JP3202029B2 - Microdetermination of interleukin 5 - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a monoclonal antibody against human interleukin 5 (referred to as human IL-5), a method for measuring human IL-5 by a sandwich method using the monoclonal antibody, and a method for measuring the same. It relates to the measurement kit to be used.

[0002]

2. Description of the Related Art IL-5 is a T cell-replacing factor (T cell-repla
cing factor), first activated B cells
The final step to differentiate into immunoglobulin-secreting cells
(J. Immunol.125, 26
46, 1980, Kiyoshi Takatsu et al.). Recently, mice and chicks
CDNA encoding IL-5 was isolated (Nature
324, 70, 1986, Tatsuo Kinashi et al .; Cell14, 9148, 1
986, Chihiro Azuma et al.) Purified IL-5 is available
Has enabled extensive research, and this
IL-5 has various biological activities, such as differentiation of neutrophils.
Induction (J.Exp.Med.167, 43, 1988, Yuji Yamaguchi
Sci. USA), stimulation of B cell proliferation (Proc. Natl. Acad. Sci. USA8
Three, 437, 1986, Colin J. Sanderson et al.), Cytotoxicity (cyto
Toxic) Enhancement of T cell formation (Proc. Natl. Acad. Sci. USA84,
 4234, 1987, Kiyoshi Takatsu et al.)
Was revealed. However, normal conditions and disease states
The exact role of IL-5 in the disease state has not been elucidated.

For the measurement of IL-5, a bioassay based on its ability to induce IgM secretion in murine B-cell lymphoma BCL1 cells has been used, but this method has low sensitivity and is a routine assay. not appropriate. Moreover, BCL1 cells also respond to other cytokines such as IL-4 and GM-CSF. Therefore, for the biological research and practical use of IL-5, a method for measuring IL-5 with high specificity, high sensitivity and reliability, which is an alternative to the above method, is required.

[0004]

Accordingly, the present invention provides a method for measuring IL-5 which can be used on a daily basis, has high specificity for IL-5, and has high sensitivity, and implementation of the method. And a monoclonal antibody against IL-5, which is the premise of this technology.

[0005]

Means for Solving the Problems The present inventor has succeeded in obtaining a hybridoma producing an anti-IL-5 monoclonal antibody useful for measuring IL-5, and has a high sensitivity and specificity for IL-5. We succeeded in developing a sandwich measurement method and a kit therefor. Therefore, the present invention relates to IL-
5, a monoclonal antibody producing said monoclonal antibody, said monoclonal antibody,
Provided are a method for measuring IL-5 by a sandwich method using a polyclonal antibody against IL-5, and a measurement kit for use in the method.

[0006]

DETAILED DESCRIPTION As an immune system for producing a hybridoma that produces the monoclonal antibody of the present invention, any IL-5-containing preparation can be used. Preferably, recombinant interleukin 5 is used. The production method is described in detail in, for example, JP-A-63-185387.
The Escherichia coli into which the expression vector containing the gene encoding 5 has been introduced has been deposited as Microengineering Research Co., Ltd. No. 1477 (FERM BP-1477).

The production of hybridomas is described in Milstein et al., N.
, 256 , 495, 1975. A specific method is described in Example 1.

Preparation of the monoclonal antibody can be carried out by culturing the hybridoma in a conventional medium or by infecting the peritoneal cavity of an animal and collecting ascites, the latter method being more preferred. In this method, a hybridoma is first inoculated into the abdominal cavity of an animal such as a BALB / c mouse, and ascites is collected from the animal. Next, the ascites is clarified according to a conventional method such as centrifugation, and then applied to a carrier on which protein A that specifically adsorbs immunoglobulin is immobilized, for example, protein A-Sepharose CL4B, and the adsorbed antibody is subjected to a conventional method. The purified monoclonal antibody is obtained by elution according to

