-900506 substance with the 900506 substance.

(III) Consisting of a first antibody that recognizes a test substance and an immobilized second antibody that recognizes the first antibody, the test substance contained in the sample and the enzyme-labeled test substance are A highly sensitive enzyme-linked immunosorbent assay ( indirect method).

(■) Components include an antibody that recognizes the antigenic determinant in the FR-900506 substance and an enzyme-labeled FR
FR-90 characterized by containing -900506 substance
Test kit for detecting 0506 substance.

The present invention will be explained in detail below.

(I) Antibody that recognizes antigenic determinant in FR-900506 substance The above antibodies include polyclonal antibodies and monoclonal antibodies.

Polyclonal antibodies have heavy chain
IgG, IgA, IgM,
There are classes such as IgD and IgE, and each of these classes has several subclasses.
The antibody of the present invention can be of any type as long as it recognizes the antigenic determinant in the FR-900506 substance, and a particularly preferred class is IgG.

Polyclonal antibodies are isolated and purified from the antiserum obtained by administering an immunogenic substance (for example, FR-900506 substance) to animals for immunization.

Immunological manipulations are performed by conventional methods.

The animal to be immunized is not particularly limited, but rabbits, guinea pigs, rats, mice, goats, etc. are usually used, with rabbits being particularly preferred.

Substances that serve as immunogens (for example, FR-900506 substance) usually increase immunogenicity, so for example, bovine serum albumin (
It is used in combination with a carrier such as BSA (hereinafter referred to as BSA), gelatin, or hemocyanin. The immunogenic substance is FR-90
0506 substance, a conjugate with BSA (BSA
-FR-900506 substance conjugate) is, for example, R-90
0506 substance is converted into a half ester of dicarboxylic acid such as succinic acid, and then treated with N-hydroxysuccinimide or the like in the presence of a condensing agent such as dicyclohexylcarbodiimide to lead to an active ester of the half ester,
It can be further obtained by reacting with BSA.

Polyclonal antibodies are isolated and purified from the obtained antiserum by conventional procedures such as salting out with ammonium sulfate, centrifugation, dialysis, and column chromatography.

Monoclonal antibodies also have classes and subclasses similar to polyclonal antibodies depending on their H chains, but the monoclonal antibodies of the present invention can be of any type as long as they recognize the antigenic determinant in the FR-900506 substance. A particularly preferred class that can be used is IgG.

Monoclonal antibodies are usually produced by cell fusion methods, but they can also be produced by genetic engineering techniques.

Antibody-producing cells (e.g., anti-FR-900506 substance antibody-producing cells) used in the cell fusion may contain substances with increased immunogenicity (e.g., BSA-FR-90050).
These include lung cells, lymph node cells, peripheral lymphocytes, etc. of animals (eg, mice, rats, rabbits, goats, etc.) that have been immunized with a 6-substance conjugate. Furthermore, antibody-producing cells produced by reacting an immunogen in a culture medium with the aforementioned cells or lymphocytes taken out in advance can also be used. In addition, when using the latter procedure, human-derived antibody-producing cells can also be prepared. Antibody-producing cells and myeloma cells may be derived from different animal species as long as they can be fused, but it is preferable to use cells derived from the same species.

The production of monoclonal antibodies by cell fusion method has been described, for example, by Köhler and Milstein.
The method is carried out in a conventional manner according to the basic method of John Stein, 1996 [Nature, Vol. 256, p. 495 (1975)].

Particularly preferably, lung cells obtained from a mouse immunized with a BSA-FR-900506 substance conjugate are fused with mouse myeloma cells to produce a /S hybridoma,
From among them, the eupridomas producing monoclonal antibodies specific to the FR-900506 substance are selected. The hybridoma is grown in the peritoneal cavity of a mouse, and an IgG fraction of anti-FR-900506 monoclonal antibody is obtained from the ascites fluid.

