JPH02227092A - Monoclonal antibody and determination of amp deaminase using the same - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody by immunizing an animal with an AMP deaminase purified from sheep brain, fusing the obtained spleen cell of the animal to a myeloma cell and culturing the obtained hybridoma. CONSTITUTION:AMP deaminase separated from sheep brain is purified by various column chromatography processes until the enzyme becomes a simple substance judged by electrophoresis. The purified deaminase is used as an immunogen and an animal is immunized with the enzyme. The spleen cell of the animal is collected and fused with a myeloma cell to obtain a hydridoma. The hybridoma is cultured and the objective monoclonal antibody is separated from the supernatant liquid of the cultured product or the hybridoma is transplanted into an animal body and the monoclonal antibody is separated from the ascites. The obtained monoclonal antibody can be used for the quick detection and determination of AMP deaminase in body fluid, etc.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention provides a monoclonal antibody against sheep brain-derived AMP deaminase, and the use of this monoclonal antibody to detect AMP deaminase in specimens such as tissues and body fluids. Regarding measurement methods.

[Conventional technology and its problems]

A-needle deaminase exhibits properties as a typical allosteric enzyme. Until now, interest in this enzyme has focused on its various regulatory mechanisms, and little has been done to elucidate its physiological role.

On the other hand, in recent years, through the investigation of the distribution and localization of in vivo macromolecules in organs or cells using immunostaining methods,
Elucidation of the physiological role of these macromolecules in living organisms is actively being carried out.

However, a monoclonal antibody against AMP deaminase with high specificity has not been obtained to date, and the physiological significance of this enzyme has not been determined from the distribution and localization of the enzyme in tissues or cells, that is, from the morphological aspect. The explanation had not yet been made.

[Means to solve the problem]

Under these circumstances, the present inventors conducted various studies to elucidate the physiological significance of A-needle deaminase from a morphological perspective, and as a result, they newly created a monoclonal antibody against sheep brain-derived AMP deaminase. By using this, we succeeded in specifically measuring AMP deaminase in tissues and body fluids, and completed the present invention.

That is, the present invention provides a novel sheep brain-derived AMP deaminase and a method for measuring A-needle deaminase in a test subject using the same.

The monoclonal antibody against sheep brain-derived A needle deaminase of the present invention is prepared, for example, as follows. That is, first, using various column chromatography methods, AMP deaminase from sheep brain was electrophoretically purified until it became single, and this was used as an immunogen.
Immunize the animal. Next, the animal's anchor cells are collected and fused with myeloma cells to obtain hybridomas.

Examples of immunized animals include mice and rats. Known methods such as a method using polyethylene glycol, a method using Sendai virus, or a method using electric pulses can be used for cell fusion.

When screening for hybridomas, be sure to
Care must be taken to obtain only monoclonal antibody producing cells against needle deaminase.

To this end, it is desirable that the A-needle deaminase derived from sheep brain used as an antigen at the time of immunization and screening be a single pure product in terms of electrophoresis.

The monoclonal antibody of the present invention can then be obtained by culturing the obtained hybridoma and collecting the monoclonal antibody from the culture supernatant, or by transplanting the hybridoma into an animal and collecting the monoclonal antibody from its ascites. sexual antibodies are obtained. Here, ammonium sulfate salting out or the like may be used to isolate the monoclonal antibody of the present invention from the culture supernatant or ascites.

The monoclonal antibody of the present invention thus obtained has the following properties.

(i) Antibody class: IgM (ii) Antibody titer: 2,000 times (iii) Cross-reactivity: Cross-reactive with AMP deaminase in human brain, human spinal cord, human heart, human liver, and rat brain.

To measure AMP deaminase in tissues using the monoclonal antibody of the present invention obtained as described above, an immune reaction is performed by adding a monoclonal antibody against sheep brain-derived AMP deaminase to a subject. .

Next, this reaction solution is reacted with an enzyme-labeled goat anti-mouse IgM antibody, followed by color development using an appropriate coloring agent and examination using an optical microscope.