Preparation of a polyclonal antibody can also be performed according to a conventional method. For example, purified recombinant human IL-5
Is suspended in Freund's complete adjuvant, which is injected into animals, for example rabbits, for immunization and boosting.
Blood is collected 1 to 2 weeks after the final booster, serum is obtained according to a conventional method, and a protein fraction containing the target polyclonal antibody is obtained, for example, as ammonium sulfate 0 to 50% saturated fraction by ammonium sulfate salting out. This is then combined with a carrier-bound Protein A, such as Protein A-Sepharose CL
Adsorb immunoglobulin by applying to -4B. Next, the bound immunoglobulin is eluted according to a conventional method, and then applied to a carrier on which IL-5 is immobilized, for example, Sepharose 4B on which IL-5 is immobilized, and only a polyclonal antibody against IL-5 is adsorbed. It is eluted according to a conventional method to obtain a purified polyclonal antibody.

[0010] The sandwich measurement method of the present invention can be performed according to a conventional method. That is, first, a monoclonal antibody against IL-5 is adsorbed and fixed on the surface of a suitable solid support, and then this antibody is brought into contact with a sample expected to contain human IL-5. This allows humans in the sample
IL-5 specifically binds to the monoclonal antibody immobilized on the solid support, resulting in human IL-
5 is immobilized on a solid support via a pre-immobilized monoclonal antibody.

Next, the above carrier is used as a secondary carrier by IL-5.
In contact with a solution containing a polyclonal antibody against As a result, the polyclonal antibody binds to a second epitope of IL-5 that has not been used for binding to the monoclonal antibody, and the polyclonal antibody binds to the solid support via the previously immobilized monoclonal antibody and IL-5. Bind, in which case the immobilized amount of polyclonal antibody is the amount of IL-5 in the sample, ie, immobilized
Reflects the amount of IL-5.

Thus, the amount of IL-5 in a sample can be determined by measuring the amount of immobilized polyclonal antibody. The method for measuring the amount of the immobilized polyclonal antibody will be described later in detail.

In the above method, the solid carrier includes:
For example, a microtiter plate such as a polyvinyl chloride microtiter plate or a polystyrene microtiter plate can be used. Immobilization of the monoclonal antibody can be performed, for example, by diluting the monoclonal antibody into an appropriate buffer such as a carbonate buffer or a phosphate buffer, applying the diluted solution to the surface of the solid support, and then heating the solution at 4 ° C. to 37 ° C. for 30 minutes. This can be done by incubating for at least a minute.

Next, the solid carrier is washed with a washing solution such as Tween.
Washing is performed several times with a buffer containing a surfactant such as 20 such as a phosphate buffer, a Tris buffer, a borate buffer or the like to remove unadsorbed monoclonal antibodies.

Next, the solid support is treated with a blocking buffer to block free binding groups on the surface of the solid support. As a blocking buffer, for example, 1 to several%
Buffers containing bovine serum albumin (BSA), ovalbumin, skim milk, etc., for example, Tris buffer, phosphate buffer, borate buffer, etc. can be used. This can be done by incubation for more than one minute.

Next, the blocking buffer is removed by washing the solid carrier. This washing can be performed in the same manner as the washing after fixing the monoclonal antibody. Thus, a solid carrier is prepared.

Next, the solid carrier is brought into contact with a sample diluted in a suitable buffer, for example, a phosphate buffer, a Tris buffer, a borate buffer or the like. This contact is 4
It can be carried out by incubation at 30 ° C. to 37 ° C. for 30 minutes or more. Next, the solid carrier is washed as described above.

Next, the solid carrier is contacted with a buffer containing a secondary antibody, that is, a polyclonal antibody against IL-5. As the buffer in this case, for example, a phosphate buffer, a Tris buffer, a borate buffer, or the like can be used. This contact is performed by incubation at 4 ° C. to 37 ° C. for 30 minutes or more. Next, the solid carrier is washed as described above.