(I[)FR-900506 Enzyme immunoassay method (direct method) Enzyme immunoassay using direct method is PR-900506
An antibody that recognizes an antigenic determinant present in a substance is immobilized, and the immobilized antibody is competitively reacted with a specimen containing the FR-900506 substance and an enzyme-labeled FR-900506 substance, and the immobilized antibody is reacted with the FR-900506 substance labeled with an enzyme. This is done by detecting the bound enzyme-labeled FR-900506 substance. The antibody that recognizes the antigenic determinant present in the FR-900506 substance means the antibody described in invention (1). Either a polyclonal antibody or a monoclonal antibody can be used, but it is more selective and Monoclonal antibodies are preferred because there is no difference in their properties between lots. Examples of in-phase materials for immobilization include plates (immunoplates, etc.), particles (immunobeads, etc.)
, polystyrene poles, test tubes, etc. are used, but immunoplates are preferred from the viewpoint of easy operability. FR-90
Enzymes that label 0506 substances include enzymes commonly used in fermentation immunoassays. For example, peroxidase, β-D-galactosidase, alkaline phosphatase, glucose oxitase, acetylcholinesterase, glucose-6-phosphate dehydrogenase, malate dehydrogenase or urease can be used.
A preferred enzyme is peroxidase (hereinafter referred to as POD).

The enzyme-labeled FR-900506 substance is prepared according to a conventional method. For example, when using a crosslinking agent, the invention (I)
) A half ester made from the FR-900506 substance described in ) and a dicarboxylic acid such as succinic acid is reacted with N-hydroxydisuccinimide, etc., and an enzyme that can be used for labeling POD etc. is added to the active ester of the half ester produced. Obtained by reaction.

Detection of the enzyme labeled substance bound to the immobilized antibody is carried out by measuring the activity of the enzyme used for labeling by a conventional method. For example, when the enzyme used for labeling is POD, an enzyme substrate solution containing O-phenylenediamine and hydrogen peroxide is used to detect the POD bound to the immobilized antibody.
D-FR-900506 By measuring the degree of color development of the oxidized substrate according to the amount of the substance label, FR
-900506 substance is quantified.

This direct method allows trace concentrations of FR-900506 substance (
10-1 to 1103 n/mQ) can be quantitatively and qualitatively measured with high sensitivity and ease.

(I[) Enzyme immunoassay (indirect method) Enzyme immunoassay using an indirect method uses a first antibody that recognizes the test substance and an immobilized second antibody that recognizes the first antibody. A test substance contained in a sample and an enzyme-labeled test substance are competitively reacted with one antibody, and the enzyme-labeled test substance bound to the first antibody is bound to the second antibody obtained. This is done by detecting substances.

This indirect method can measure various substances as test substances, including peptides, steroids, prostaglandins, polysaccharides, or macrocyclic compounds, but it is not suitable for measuring the concentration of macrocyclic compounds, more specifically FR-900506. Can be used.

The first antibody can be either a polyclonal antibody or a monoclonal antibody as long as it can recognize the test substance, but monoclonal antibodies are preferred because they are more selective and there are no differences in their characteristics between manufacturing lofts. preferable.

The first antibody is prepared in the same manner as described in invention (I), and when the test substance is the FR-900506 substance, the antibody fully described according to invention (I) is used as the first antibody.

As the second antibody that recognizes the first antibody, an antibody prepared by a conventional method using the first antibody or an antibody of the same type as the first antibody as an antigen, or a commercially available antibody can be used. Any antibody, polyclonal or monoclonal, can be used as long as it does not interfere with the antigen-antibody reaction between the antibody and the test substance and recognizes the first antibody. Preferably, when an IgG class antibody obtained from a rabbit is used as the first antibody, a goat anti-rabbit IgG is used as the second antibody, and when an IgG class antibody obtained from a mouse is used as the first antibody. uses rabbit anti-mouse IgG as the second antibody.

The same phase to be immobilized, the enzyme to label the test substance, and the method for detecting the enzyme are the same as those described in Invention (I[)].

In this indirect method, the detection limit value of the test substance can be changed by adjusting the amount of the first antibody recognized by the immobilized second antibody. That is, in the case of quantifying the FR-900506 substance described as a specific example of the present invention, 10-"
It has been shown that it is possible to quantitatively and qualitatively measure concentrations down to extremely small concentrations on the order of ng/mQ with high sensitivity and ease.

(IV) Test kit This test kit consists of an antibody that recognizes the antigenic determinant present in the FR-900506 substance and an enzyme-labeled FR.
-900506 substance.

The 'antibody that recognizes the antigenic determinant present in the FR-900506 substance' refers to the polyclonal antibody or monoclonal antibody described in (1) above, but monoclonal antibodies are preferred. Note that the antibody can be supplied in either a solid state or a solution state.

