JPH06189785A - Production of anti-d@(3754/24)+)-6-erithro-neopterin monoclonal antibody - Google Patents

Abstract

PURPOSE:To provide a method for production of D(+)-6-erithro-neopterin monoclonal antibody, capable of producing this antibody according to a ready method without requiring complicated chemical synthesis of a D(+)-6-erithro- neopterin derivative as an intermediate and without requiring an operation for absorption of the antibody. CONSTITUTION:This production method comprises allowing a crosslinking reaction to take place between D-(+)-6-erithro-neopterin and an immunologically activating protein or a sulfur-containing protein, immunizing an animal with the resultant cross-linked material as an immunogen and inducing the anti-D (+)-6-erithro-neopterin monoclonal antibody from the immunized animal.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing an anti-D (+)-6-erythro-neopterin monoclonal antibody and an immunoassay method using the monoclonal antibody.

[0002]

2. Description of the Related Art D (+)-6-erythro-neopterin is released from activated macrophages and is clinically used as an index of enhanced cellular immunity, and it is diagnosed and clinically diagnosed by viral and bacterial infections, cancer and organ transplantation. Has been attracting attention as a dynamic indicator.

Even if D (+)-6-erythro-neopterin was directly immunized for the purpose of obtaining an antibody against D (+)-6-erythro-neopterin, the molecular weight of D (+)-6-erythro-neopterin was It is known that antibodies cannot be obtained due to their small size. Therefore, D (+)
A method has been developed in which a derivative obtained by binding a spacer to -6-erythro-neopterin is chemically synthesized, and an antiserum is obtained by immunizing a rabbit or the like with an antigen binding to bovine serum albumin or the like with glutaraldehyde or the like as an antigen. (Tokuhei 2
-55434).

However, in order to obtain an immunogen chemically synthesized by this method, complicated procedures and skill are required, and it is generally difficult to utilize it, and D
In order to obtain an antibody against (+)-6-erythro-neopterin, it is necessary to remove the nonspecific component by a complicated absorption operation.

Further, Weyrer et al. Attempted to produce a monoclonal antibody using the antigen chemically synthesized by this method, but could not obtain a practical monoclonal antibody (HYBRIDOMA, 9, 71, 1990).

[0006] Conventionally, for the detection of D (+)-6-erythro-neopterin, an immunogen produced by chemical synthesis, which requires skill as described above, is used, and an antibody purified from antiserum of rabbit or the like is subjected to a complicated absorption procedure. It has been used, but a practical method for producing a monoclonal antibody and a method for using it have not yet been obtained.

[0007]

As described above, as described above,
To obtain an antibody against D (+)-6-erythro-neopterin, as disclosed in Japanese Patent Publication No. 2-55434, the chemical synthesis of a derivative of D (+)-6-erythro-neopterin is carried out, and thereafter, , Bound to bovine serum albumin had to be used. However, that method requires complicated procedures, and it has not been possible to obtain a practical monoclonal antibody from the immunogen. Furthermore, from antisera obtained from rabbits, etc., D
To obtain an antibody specific for (+)-6-erythro-neopterin, a complicated absorption operation is required.

Therefore, an object of the present invention is to eliminate D (+)-6-erythro-neopterin derivatives by these complicated chemical syntheses, and to perform D (+)-which does not require antibody absorption operation by an easy method. A method for producing a monoclonal antibody of 6-erythro-neopterin. Another object of the present invention is to provide an immunoassay method using the monoclonal antibody produced by this method.

As a result of earnest research, the inventors of the present application have found that D
By using (+)-6-erythro-neopterin directly bound to an immunostimulatory protein or a sulfur-containing protein as an immunogen, an anti-D (+)-6-erythro-neo having a high affinity by a simple operation is used. The present invention has been completed by finding that a pterin monoclonal antibody can be obtained.