Next, the detection and measurement of the secondary antibody bound and immobilized as described above can be performed according to any conventional method. The secondary antibody itself can be labeled with, for example, a radionuclide, a fluorescent substance, an enzyme, or the like, and directly detected and measured. However, in a preferred method of the present invention, a tertiary antibody specific for the secondary antibody is used, and this tertiary antibody is labeled by various methods.

The tertiary antibody is an antibody of an animal of a species different from the species of the animal used for preparing the secondary antibody, wherein the antibody against the immunoglobulin of the animal species used for preparing the secondary antibody or a fragment thereof is used. Used. For example, when the secondary antibody is prepared using rabbits, a goat antibody against rabbit immunoglobulin can be used as the tertiary antibody.

For labeling the tertiary antibody, a conventional labeling substance such as a radionuclide such as ¹²⁵ I, ³ H, ¹⁴ C; a fluorescent substance such as fluorescein isothiocyanate (F
ITC), rhodamine, Texas Red; enzymes such as horseradish peroxidase, alkaline phosphatase, urease and the like can be used. In a preferred embodiment of the present invention, an enzyme is used as a label for the tertiary antibody, and the sandwich method of the present invention is performed as an enzyme-linked immunosorbent assay (ELISA). For detection of an enzyme, a substrate corresponding to the enzyme,
Preferably, a chromogenic enzyme substrate is used. For example, when peroxidase is used as an enzyme, hydrogen peroxide is used as the substrate, and as a chromogenic reagent, for example, orthophenylenediamine, 3,3 ', 5,5'-tetramethylbenzidine is used. Are used.

The present invention also provides a kit for measuring IL-5, which kit comprises at least a monoclonal antibody against IL-5 and a polyclonal antibody against IL-5. The monoclonal antibody may be in solution or lyophilized form, or may be immobilized on a solid carrier. Where the kit comprises a monoclonal antibody immobilized on a solid support, the solid support is preferably after being treated with a blocking agent.

[0023] The measurement kit can further include, as a third element, a labeled tertiary antibody that specifically binds to the polyclonal antibody (secondary antibody). In this case, when the label of the tertiary antibody is an enzyme, a coloring reagent containing a substrate of the enzyme can be further included in the kit. However, a commercially available product can be used as the enzyme-labeled tertiary antibody and the corresponding coloring reagent, and they are not essential elements of the kit of the present invention.

The assay kit of the present invention can also contain, as optional components, a buffer for diluting a sample, a buffer for diluting various reagents, a washing solution, and the like.

Next, the present invention will be described more specifically with reference to examples.

Example 1 Preparation of hybridoma Hybridoma was prepared according to Milstein et al., Nature, 256 ,
495, 1975. That is, for example, purified recombinant human IL
-5 is suspended in Freund's complete adjuvant, and the mice (BALB / c) are intraperitoneally injected three times every 7 to 10 days to immunize the mice. Three days after the final immunization, spleen cells (splenocytes) of the mouse and mouse myeloma cells were fused using polyethylene glycol, and HAT medium (RPMI
Fusion cells (hybridomas) with 1640 + 10% serum in medium containing hypoxanthine, aminopterin and thymidine)
After selective culturing, primary screening was performed on the culture supernatant by reaction with human IL-5 using ELISA.

Hybridomas that were determined to be positive in the above screening were further subjected to subcloning by the limiting dilution method to obtain a single clone (monoclone).
And The subclass of the antibody (ie, MoAb) produced by the single cloned hybridoma was identified using a commercially available mouse globulin identification kit.

As described above, four hybridoma clones D138, C213, D171 and E060 were obtained. In this specification, for the sake of convenience, the labeling of the hybridoma and the labeling of the monoclonal antibody produced by the hybridoma are performed using the same symbols. The hybridoma D138 has been deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as Micromachining Article No. 3289 (FERM BP-3289).