The enzyme-labeled FR-900506 substance is as described in (I[) above, but preferably it is a peroxidase-labeled FR-900506 substance.

Note that the compound can also be supplied in either a solid state or a solution state.

This test kit may contain materials used in performing the highly sensitive enzyme immunoassay method of the present application. For example, a known amount of the FR-900506 substance required for quantitatively measuring the FR-900506 substance may be included as a standard sample. Another example is "FR-900" as shown in the present invention (I[[)].
Examples include antibodies that recognize antigenic determinants present in 506 substances.

Yet another example is the enzyme substrate used as a label for the FR-900506 substance.

Example 1. Anti-FR-900506 substance Polyclonal antibody 2 1 1) Synthesis of active ester of succinic acid-FR-900506 substance half ester FR-900506 substance ↓ Succinic acid-FR-900506 substance half ester■ FR-900506 substance (248 mg) was mixed with pyridine (
7 mm), and succinic anhydride (145 mg
) and 4-dimethylaminepyridine (7-) and stirred at room temperature for 18 hours. The reaction solution was concentrated to dryness under reduced pressure, and the residue was subjected to silica gel column chromatography and developed with ethyl acetate to obtain succinic acid-FR-900506 substance half ester (90 mg). This half ester (
N-hydroxysuccinimide (12.6 mg) was dissolved in a solution of N-hydroxysuccinimide (12.6 mg) dissolved in ethyl acetate (tomQ), dicyclohexylcarbodiimide (22 mg) was added thereto under stirring under water cooling, and the mixture was stirred overnight at room temperature. . The precipitate was filtered off and the filtrate was evaporated under reduced pressure. The residue was purified by silica gel thin layer chromatography and developed with ethyl acetate to obtain the active ester of the above compound (74,1
mg).

IRν (knee): 1813. 1784.
1735. 1640°1200 ctn-” 2) Preparation of BSA-FR-900506 substance conjugate The active ester (37mg) of the succinic acid-FR-900506 substance half ester obtained above was mixed with dioxane (4m
BSA (manufactured by Almor Pharmaceutical Co., Ltd.) is added to the solution dissolved in m).
(30.1mg) in 0.05M phosphate buffer (pH 7)
, 3 > (10 + 1111) and add the solution dissolved in 4
The BSA-FR-900506 substance conjugate is prepared by stirring at °C for 3 days and dialyzing the reaction mixture against 0.05M phosphate buffer (pH 7.3) for 24 hours.

2O- 3) Preparation of POD-FR-900506 substance label 1)
Horseradish peroxidase was added to a dioxane solution (IOA) of the active ester (0.48 mg) of the half ester of succinic acid-FR-900506 substance obtained in
a, rypa VI + manufactured by Sigma) (10 mg) in dioxane-0.5% sodium hydrogen carbonate (1: lv
/v) Add the mixed solution (0.36n+u) and incubate at 4℃ for 23 minutes.
5 hours of indirect f. Add to this 0.1%w/v gelatin-0
．． After adding 0.05M phosphate buffer (pH 7.0) (in order of 1.77), dialysis is performed against 0.05M phosphate buffer (pH 7.0) to obtain a POD-FR-900506 substance label solution. .

4) Preparation of anti-FR-900506 substance polyclonal antibody 2) Prepare the obtained BSA-FR-900506 substance conjugate (1.6 mg as BSA amount) in phosphate buffered saline (hereinafter referred to as PBS solution) (2) , 5119) was emulsified in an equal amount of Freund's complete adjuvant, and the foot pads of seven New Sealant White rabbits (female) were subcutaneously injected with five doses each for immunization. After 2 weeks and 3 weeks,
A booster immunization is performed by injecting 5 mfL of a PBS solution of the BSA-FR-900506 substance conjugate in the same amount as above and emulsifying it with an equal amount of Freund's incomplete adjuvant into the foot pad and subcutaneously.

