JP2996683B2 - Monoclonal antibody, measuring method using the same, and purification and concentration method - Google Patents

Description

The present invention relates to monoclonal antibodies, and more particularly to ω
-3 relates to a monoclonal antibody against a compound having a double bond. The present invention also relates to a hybridoma cell line that produces such a monoclonal antibody, an immunoassay using the monoclonal antibody, an immunohistochemical search method, and a purification and concentration method.

[Conventional technology]

Eicosapentaenoic acid (hereinafter abbreviated as EPA), one of the highly unsaturated fatty acids, has been suggested to be related to adult diseases such as myocardial infarction and arteriosclerosis in humans and experimental animals, and has attracted attention as a functional food and pharmaceutical However, detailed in vivo behavior has not been elucidated yet. EPA is said to produce various prostanoids in the same way as the arachidonic acid cascade, but its analysis has not been performed sufficiently due to its small amount and short half-life.

[Problems to be solved by the invention]

However, since human EPA and related polyunsaturated fatty acids are closely related to the onset of various diseases in humans, quantitative analysis of the behavior of these polyunsaturated fatty acids, that is, compounds having an ω-3 double bond, in vivo is performed. Is required,
In addition, there is a need to study the relationship between quantitative variation of compounds having an ω-3 double bond and diseases. In view of these, the present inventors have conducted intensive studies and completed the present invention.

[Means for solving the problem]

The present inventors have conducted intensive studies to analyze polyunsaturated fatty acids in vivo and related substances, that is, compounds having an ω-3 double bond, and have obtained a conjugate of a compound having an ω-3 double bond and a protein. As a result of preparing an anti-ω-3 double bond monoclonal antibody as an immunogen and conducting various studies, a monoclonal antibody having specific reactivity with a compound having an ω-3 double bond was obtained, thereby completing the present invention. Reached.

1. A monoclonal antibody that exhibits high reactivity with an ω-3 double bond, has no reactivity with a double bond other than ω-3, or is below the detection limit, and belongs to the class of IgG.

2. The monoclonal antibody according to 1 above, wherein the ω-3 double bond is a constituent of an unsaturated aliphatic compound.

3. The monoclonal antibody according to the above 1, wherein the ω-3 double bond is a constituent of unsaturated fatty acids and metabolites thereof.

4. The monoclonal antibody according to the above 1, wherein the ω-3 double bond is a constituent of eicosanoid and a metabolite thereof.

5. The monoclonal antibody according to the above 1, wherein the ω-3 double bond is a constituent of a prostanoid and a metabolite thereof.

6. Fusion of spleen cells and myeloma cells capable of producing a monoclonal antibody capable of recognizing ω-3cis double bond obtained using a conjugate of a compound having an ω-3 double bond and a carrier protein as an immunogen, Hybridoma obtained by

7. Fusion of spleen cells and myeloma cells capable of producing a monoclonal antibody capable of recognizing ω-3 cis double bond obtained using a conjugate of a compound having ω-3 double bond and carrier protein as an immunogen And culturing the hybridoma cells obtained by cloning to produce the monoclonal antibody according to any of 1 to 5 above.

8. The method for producing the monoclonal antibody according to the above 7, wherein the spleen cells are mouse spleen cells, and the myeloma cells are mouse-derived myeloma cells.

9. A composition for immunological measurement or detection comprising a carrier acceptable for measurement and the monoclonal antibody according to any one of 1 to 5 above.

10. A method for purifying and concentrating a compound having an ω-3 double bond from a biological sample containing a compound having an ω-3 double bond, wherein the monoclonal antibody according to any one of the above 1 to 5 is used. Purification of the ω-3 double bond,
Concentration method.

It is about.

Preferably IgG are as a class of the monoclonal antibodies of the present invention, it is practically advantageous since excellent and have high antibody titers to inter alia the IgG ₁ antibody specificity. The immunological search method and assay method using this antibody are based on ω
It is useful for analyzing compounds having a -3 double bond.

The method for producing a monoclonal antibody capable of recognizing the compound having an ω-3 double bond of the present invention is as follows.