Example 2 Preparation of Monoclonal Antibody The obtained hybridoma was inoculated into the abdominal cavity of a BALB / c mouse, and ascites was collected. 20 ml of the ascites fluid obtained as described above was clarified by centrifugation, and then a 35 ml protein A-Sepharose CL-4B column (Pharmacia LKB Biotechnology,
After the column was washed with 1.5 M glycine buffer (pH 8.9) containing 3 M NaCl, the monoclonal antibody adsorbed on the column was washed with 0.1 M citrate buffer (pH 4.0). Eluted. The same operation was performed for other hybridomas to obtain corresponding monoclonal antibodies.

The four types of monoclonal antibodies D138, C213, D171, and E060 obtained as described above were
_{gG 2a, IgG 1, IgG 2a} , and belongs to the immunoglobulin subclass IgG _1. The binding of these antibodies is specific for human IL-5 and cross-reacts with other cytokines, such as interferon gamma, IL-4, TNF-α and GM-CSF, and mouse recombinant IL-5. Not shown. Furthermore, the epitopes (antigenic determinants) recognized by these antibodies are all peptide sites.

Example 3 Preparation of polyclonal antibody 50 μg of recombinant IL-5 was suspended in Freund's complete adjuvant, and the rabbit was immunized three times every 3 to 5 weeks, and blood was collected 7 days after the final immunization. , And serum was obtained according to a conventional method. After ammonium sulfate was added to 20 ml of the serum thus obtained to make it 50% saturated, the precipitate of the protein fraction was recovered by centrifugation, and ammonium sulfate was removed by dialyzing against PBS. Next, the protein fraction was applied to a protein A-Sepharose CL-4B column to adsorb immunoglobulin, and the immunoglobulin fraction was eluted in the same manner as in the purification of the monoclonal antibody.

Next, this immunoglobulin fraction was treated with IL-5-fixed Sepharose 4B (IL-5 Sepharose 4B;
200 μg hI using CNBr-activated Sepharose 4B manufactured by Pharmacia and bound to IL-5 according to a conventional method;
(L-5 / 0.3 g Sepharose) to specifically adsorb the antibody against IL-5, which was then eluted with 0.1 M glycine buffer (pH 2.5). Thus, IL-5
A purified polyclonal antibody against was obtained.

Example 4 Measurement of IL-5 by ELISA Method 96-well polyvinyl chloride microplate (Falcon
In the central 60 wells of 3912), monoclonal antibody (10) dissolved in buffer A (0.1 M carbonate buffer, pH 9.5) was added.
(μg / ml) solution, cover the microplate with a plastic cover (Falcon 3913), and incubate at 4 ° C. overnight.

After the solution was poured from the wells of the microplate, 200 μl of buffer B (0.1% Tween) was added to the wells.
phosphate buffer containing en 20) and after 3 minutes,
The liquid was removed as described above. This was repeated three more times to wash the wells.

Next, 200 μl of a solution of 1% bovine serum albumin in phosphate buffer was added to each well, and the wells were incubated at room temperature for 30 minutes to block non-specific binding sites in the wells. Next, it was washed three times as described above. A solution of IL-5 diluted in buffer C (buffer B containing 10% fetal bovine serum) (concentration 500-7.8 pg /
ml) (standard solution) or 100 μl of test sample diluted in buffer B was added to the wells, covered, incubated overnight at room temperature, and washed 5 times as described above.

Next, a polyclonal antibody (secondary antibody) against IL-5 (concentration 40 ng / ml) diluted in buffer D (buffer B containing 3% polyethylene glycol 6000) was added.
00 μl was added to each well, covered, and incubated at room temperature for 4 hours. Next, 2
Washed twice. Next, 100 μl of a solution of a labeled tertiary antibody conjugated with a peroxidase to a goat anti-rabbit immunoglobulin antibody in buffer D was added and covered and incubated for 4 hours at room temperature. Next, it was washed five times by the above method.