Furthermore, 3 weeks after the booster immunization, a final immunization is performed in the same manner. Nine days after the final immunization, whole blood was collected, and the obtained antiserum (
340ml) with an equal volume of PBS solution and an equal volume of 100ml
Add % saturated ammonium sulfate and salt out. The resulting precipitate was collected by centrifugation (10, OOOrpmX 10 minutes) and diluted with 20mM
DEAE cellulose (DE52, manufactured by Whatman Chemical Separations) dissolved in phosphate buffer (pH 8,0) (200 mQ), thoroughly dialyzed against the same buffer, and equilibrated with the same buffer. ),
Anti-FR-900506 substance polyclonal antibody (5,6
A pass-through fraction (IgG fraction) containing g) was obtained (the amount of antibody was calculated from the absorbance value at a wavelength of 280 nm).

Note that the PBS solution used in this example consists of 1.
It means a solution containing the following composition in 0ρ, and the same applies to the following examples.

NaC18,Og Na2HPO41,15g KCl 0.2gKH2PO40
, 2g Example 2. Anti-FR-900506 monoclonal anti-stand ■
1 1> Preparation of anti-FR900506 substance monoclonal antibody-producing hybridoma BSA-FR-90050 obtained in Example 1, 2)
PBS solution of 6 substance conjugate (BSA amount 50x) (
0,211111> in an equal amount of Freund (Freund
) in complete adjuvant and injected intraperitoneally into BALB/c mice (female).

Thereafter, the same amount of BSA-FR-900506 substance conjugate in PBS was emulsified with an equal amount of Freund's incomplete adjuvant and was intraperitoneally injected twice at appropriate intervals to give the second and third injections. Perform immunization. Furthermore, as a fourth immunization, 10 times the amount of BSA-FR-900 was administered.
P of 506 substance conjugate (soosg as BSA amount)
BS solution (0,2IlIQ) was added to Freun
d) was emulsified in an incomplete adjuvant and injected subcutaneously, the BSA-FR-900506 substance conjugate (BSA amount: 200 ( ) in PBS solution (0, z
mQ) was injected intravenously through the tail vein, and the lungs were removed 3 days later.

This lung is loosened with a bottle set, and the obtained lung cells and mouse myeloma cells P3 X 63Ag8U.1 are fused according to the following method.

That is, lung cells were suspended in Dulbetsko's modified Eagle's Minimum Essence Medium (minimum essential medium, hereinafter referred to as D-MEM), and red blood cells in the suspension were mixed with 0.83% ammonium chloride solution (9 volumes). 17
Disrupt by treating with a mixture of M tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (pH 7.65, 1 volume) at 4°C for 5 minutes, and remove by centrifugation. Mouse myeloma cells and lung cells cultured in D-MEM supplemented with 10% fetal bovine serum were washed several times with D-MEM. Add a suspension of lung cells (2 x 108 cells) to a suspension of mouse myeloma cells (4 x 107 cells). , mix the mixture with 50111
Mix well in a 9 plastic tube (Corning Glass Works 50IIIQ Corning centrifuge tube).

The medium was removed by centrifugation and the cells were incubated in a water bath at 37 °C.
Warm to. Add 45% polyethylene glyfur (manufactured by Merck, average molecular weight 4.000) to the cells while shaking.
) solution (1 hour) was gradually added over a period of 1 minute and the mixture was left at room temperature for 5 minutes. Add D- to the reaction mixture for 5 minutes.
After quenching the MEM (15 mA) to stop the cell fusion reaction and adding a large amount of D-MEM, the mixture is centrifuged and the supernatant is removed. The residue contained 15% fetal bovine serum (manufactured by Centaurus, Lot 757), 2mM glutamine,
2-mercaptoethanol 2 x 10-5M, streptomycin sulfate 1100N/mQ, penicillin G 100U
/mQ, a complete medium (hereinafter referred to as CM) consisting of D-MEM supplemented with gentamicin sulfate 80x/mu and fungizone (amphotericin B1 manufactured by Gibb Collaborative Laboratories).
Add.

After mixing the mixture a little, the resulting fused cell suspension was dispensed into 10 24-well plates (manufactured by Nunc Co., Ltd.) in 1-well portions so that the number of lung cells was 1 x 106 per well. After culturing for 1 day at 37°C in % carbon dioxide gas, aminopterin (4X10"M), thymidine (1,6X 10"M)
=M) and hypoxanthine (IXIO-'M>) and 1 mQ of CM (HAT medium) are added to each well. After 1 day, half of the medium is removed from each well by suction.
Add T medium. Continue to replace the medium every 2 or 3 days thereafter.