Animals are immunized with cis-3-hexen-1-ol and their spleen cells are harvested. In this step, cis-
Since 3-hexen-1-ol cannot be used alone as an immunogen, it cannot be used as an immunogen after being bound to an appropriate carrier protein. As the carrier protein, a protein used for the purpose of enabling the immunocompetent cell to recognize a substance serving as a hapten, for example, keyhole limpet hemocyanin, albumin, etc., may be used, but other proteins or hapten may be used. It does not hinder the use of. It does not prevent the use of other known compounds as the compound having an ω-3 double bond.

Next, in the fusion of the spleen cells and myeloma cells (myeloma) of the obtained immunized animal, any of a known method using polyethylene glycol, a fusion method using Sendai virus, and a method using electric pulse may be used. . The culture supernatant of the obtained hybridoma was subjected to ELISA using a conjugate of a compound having an ω-3 double bond as an immunogen and a carrier protein, EIA using a test tube, and further using a radiolabel. The reactivity was examined by direct or inhibition test by RIA method, and from the positive hybridoma,
A monoclonal antibody is prepared by the limiting dilution method. still,
The spleen cells and myeloma cells for producing a monoclonal antibody via a hybridoma as described above are preferably from the same animal species, and mouse spleen cells and mouse-derived myeloma cells are particularly suitable for practical use.

The hybridoma MH-N according to the present invention is manufactured by
Deposit No. 08 (FERM BP-2808) with the Research Institute of Microbial Industry and Technology.

This monoclonal antibody-producing hybridoma can be frozen and stored, and can be cultured in a large amount by an appropriate method. The antibody can be obtained from the culture supernatant, or the hybridoma can be transplanted into an animal to form a tumor, and the desired monoclonal antibody can be obtained from the ascites or serum. Purification of the monoclonal antibody of the present invention is performed by salting out and using an affinity column or DEAE column.

The present invention relates to a monoclonal antibody capable of recognizing such a compound having an ω-3 double bond. 1. It has high reactivity with an ω-3 double bond and has no reactivity with a double bond other than ω-3. Or less than the detection limit, 2. has high reactivity to fatty acids called ω-3 such as EPA, and does not recognize reactivity with fatty acids other than ω-3 such as ω-6 or is below the detection limit. Yes, 3. EPA metabolite in vivo
It has high reactivity with type 2 eicosanoids and no reactivity with type 2 or type 1 eicosanoids or below the detection limit.
Prostaglandins, leukotrienes and hydroxy fatty acids derived from arachidonic acid and dihomo γ-linolenic acid, respectively. 4. The corresponding antigen can be detected in human and animal biological components, such as serum or urine.
2. It is possible to separate and purify type 3 eicosanoids from type 2 eicosanoids by utilizing the properties of the above 2 and further concentrate them, which can be a useful means for trace analysis such as GC / MS. It has been found that EPA can be used as a marker for the prevention of adult diseases such as cerebral thrombosis and myocardial infarction.

That is, the present invention uses a monoclonal antibody capable of recognizing a compound having an ω-3 double bond in a method of measuring, purifying, isolating, enriching, or microquantifying an in vivo component by an immunological method using an antibody. The present monoclonal antibody can be used for basic research and clinical application by utilizing the above-mentioned properties.

In the measurement method of the present invention, a direct method, a competitive inhibition method, a sandwich method, a competitive inhibition method using a sandwich, and the like are used.It is preferable to immobilize a biological sample or a monoclonal antibody on a carrier, and the immobilization method is A known method can be adopted. As the carrier, a solid-phase ball, bead, gear, microplate or the like using, for example, polystyrene, polyethylene, polyacrylate, Teflon, polyacetate, polyacetal, latex or the like is preferably used.

Further, the monoclonal antibody is used after being labeled, but the method and means are not limited at all, and known methods and means, for example, an anti-immunoglobulin antibody or a staphylococcus labeled with a radioactive substance or an enzyme or a fluorescent substance It can be measured by a secondary reaction with protein A. As the labeling agent, horseradish peroxidase substrate as 2,2 Ajinoji - [3 ethylbenzthiazoline sulfonic acid] ammonium acid _{(ABTS) -H 2 O 2,} 5-
Aminosalicylates -H ₂ O _2, o-phenylenediamine -H
₂ O ₂ , 4-aminoantipyrine-H ₂ O _{2 and the} like, as a substrate of β-D-galactosidase, β-D-galactopyranoside, o-nitrophenol-β-D-galactopyranoside, etc. be able to. In addition to these reagents, known reagents such as a solubilizer, a detergent, and a reaction terminator are used for the measurement.