That is, the present invention immunizes an animal by using D (+)-6-erythro-neopterin cross-linked with an immunostimulatory protein or a sulfur-containing protein as an immunogen and immunizes the animal with anti-D (+)-6. -Anti-D (+)-consisting of inducing erythro-neopterin monoclonal antibody
A method for producing a 6-erythro-neopterin monoclonal antibody is provided. In addition, the present invention utilizes the specific reactivity between the anti-D (+)-6-erythro-neopterin monoclonal antibody produced by the method of the present invention and D (+)-6-erythro-neopterin. An immunoassay method comprising detecting or measuring D (+)-6-erythro-neopterin.

The present invention will be described in more detail below.

In the present invention, "immunostimulatory protein"
Is a protein that enhances antibody production, and examples thereof include muramyl dipeptide, tubercle bacillus, and pertussis. The sulfur-containing protein is a protein containing sulfur, and preferable examples thereof include γ-globulin, macroglobulin, thyroglobulin, mercaptoalbumin, albumin and fibrinogen.

Crosslinking of the immunostimulatory protein or sulfur-containing protein with D (+)-6-erythro-neopterin can be carried out by reacting them with a crosslinking agent. When the immunostimulatory protein is used, glutaraldehyde, carbodiimide or the like can be used as the cross-linking agent. In this case, immunostimulatory protein, D (+)
The mixing ratio of -6-erythro-neopterin and the cross-linking agent is not particularly limited, but is 1:10:10 to 1: 1.
0 ² : 10 ³ is preferable, and the reaction is preferably 12 ° C.
It is carried out at ~ 37 ° C for about 1 to 20 hours. When a sulfur-containing protein is used, succinimide or maleimide can be used as the cross-linking agent. In this case, the mixing ratio of the immunostimulatory protein, D (+)-6-erythro-neopterin and the cross-linking agent is not particularly limited,
It is preferably about 1:10 ² : 10 ² to 1:10 ³ : 10 ³ and the reaction is preferably carried out at 12 ° C to 37 ° C for about 1 to 20 hours.

The animal is then immunized with D (+)-6-erythro-neopterin, which has been cross-linked with the protein as described above, as an immunogen. The method of immunization can be performed by a conventional method. For example, an animal such as a mouse, a rabbit, a rat, and a goat can be immunized by repeatedly inoculating it with an adjuvant several times every 2-3 weeks.

Next, a monoclonal antibody is induced from the immunized animal. This can be done by conventional methods well known in the art. That is, antibody-producing cells such as spleen cells and lymphocytes are collected from the immunized animal and fused with the tumor cell line. As the tumor cell line, a myeloma cell having an appropriate selection marker such as hypoxanthine-guanine-phospholipidyltransferase deficiency or thymidine kinase deficiency can be used. Cell fusion is described, for example, in Kohler, G. and Milstein, C.
(Nature 256,495,1975) or a method similar thereto. For the cells obtained by cell fusion, the antibody titer in the culture supernatant is measured by, for example, an enzyme immunoassay, and a hybridoma producing an anti-D (+)-6-erythro-neopterin antibody is selected. The selected hybridoma is further cloned by, for example, the limiting dilution method to obtain a clone. This clone is
For example, it can be transplanted into the abdominal cavity of BALB / c mouse to which pristane has been administered in advance, and ascites containing a high concentration of monoclonal antibody can be collected 7 to 14 days later.

The enzyme immunoassay for measuring the antibody titer can be carried out, for example, as follows.
That is, the specimen was added to the microplate coated with the above-mentioned neopterin cross-linked product, and after the reaction for a certain time and washing,
Add peroxidase-labeled anti-mouse immunoglobulin,
The reaction is continued for a certain period of time. After washing unreacted labeled antibody, O-phenylenediamine solution containing hydrogen peroxide was added and allowed to react for a certain period of time.
The absorbance of is measured.