Solid phase BSA in 161 wells where hybridomas grew
Shows reactivity to FR-900506 substance conjugate and R
- Select 14 wells that show specific reactivity to the 900506 substance. Of the 14 wells, 8 wells with high antibody titers were placed in a feeder layer (5 x 106 cells/mQ) of BALB/c mouse thymocytes.
Using a 96-well flat-bottomed microplate (manufactured by Nunc), cloning was performed by the limiting dilution method to obtain three types of hybridomas that produced monoclonal antibodies specific to the FR-900506 substance.

The antibody assay in each well is performed by selecting clones that react with the solid-phase BSA-FR-900506 substance conjugate as shown below, and whose reaction is antagonized by an excessive amount of the FR-900506 substance. Do, that is, B
The reactivity of the SA-FR-900506 substance conjugate was confirmed by coating the conjugate in the same phase (solid phase coating conditions were 20 Pg/l1lu PBS solution at 100 kg/l).
Add culture medium (Loom) to each well (add wells and leave to stand at 37°C for 2 hours), and leave to stand at 37°C for 2 hours. After removal by suction and washing, dilute POD-labeled anti-mouse IgG (manufactured by Miles, code 61-204.1) 2000 times with a PBS solution containing 1% BSA (hereinafter referred to as 1% BSA-PBS solution) (100 ml). was added and left at 37°C for 1 hour. After suction removal and washing, P is applied using the conventional coloring method.
Measure OD activity. In addition, to confirm whether the antibody specifically reacts with the FR-900506 substance, use BSA
- FR-900506 substance conjugate was added to each well coated with solid phase, and FR was prepared to a concentration of 100Pt/mQ.
-900506 substance in 1% BSA-PBS solution (50
After reaction by adding culture solution (100 taQ), the same reaction and color development procedure is performed using PODm-recognizing mouse IgG, and the color development can be suppressed by adding excess FR-900506 substance. Confirm by

2) Isolation and Purification of Monoclonal Antibodies 2, 6.10.10.14-Tetramethylpentadecane was administered to a BALB/c mouse (female) that had been made immunosuppressed. Three types of hybridomas were transplanted at 1 x 107 cells per mouse, and 10 to 1
Ascites fluid is collected after 4 days. After fractionation with 50% saturated ammonium sulfate, it is dialyzed against 10 mM phosphate buffer (pH 8.0). Subjected to DEAE cellulose column chromatography equilibrated with 10mM phosphate buffer (pH 8,0),
0-150m in 10mM phosphate buffer (pH 8,0)
M was eluted using a continuous sodium chloride concentration gradient method to obtain a fraction of IgG produced by each hybridoma. The subclass of each IgG was determined by Ophthalony's double immunodiffusion method (Table 1).

Table 1 1) Adsorption operation of antibodies to the same phase Dispense 200 d of monoclonal or polyclonal antibody solution (2/nil, PBS solution) into each well of Eliza Plate S (MS-3496F, manufactured by Sumitomo Bakelite) and leave it at 4°C overnight.

After collecting the antibody solution, wash the plate three times with PBS solution.

2) Operation for preventing non-specific adsorption Fill each of the above plates with a 1% BSA-PBS solution, leave it at 37°C for 30 minutes, and then remove the PBS solution by suction.

3) Antigen-antibody reaction (1) Dispense 100 volumes of standard FR-900506 substance solution diluted with 1% BSA-PBS solution containing 10% normal plasma into each well.

(2) POD-FR-90 prepared in Example 1-3)
0506 substance label solution was diluted 2×10 5 times with 1% BSA-PBS solution, and 100 volumes were dispensed into each of the above wells.

(3) Mix each well with a plate mixer for 10 seconds and leave at 4°C overnight.

4> PBS containing enzyme reaction 005% Twaen 20
After washing each well above twice with the solution and twice with PBS solution, enzyme substrate solution (200) 1 prepared as below was added.
11) was dispensed into each well, and allowed to react at room temperature for 30 minutes. Prepare the enzyme substrate solution immediately before use.

Enzyme substrate solution: O-phenylenediamine (100mg) and 30% hydrogen peroxide solution (50ml) were added to Macvine (Mcl) at pH 5.4.
lvaine) buffer solution (0.1M disodium hydrogen phosphate solution added with 0.1M citric acid to adjust pH to 5.4) (toomQ).