0.0027% in 0.1 M acetate buffer (pH 5.5)
Freshly prepared substrate solution containing hydrogen peroxide and 0.55 nM 3,3 ', 5,5'-tetramethylbenzidine
After adding 100 μl to each well and incubating at room temperature for 10 minutes to carry out the enzyme reaction, the reaction was stopped by adding 100 μl of 1N HCl. Next, the absorbance at 450 nm was read by an ELISA plate reader.

When D138, C213, D171, and E060 were used as monoclonal antibodies to measure IL-5 standard samples at various concentrations according to the above method, the measurement sensitivity was as shown in Table 1 below. .

[0038]

[Table 1]

By using the monoclonal antibody D138 or D171 as the primary antibody (capture antibody), the detection limit is 7.8.
High sensitivity measurement of pg / ml, which cannot be obtained by the conventional method, can be performed. As an example, when the monoclonal antibody D138 is used in FIG.
The calibration curve between the absorbance in nm is shown.

Next, in order to examine the specificity of the sandwich method of the present invention, various cytokines, cell culture supernatant, and human serum were measured using D138 as a capture antibody, and these were detected by the method of the present invention. I checked whether it would be done. Table 2 shows the results.

[0041]

[Table 2]

As is clear from the table, the assay of the present invention did not detect human cytokines other than IL-5, and did not detect mouse IL-5. In addition, no component in the serum of a healthy person was detected. Therefore, the sandwich assay of the present invention has extremely high specificity for human IL-5.

Next, the simultaneous reproducibility of the measurement method of the present invention (the degree of variation when a plurality of measurements are made on the same sample at the same time) and the daily reproducibility (the same sample was measured on different days) The difference in the measurement results in each case) was examined.

Three kinds of samples A, B and C having different concentrations of IL-5 were measured in quintuple, while the same sample was
Measurements were taken five times on separate days, once a day. Table 3 shows the average value and the standard deviation and the ratio of the standard deviation to the average value (CV%) for each sample.

[0045]

[Table 3]

As described above, both the simultaneous reproducibility and the daily reproducibility were good.

[Brief description of the drawings]

FIG. 1 shows an example of the relationship between human IL-5 concentration and absorbance when human IL-5 is measured by sandwich ELISA using monoclonal antibody D138 against human IL-5 as a capture antibody.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12P 21/08 C12R 1:91) (72) Inventor Toshihiro Nakanishi 1-1-1 Wakayamadai, Shimamotocho, Mishima-gun, Osaka Suntory (56) References JP-A-59-39832 (JP, A) JP-A-1-307666 (JP, A) JP-A-1-59068 (JP, A) European Patent Application Publication 367596 ( EP, A 1) Biochem. , Vol. 106, (1989), p. 23-28 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12P 21/08 C12N 5/12-5/28 G01N 33/53 G01N 33/577 C12N 15/06-15/08 BIOSIS (DIALOG) ) MEDLINE (STN) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A method for measuring human interleukin 5 without substantially reacting with mouse interleukin 5.
A monoclonal antibody against human interleukin 5 having a detection limit of 500 pg / mL or less . 2. The method according to claim 1, wherein said detection limit value is 31 pg / mL or less.
The monoclonal antibody according to claim 1. [Claim 3] Hybridoma (Jikoro No. 3289)
The monoclonal antibody according to claim 1 or 2 , which is produced by: 4. A sandwich assay using the monoclonal antibody against human interleukin 5 and the polyclonal antibody against human interleukin 5 according to any one of claims 1 to 3. Measurement method. 5. A human comprising the monoclonal antibody against human interleukin 5 and the polyclonal antibody against human interleukin 5 according to any one of claims 1 to 3, which may be immobilized on a solid carrier. Kit for measuring interleukin 5. 6. A hybridoma that produces the monoclonal antibody according to any one of claims 1 to 3 .