By the method of the present invention, a monoclonal antibody having a high specificity for D (+)-6-erythro-neopterin can be obtained. This monoclonal antibody can be used in an immunoassay for detecting D (+)-6-erythro-neopterin. Immunoassay methods themselves are well known in the art, and the above monoclonal antibody can be applied to any immunoassay method. That is, if the classification is performed based on the labeling agent, it can be applied to enzyme immunoassay, fluorescent immunoassay, radioimmunoassay, immunoassay using biotin, and the like. The sandwich method,
It can be applied to the agglutination method, the competition method, and the like. According to the immunoassay method of the present invention, anti-D (+)-6-erythro-
Since the neopterin monoclonal antibody has high specificity and affinity for D (+)-6-erythro-neopterin, D (+)-6-erythro-neopterin can be detected or measured with high sensitivity.

Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

[0019]

Example 1 M. tuberculosis was suspended in PBS to a concentration of 5 mg / ml, and 10 ⁸ B. pertussis were suspended, and then 2 × 10 ^-2 M D (+)-6-erythro was suspended. -200 μl neopterin was added. 150 μl of 25% glutaraldehyde was added thereto and reacted at 37 ° C. for 3 hours. The reaction solution was dialyzed against PBS.

1 volume of this and 1 volume of complete adjuvant were mixed, and 100 μl each was added to BALB / c mice at 2-week intervals.
I was immunized twice. Four days after the final immunization, the mouse spleen was taken out, and its splenocytes and mouse myeloma P3X63.Ag8.653 and 1 were isolated.
Mixed at a ratio of 0: 1 and Kohler et al. (Nature 256,49
5,1975), cell fusion and HAT selection were performed.

The antibody titer in the culture supernatant of the well in which the growth was observed was measured by the enzyme immunoassay and further cloned by the limiting dilution method. Enzyme-linked immunosorbent assay was performed by dissolving γ-globulin in PBS at 5 mg / ml,
Add 5 μl of mercaptoethanol and add 2 × 10 ^-2 M D
200 μl of (+)-6-erythro-neopterin was added, and 200 μl of 2 × 10 ^-2 M m-maleimidobenzoyl-N-hydroxysuccinimide ester was added, and the mixture was mixed at 37 ° C. for 3 days.
What was reacted for a time was dialyzed against PBS and adsorbed on a microplate as a neopterin crosslinked product. Add the culture supernatant of the wells and incubate at 37 ℃ for 1 hour, then wash,
Add peroxidase-labeled anti-mouse immunoglobulin,
Further, the mixture was reacted at 37 ° C for 1 hour. After washing unreacted labeled antibody, 3mg / ml O-phenylenediamine solution containing 0.002% hydrogen peroxide was added and reacted at room temperature for 30 minutes, then 1N
The reaction was stopped by adding sulfuric acid, and the absorbance at 490 nm was measured.

The obtained clone (designated as 10E-4-5) was BA to which 0.5 ml of pristane was previously administered.
LB / c mice were transplanted into the abdominal cavity and 7 to 14 days later, ascites fluid containing a high concentration of monoclonal antibody was collected. These ascites were purified to IgG by ion exchange chromatography after ammonium sulfate fractionation method (45% saturation).

Example 2 Thyroglobulin was dissolved in PBS to a concentration of 5 mg / ml, 5 μl of 2-mercaptoethanol was added, and 2
× 10 ^-2 M D (+)-6-erythro-neopterin 200 μm
1 was added. 2 × 10 ^-2 M succinimidyl-4 there
200 μl of-(N-maleimidoethyl) cyclohexane-1-carboxylate was added and reacted at 37 ° C for 3 hours. The reaction solution was dialyzed against PBS.

The 1 volume and 1 volume of complete adjuvant were mixed, and cell fusion was carried out in the same manner as in Example 1 to obtain a clone (designated as 12A-3-4). Ascites of the obtained clone was collected in the same manner as in Example 1 and purified to IgG.