5> Reaction termination operation Dispense 4N sulfuric acid solution (50sQ) into each well above,
Stop the reaction.

6) Using the measurement substrate solution as a control, measure the absorbance of the reaction solution at a wavelength of 492 nm using Multiscan (trade name, manufactured by Titertic).

According to the above measurement method, the anti-FR-900 obtained in Example 1
506 substance polyclonal antibody and anti-F obtained in Example 2
Table 2 shows the results when using R-900506 substance monoclonal antibodies (3 types) and using normal peagle-sized (male) plasma as the normal plasma.

Table 2 Enzyme immunoassay results of FR-900506 substance by direct method Po-Ab - Anti-FR-900506 polyclonal antibody Mo-Ab (1) --- Monoclonal antibody F
R-900506-1-40-56Mo-Ab(2)・
...//FR-900506-1-53-19Mo-A
b(3)...//FR-900506-1-60-4
6 column 4, l method of FR-900506 (5
Yiri 1 1) Adsorption operation of antibody to the same phase Second antibody solution (3pg/+nQ% PBS solution) 200
4 portions were dispensed into each well of Elisa Plate H (MS-3596F, manufactured by Sumitomo Bakelite Co., Ltd.) and left overnight at 4°C. After collecting the antibody solution, wash the plate three times with PBS solution.

2> Operation to prevent non-specific adsorption Add 1% BSA-PBS solution to each plate above.
After filling and leaving at 37°C for 30 minutes, remove the PBS solution by suction.

3> Antigen-antibody reaction (1) Dilute with 1% BSA-PBS solution (5x10'-fold dilution if the first antibody is a polyclonal antibody, 2x10'-fold dilution if the first antibody is a monoclonal antibody)
POD-FR-900506 substance labeled solution 100
Dispense aliquots into each of the above wells.

(2) 100JJ of standard FR-900506 substance solution diluted with 1% BSA-PBS solution containing 10% normal plasma
(3) First antibody solution prepared with 1% BSA-PBS solution (if the first antibody is a polyclonal antibody, dispense 400n
g/mQ, if the first antibody is a monoclonal antibody, L
ong/ml) 50S is added to each well.

(4) Stir each well with a plate mixer for 10 seconds and leave at 4°C overnight.

4) Enzyme reaction Same as Example 3-4>.

5> Stopping the reaction Same as 5) of Example 3.

6〉Measurement Same as 6) of Example 3.

According to the above measurement method, the anti-FR-900 obtained in Example 1
Table 3 shows the results when the 506 substance polyclonal antibody and the monoclonal antibody FR-900506-1-60-46 obtained in Example 2 were used as the first antibody, and normal peagle-sized (male) plasma was used as the normal plasma. .4.

Table 3 Results of enzyme immunoassay of FR-900506 substance by indirect method using anti-FR-900506 substance polyclonal antibody Table 4 Monoclonal antibody FR-900506-1-
If the first antibody is an anti-FR-900506 polyclonal antibody, the second antibody should be goat anti-rabbit IgG (Miles antiserum). (Code 64-331-1) was purified with DEAE cellulose after salting out ammonium sulfate),
Also, the first antibody is monoclonal antibody FR-900506
-1-60-46, use rabbit anti-mouse = 36-IgG (manufactured by EI Laboratory, Catalog No.
.

AF-011) respectively.

5. A solid dispersion formulation containing the FR-900506 substance with the following composition was orally administered in a single dose (1 mg/dose) to three male pegle dogs.
kg) L,, the measurement method described in Example 4 (monoclonal antibody PR-900506-1-6 as the first antibody)
Standard FR-90 diluted in 1% BSA-PBS solution containing 10% normal plasma according to
Instead of the 0506 substance solution, a sample (plasma) diluted 10 times with a 1% BSA-PBS solution was used to monitor the plasma concentration. The results (average value of 3 animals) are shown in Table 5.

The time in the table indicates the time elapsed after administration of the solid dispersion formulation containing the FR-900506 substance.