Next, the mode of the immunoassay according to the present invention is described using ω-
The method for measuring a compound having a triple double bond will be described as an example.

That is, a monoclonal antibody is added at 5 μg / well to a 96-well microplate or latex beads, and then left at 4 ° C. for 12 to 24 hours.

Next, non-specific adsorption of antibodies and other proteins is prevented by adding 100 μl of phosphate buffered saline (PBS) containing 1% BSA to each well. Next, 50 μl of the sample was added to each well, and after stirring well, 37 ° C.
And leave for 30 minutes. After thoroughly washing the reaction mixture with a phosphate buffered saline, a biotin-labeled monoclonal antibody (1 μg / μ) having a recognition site different from that of the monoclonal antibody against the compound having an ω-3 double bond is added to each well.
Add 50μ. A known method was used for biotin labeling of the monoclonal antibody. After stirring well, leave at 37 ° C for 30 minutes. After thoroughly washing the reaction mixture with phosphate buffered saline, a 5000-fold dilution of horseradish peroxidase (HRP) -labeled avidin is added to each well in an amount of 50 μl. After stirring well, leave at 37 ° C for 30 minutes. The reaction mixture is thoroughly washed with phosphate buffered saline, and after draining, the substrate solution (orthophenylenediamine (OPD) 1
mg / ml, pH 4.5 acetate buffer) at 100 ° C.
Let react for minutes. EIA is the absorbance at 492 nm of each well.
Ω-3 present in the sample by measuring with a reader
The compound having a double bond is quantified.

The present invention also includes a method for detecting the binding protein of a compound having an ω-3 double bond in a biological component by immunoblotting using a monoclonal antibody. The immunoblot is performed by a known method, and there is no limitation on the method and material. Specifically, the search method of the present invention separates a biological component by SDS-PAGE, transfers it to a known membrane such as nitrocellulose using electric or capillary phenomenon, and then uses a known monoclonal antibody. Is used to detect a compound having an ω-3 double bond on the membrane and to determine the possibility of pathological change, to determine the degree of progression of the disease state, and to determine the dose of a drug by the immunochemical search method described above. It is.

Further, the present invention provides a method for immunohistochemically searching for the presence of a compound having an ω-3 double bond in a tissue, wherein the monoclonal antibody is capable of recognizing the compound having a compound having an ω-3 double bond. A search method characterized by binding to a part of a tissue by an immune reaction is also included. The tissue in such an immunohistochemical search method is
An animal, particularly a human cell or tissue section, is practically preferably used.

More specifically, the search method of the present invention provides that the
A monoclonal antibody capable of recognizing a compound having a double bond is caused to act to generate an immune reaction, and the monoclonal antibody is allowed to bind to a site of the compound having an ω-3 double bond in a tissue. For example, treatment with a biotinylated antibody recognizing such a monoclonal antibody, further labeling the site of monooxygenase in the tissue by staining or the like by a method such as the action of an enzyme-labeled avidin to search for the normality of the tissue, This enables a search for an existing part. Such an immunohistochemical method is performed by a known method, and there is no limitation on the method and material.

The present invention also includes a method for separating, purifying, and concentrating a compound having an ω-3 double bond, which comprises using a monoclonal antibody capable of recognizing a compound having an ω-3 double bond. These techniques have been found to be useful for trace quantification of type 3 eicosanoids in vivo, but they can be used for the separation, purification, and concentration of any other reactive compounds.

〔Example〕

Next, the present study will be described with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) Preparation of immunogen A conjugate of cis-3-hexen-1-ol with human serum albumin (HSA) is prepared by the active ester method. For example, a succinic ester is prepared by reaction with succinic anhydride, and an N-succiniicidyl ester of cis-3-hexen-1-ol is prepared in the presence of carbodiimide, and cis-3-hexene is quantitatively reacted with HSA. An HSA conjugate of -1-ol (3cis-HSA) is obtained.