Example 3 The obtained two kinds of monoclonal antibodies were diluted with PBS, and the titers of the antibodies were compared using an existing foreign RIA reagent (IMMUtest Neopterin; HENNING BERLIN GMBH) for measuring neopterin. The results are shown in Fig. 1. The obtained monoclonal antibody showed high titer.

Example 4 When the enzyme immunoassay of Example 1 was carried out on the obtained two kinds of monoclonal antibodies, D of 2 × 10 ⁻⁴ M was prepared in advance.
(+)-6-Erythro-neopterin, 2 × 10 ^-4 M D (+)-
Table 1 shows the results of examining the specificity of the monoclonal antibody after reacting with 6-erythro-biopterin or 2 × 10 ^-4 M xantopterin at 37 ° C for 1 hour.
It was shown to. The obtained monoclonal antibody showed high specificity for D (+)-6-erythro-neopterin.

[0027]

[Table 1]

Example 5 For the purpose of detecting D (+)-6-erythro-neopterin, when the enzyme immunoassay of Example 1 was carried out on the obtained two kinds of monoclonal antibodies, they were previously diluted to various concentrations. Fig. 2 shows the results of examining the detectable neopterin concentration after reacting with the prepared D (+)-6-erythro-neopterin for 1 hour at 37 ° C. In FIG. 2, B / B ₀
Represents the reaction as an index, B ₀ represents a state in which 100% reaction has occurred, and B represents a state in which the reaction has changed due to the action of neopterin. The obtained monoclonal antibody could measure D (+)-6-erythro-neopterin with high sensitivity up to 16 nmol / L.

Example 6 For the purpose of detecting D (+)-6-erythro-neopterin, two kinds of the obtained monoclonal antibodies were combined with human tissue sections and 37
After reacting at ℃ for 1 hour, washed, peroxidase-labeled anti-mouse immunoglobulin was added, and further reacted at 37 ℃ for 1 hour. After washing unreacted labeled antibody, 0.5 mg / ml diaminobenzidine solution containing 0.002% hydrogen peroxide was added and reacted at room temperature for 5 minutes, then washed with purified water to stop the reaction, and brown under a microscope. The staining was observed. The obtained monoclonal antibody showed a unique staining image.

[0030]

INDUSTRIAL APPLICABILITY According to the present invention, D by complicated chemical synthesis
Without passing through the (+)-6-erythro-neopterin derivative,
Moreover, D (+)-is a simple method that does not require antibody absorption.
A method for producing a monoclonal antibody of 6-erythro-neopterin has been provided. Since the monoclonal antibody produced by the method of the present invention has high specificity and affinity for D (+)-6-erythro-neopterin, an immunoassay method using this antibody was used to determine D (+)-6-erythro-
It has become possible to detect or measure neopterin with high sensitivity.

[Brief description of drawings]

FIG. 1 is a diagram comparing the titers of a monoclonal antibody produced by the method of the present invention and an antibody in a commercially available RIA measurement kit for comparison.

FIG. 2 is a diagram showing the results of examining the detectable neopterin concentration of D (+)-6-erythro-neopterin by the monoclonal antibody produced by the method of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location G01N 33/53 S 8310-2J 33/577 B 9015-2J // (C12P 21/08 C12R 1: 91) (72) Inventor Genio Tsusue 23-2, Matsubara 2-2, Setagaya-ku, Tokyo

Claims (2)

[Claims] 1. An animal is immunized with D (+)-6-erythro-neopterin cross-linked with an immunostimulatory protein or a sulfur-containing protein as an immunogen, and anti-D (+) is obtained from the animal.
A method for producing an anti-D (+)-6-erythro-neopterin monoclonal antibody, which comprises inducing a 6-erythro-neopterin monoclonal antibody. 2. An anti-D produced by the method of claim 1.
By detecting or measuring D (+)-6-erythro-neopterin by utilizing the specific reactivity between the (+)-6-erythro-neopterin monoclonal antibody and D (+)-6-erythro-neopterin An immunoassay method comprising.