Composition of solid dispersion formulation (per 1.0g) FR-9005
06 substance 0.2g Hydroxypropyl methyl cellulose 2910 (TC-5R) 0.
2g croscarmellose sodium (AC-Di-5ol) 0.2
g milk @ 0.4g Table 5
．． Results of plasma concentration monitoring of FR-900506 substance using indirect method Constituent components (for 1000 samples) (1) First antibody (anti-FR-900506 substance monoclonal antibody) solution (5004) 1: 1% BSA-PBS and adjusted to lx/mA (
2) POD-FR-900506 substance solution (500 kg) 2): The solution obtained in 3) of Example 1 was diluted with 1% BSA-
PR54 trowel diluted 1000 times (3) FR-900506 substance standard solution (1 mm) 3
).

Prepared with methanol to 1 mg/mQ (4)
Second antibody (rabbit anti-mouse IgG) solution (600,
,)4): Prepared at 1 mg/mQ in PBS1)...1
Use after diluting 100 times with %BSA-PBS.

2> Use by diluting 200 times with 1% BSA-PBS.

3) Dilute to the required concentration with 1% BSA-PBS and use.

4)...Dilute 333 times with PBS and use.

The test kit (I>) is used according to the operating procedure of Example 4 in carrying out the indirect method.

Example 73 Test for indirect method Components (for too much specimen)> (1) First antibody (anti-FR-900506 substance monoclonal antibody) solution (1+all) 1).

1% RAS - Prepared at 1 palm/body with PBS.

(2) POD-FR-900506 substance solution (700
2): Add 1% BSA to the solution obtained in 3) of Example 1.
- Diluted 1000 times with PI6.

(3) FR-900506 substance standard solution (1 mu each
): in methanol, 100.50.20.10, respectively.
．． 5.2.1.0.5.0.2 or Ong/ml
(4) Second antibody (rabbit anti-mouse IgG) solution (1 relative) 3): Prepared at 1 mg/mQ in PBS 1>...1
PBS containing % BSA and 0.05% Tween 20
(Hereafter, 1% BSA-0,05% Tween 2O-PBS
(abbreviated as )) and dilute it 50 times before use.

2)...1% BSA-0.05% Tween 20-
Use after diluting 300 times with PBS.

3>...The test kit (■), which is diluted 200 times with PBS, is used in the indirect method according to the column route or extraction route as described below.

[Column route 1, measurement sample (plasma or serum) (1oon
) to (1,IN hydrochloric acid (1 mm) and methanol (1 mm)
0)LQ) and stir for 2-3 minutes.

1', Preparation of standard solution of FR-900506 substance Normal plasma or normal serum (100S) was added with 0. IN
Add hydrochloric acid (1 mm) and FR-900506 substance standard solution (10 volumes) and stir for 2-3 minutes.

2. Column treatment i) Activate the Seppupak column (trade name: C18 cartridge column, manufactured by Waters Associates (USA)) with methanol (511111), and then wash with 4% acetic acid solution (20 mQ).

i) Apply the measurement sample or standard solution of 1 or 1' to the Sep-vac force ram.

i) The Seppupak column was soaked in 4% acetic acid solution (20m+
,).

~) FR-900506 substance in methanol (3mm)
and collect in a centrifuge tube.

3. Dry with a stream of nitrogen gas.

4, 1% BSA of POD-FR-900506 substance
Dissolve the residue in 0.05% Tween 2O-PBS solution #200.

5. Add the second antibody solution (20ON) to each well of a 96-well flat-bottomed microtiter plate, and stir and react overnight at 4°C.

6. Aspirate the second antibody solution from each well.

7. Wash 3 times with PBS.

8. Immediately after the third wash, add 1% BSA-0.05% Tween 20-PBS to each well of the plate.
Add 300S of non-specific adsorption prevention solution.

9. Leave at room temperature for 1 hour.

10. Aspirate and remove the non-specific adsorption prevention solution from the wells;
Immediately proceed with the measurement sample or standard solution obtained in step 4 (
18(laQ) is added to the well.

This operation is performed for each well.

11. Add the first antibody (50S) to each well.

12. Incubate the plate at 4°C overnight with stirring.

After removing the 13 solution by aspiration, the plate was washed twice with 0.05% Tween 20-PBS, and then washed twice with PBS.
Wash twice.

14. Immediately add the substrate solution (2) described in Example 3-4>.
00d) was added to each well, and stirred and reacted at room temperature for 15 minutes.

Add 15,4 N sulfuric acid solution to each well to stop the enzyme-substrate reaction.