(2) Preparation of monoclonal antibody 3cis-HSA obtained in (1) was mixed with Freund's Complete adjuvant in an equal amount,
After the emulsion was formed, 30 μg per mouse was subcutaneously administered to BALB / c mice. From the second time on, sterile saline 0.2m
30 μg of 3-cis-HSA was dissolved in l, and intravenously administered three times at 10-day intervals. The final immunization was performed using the same solution and the same volume of 3 cis-HSA.
Was administered intravenously.

Three days after the final immunization, spleen cells excised from hyperimmunized mice were fused with BALB / c mouse-derived myeloma cell line SP2 / 0-Ag14 using polyethylene glycol (PEG) 4000. The cells were added to a 96-well plate at 100 μ / well, and after 24 hours, half of the medium was replaced with HAT medium, and the medium was replaced every two days. After 7-10 days, the growth of HAT resistant hybridomas is observed. At this time, the medium was changed to HT, cultured for about 10 days, and then replaced with a hybridoma growth medium.

Screening of antibody-producing cells is performed by ELISA using 3cis-HSA used for immunization as an antigen,
A hybridoma that strongly reacts with the antigen can be selected, and a trans-3-hexen-1-ol HSA conjugate (3 trans-HSA) can be prepared by the same method as 3cis-HSA using a carrier protein, a spacer and a trans double bond. Clones having reactivity to were excluded. Further, by using 3cis-HSA as an antigen, adding various concentrations of cis-3-hexen-1-ol at the time of adding the hybridoma culture supernatant, and selecting a hybridoma that produces an antibody that receives a dose-dependent inhibitory effect thereof, Antibodies with specificity for compounds with an ω-3 double bond were selected. The cell line selected here is cloned by the limiting dilution method,
A monoclonal antibody-producing hybridoma was established. still,
For measurement of the ω-3 double bond, a hybridoma was transplanted into a BALB / c mouse pre-administered with pristane, and ascites was collected on the 10th to 15th days, and a monoclonal antibody purified by a 50% saturated ammonium sulfate salting-out method was used. Was.

(3) Class of Monoclonal Antibodies against nature omega-3 double bond of monoclonal antibody (subclass) was IgG _1. The antibody titer was 5000-fold (when the antibody purified from mouse ascites was diluted in two steps and subjected to ELISA for reaction with purified 3-cis-HSA, the dilution ratio at which the highest absorbance was maintained was defined as the antibody titer). Was.

Example 2 Microquantification of in vivo eicosanoids of a monoclonal antibody having specificity for a compound having an ω-3 double bond using an immunoaffinity column.

(1) Preparation of immunoaffinity column Approximately 1 g of a column carrier such as Affigel 10 was activated by a known method, and approximately 10% of monoclonal antibody purified from ascites was obtained.
The antibody-bound carrier obtained by quantitative reaction with
m, packed in a column container having a diameter of 0.5 cm to prepare an immunoaffinity column for the compound having an ω-3 double bond. The carrier used and the activation method may be any known method.

(2) Microquantification of eicosanoids in vivo From 1 ml of human plasma, Chem Elut1001 (Analyticheminter
natinal) and then Sep-
Obtaining eicosanoids fraction using Pak C ₁₆ column (manufactured by Waters). However, since the amount of type 3 eicosanoids was very small and the chemical properties were similar to those of type 2 eicosanoids, the separation was difficult. Therefore, Embodiment 2
When 5 ml of the eicosanoid fraction is added to the immunoaffinity column prepared in (1), the type 3 eicosanoid having an ω-3 double bond binds to the antibody in the column, and the type 2 eicosanoid passes. Further, a 95% solution of methanol is added to the column to recover the eluted type 3 eicosanoid, and a type 3 eicosanoid in human plasma, {17-6-keto-PGF1α, measured by a known method such as GC-MS or the like.
Was 6 pg / ml. By this method, ω-
A trace amount of type 3 eicosanoid, which is one of the trace components having three double bonds, can be measured in minute amounts, and a detailed study of the biological activity of the molecule in vivo has become possible. The purification of the eicosanoid fraction, the elution conditions of the immunoaffinity column, and the like can be performed by any of the known methods.

Example 3 (1) Separation, purification and concentration of type 3 eicosanoid Bovine aortic endothelial cells were homogenized and then mixed with 1 mg of EPA at 37 ° C.
Incubate for 30 minutes to obtain a fraction containing Δ17-6-keto-PGF1α. The fraction is subjected to extraction of total lipids using Chem Elut1010, and an eicosanoid fraction is obtained using a Sep-Pak C18 column. 10 ml of the mixture was added to the immunoaffinity column prepared in (1) of Example 2, and the eicosanoid type 3 was recovered with 10 ml of a 95% methanol solution, and 180 μg of Δ17-6-keto-PGF1α was obtained by a method such as HPLC. .

By this method, the separation and purification of type 3 eicosanoids,
Concentration became possible, and preparation of type 3 eicosanoid became easy.

Example 4 Inhibition ELISA between a fatty acid having an ω-3 double bond and another fatty acid was examined. As antigen on ELISA plate
3cis-HSA was fixed at 10 μg / well, and ω-3 was used as an inhibitor.
Using EPA, DHA, α-linolenic acid having a double bond and other arachidonic acid, linoleic acid, and oleic acid, the reactivity was examined by the reduction rate of OD405 when the non-addition was set to 100%. A similar study was also made for prostaglandins. Namely, prostaglandin E ₃ having an ω-3 double bond, Δ17-6-keto-PGF _1α and prostaglandin E ₂ having an ω-6 double bond, 6-keto-PGF
_1α was performed.

As a result, as shown in FIG. 1, a strong inhibitory effect was observed for fatty acids having an ω-3 double bond. On the other hand, ω−
Almost no inhibitory effect was observed for fatty acids other than the triple bond, especially oleic acid. Moreover, it observed high inhibitory effect for as Δ17-6- keto-PGF _{l [alpha]} shown in FIG. 2, the inhibitory effect on 6-keto-PGF _{l [alpha]} was low.

〔The invention's effect〕

The present invention provides a novel monoclonal antibody that specifically recognizes an ω-3 double bond, and uses the same to measure and search for compounds having an ω-3 double bond, such as eicosanoid-related substances, in a living body. Methods and purification and concentration methods can be provided.

[Brief description of the drawings]

1 and 2 are diagrams showing the inhibitory effect on various fatty acids.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI G01N 33/577 C12N 5/00 B // C12N 15/02 15/00 C (C12P 21/08 C12R 1:91) (72) Inventor Masatoshi Ishibashi 3-31-12 Shimo, Kita-ku, Tokyo Nippon Kayaku Co., Ltd.

Claims (10)

(57) [Claims] 1. High reactivity with an ω-3 double bond;
A monoclonal antibody having no reactivity with a double bond other than 3 or below the detection limit and belonging to the class of IgG. 2. The monoclonal antibody according to claim 1, wherein the ω-3 double bond is a constituent of an unsaturated aliphatic compound. 3. The monoclonal antibody according to claim 1, wherein the ω-3 double bond is a constituent of unsaturated fatty acids and metabolites thereof. 4. The monoclonal antibody according to claim 1, wherein the ω-3 double bond is a constituent of eicosanoid and its metabolite. 5. The monoclonal antibody according to claim 1, wherein the ω-3 double bond is a constituent of a prostanoid and a metabolite thereof. 6. An ω-3cis obtained by using a conjugate of a compound having an ω-3 double bond and a carrier protein as an immunogen.
A hybridoma obtained by fusing spleen cells capable of producing a monoclonal antibody capable of recognizing a double bond with myeloma cells. 7. An ω-3cis obtained by using a conjugate of a compound having an ω-3 double bond and a carrier protein as an immunogen.
Hybridoma cells obtained by fusing and cloning spleen cells capable of producing a monoclonal antibody capable of recognizing a double bond with myeloma cells, and culturing the hybridoma cells.
6. A method for producing the monoclonal antibody according to any one of items 5 to 5. 8. The method according to claim 7, wherein said spleen cells are mouse spleen cells, and said myeloma cells are mouse-derived myeloma cells. 9. A composition for immunological measurement or detection, comprising a carrier acceptable for measurement and the monoclonal antibody according to any one of claims 1 to 5. 10. A method for purifying and concentrating a compound having an ω-3 double bond from a biological sample containing a compound having an ω-3 double bond, according to any one of claims 1 to 5, wherein Ω-3 characterized by using
A method for purifying and concentrating a compound having a double bond.