16 substrate solution as a blank, and measure the absorbance of each well at a wavelength of 492 nm.

[Extraction route] 1. Measurement sample (plasma or serum) (100PA
), methanol (1 (IJI)), 0.2M phosphate buffer (pH 7,0) (1mQ) and diku
Add J Tan (6,0mQ).

1', normal plasma or normal serum (100 tJl), R-900506 substance standard solution (1ON), 0.2
Add M phosphate buffer (pH 7.0) (1.0 ml) and dichlorothane (6.0 mQ).

2. After stirring each mixture for 10 minutes, 3000 rpm
Centrifuge for 10 minutes at m.

3. Obtain the lower layer of dichloromethane (5, omQ) and dry it in a nitrogen gas stream.

3 above. The dried residue obtained in is treated according to methods similar to column routes 4 to 16 described above, and an indirect method is carried out.

Claims (23)

[Claims] (1) An antibody that recognizes the antigenic determinant present in the FR-900506 substance. (2) The antibody according to item (1), which is a polyclonal antibody. (3) The polyclonal antibody class is IgG (
2) Antibody described in section 2). (4) The antibody according to item (1), which is a monoclonal antibody. (5) The antibody according to item (4), which is an anti-FR-900506 substance monoclonal antibody produced from a hybridoma formed by cell fusion of animal anti-FR-900506 substance antibody-producing cells and myeloma cells. (6) The antibody according to item (5), wherein the anti-FR-900506 substance antibody-producing cells are lung cells. (7) Paragraph (5) or (6) where the animal is a mouse
Antibodies described in Section. (8) The monoclonal antibody class is IgG (
The antibody according to item 4), item (5), item (6) or item (7). (9) Immobilize an antibody that recognizes the antigenic determinant present in the FR-900506 substance, and immobilize the FR-900506 on the immobilized antibody.
Sample containing 0506 substance and enzyme-labeled FR-
A highly sensitive enzyme immunoassay method for FR-900506 substance, which comprises detecting FR-900506 substance labeled with an enzyme bound to an immobilized antibody by competitively reacting with FR-900506 substance. (10) The highly sensitive enzyme immunoassay method according to item (9), wherein the antibody is a polyclonal antibody. (11) The highly sensitive enzyme immunoassay method according to item (9), wherein the antibody is a monoclonal antibody. (12) Consisting of a first antibody that recognizes a test substance and an immobilized second antibody that recognizes the first antibody, the test substance contained in the sample and the enzyme-labeled test substance are A highly sensitive enzyme immunoassay method characterized by detecting a test substance labeled with an enzyme bound to the first antibody that is bound to the second antibody obtained by competitively reacting with the first antibody. (13) The first antibody is a polyclonal antibody (12)
Highly sensitive enzyme immunoassay method described in ). (14) The first antibody is a monoclonal antibody (12)
Highly sensitive enzyme immunoassay method described in ). (15) Clause (12) in which the enzyme is peroxidase;
The highly sensitive enzyme immunoassay method according to item (13) or item (14). (16) The second antibody is immobilized on the plate (
Paragraph 12), Paragraph (13), Paragraph (14) or Paragraph (15)
Highly sensitive enzyme immunoassay method described in ). (17) Paragraphs (12), (13), (14), and (15) where the test substance is the FR-900506 substance.
The highly sensitive enzyme immunoassay method according to item (16) or item (16). (18) Anti-FR-9005 as described in item 1 as a component
FR-9005 labeled with 06 substance antibody and enzyme
A test kit for detecting a FR-900506 substance, characterized in that it contains a FR-900506 substance. (19) Anti-FR-900506 substance antibody is anti-FR-90
The test kit according to item (18), which is a monoclonal antibody for substance 0506. (20) Enzyme-labeled FR-900506 substance is
The test kit according to item (18) or item (19), which is a peroxidase-labeled FR-900506 substance. (21) The test kit according to item (18), item (19), or item (20), further comprising a known amount of the FR-900506 substance. (22) Items (18), (19), and (20) further comprising an antibody that recognizes the anti-FR-900506 substance antibody.
The test kit according to paragraph or paragraph (21). (23) Hybridomas producing anti-FR-900506 substance antibodies are cultured, and anti-FR-9005
4. The method for producing a monoclonal antibody according to item (4), which comprises isolating an antibody to substance No. 06.