〔Effect of the invention〕

As described above, by using the monoclonal antibody of the present invention, the distribution and localization of AMP deaminase in tissues can be detected by immunostaining. Furthermore, by using this monoclonal antibody, it is possible to rapidly detect and quantify AMP deaminase in body fluids or tissue extracts by the BLISA method.

〔Example〕

Next, the present invention will be explained with reference to Examples.

Example 1 (1) Preparation of Immunogen AMP deaminase was electrophoretically purified from sheep brain into a single pure product by various column chromatography. That is, a frozen sheep brain is thawed and 0.
The mixture was homogenized with a phosphate buffer containing 65M potassium chloride and centrifuged to obtain a crude extract. The crude extract was then treated with phosphocellulose, AH-Sepharose, and
Each column was subjected to column chromatography using Matrix Gel Green A, and the A-needle deaminase active fraction was eluted using a potassium chloride linear concentration gradient method to obtain an enzyme active fraction. Finally, Pharmacia's FP
LC system super loin 12! The deaminase was subjected to molecular sieving to obtain electrophoretically pure A-needle deaminase.

ω) Preparation of monoclonal antibodies (+) The pure AMP deaminase obtained in (1) was mixed with Freund's complete adjuvant (Freund's comp
The mixture was mixed with an equal amount of 10 μg J per BALB/c mouse to form an emulsion, and administered to each BALB/c mouse three times at 2-week intervals.

(ii) Pile cells collected from hyperimmunized mice 3 days after the final immunization and myeloma cell line 5P210-^g14 derived from BALB/c mice were treated with polyethylene glycol (P
EG) 4000 was used for fusion. Cells were added to a 96-well plate at 100 μl/well, and after 24 hours, half of the medium was replaced with Hara (HAT) medium, and the medium was replaced every two days. Growth of pigeon-resistant hybridomas is observed after 7 to 10 days.

At this time, the medium was changed to HT, and after culturing for about 10 days, the medium was changed to hybridoma growth medium.

(iii) Screening for antibody-producing cells was performed by the two-antibody ELISA (IILIS^) method using pure HeUP deaminase used as an immunogen as an antigen. By this method, hybridomas producing monoclonal antibodies that specifically react with sheep brain-derived AMP deaminase were selected. The cell lines selected here were cloned by the soft agar method, and monoclonal antibody-producing hybridoma clones were established. For detection of AMP deaminase in tissues, a monoclonal antibody was used that was purified by ammonium sulfate salting out from the ascites fluid collected on the 10th to 15th day after the hybridoma was intraperitoneally transplanted into a mouse.

(3) Properties of monoclonal antibodies Four types of monoclonal antibodies against sheep brain-derived AMP deaminase were obtained, and the class of the antibodies was all IgM. The antibody titer was 2.000 times (antibodies purified from mouse ascites were diluted in two stages, respectively, and sheep brain-derived A
When performing BLISA to react with MP deaminase,
The dilution ratio at which the highest absorbance persisted was taken as the antibody titer). When cross-reactivity was examined, it showed reactivity with human brain, human spinal cord, human heart, human liver, and rat brain, and it was possible to detect AMP deaminase in these tissues or tissue extracts. found.

Example 2 A human spinal cord tissue section fixed with acetone was
% hydrogen peroxide to inactivate tissue-endogenous peroxidase. Then apply 1% on the tissue section surface.
Phosphate buffered saline (PBS) containing bovine serum albumin (O3^) is added and left to stand at room temperature for 1 hour to prevent non-specific adsorption of antibodies and other proteins.

Furthermore, a monoclonal antibody (500-fold dilution) against sheep brain-derived AMP deaminase was dropped onto the section surface.
Leave at 4°C for 24 hours. After thoroughly washing the section with PBS, an appropriate amount of a 400-fold dilution of biotinylated anti-mouse IgM antibody is dropped onto the section surface, reacted at 4° C. for 2 hours, and then thoroughly washed with PBS. Thereafter, it is reacted with an avitin-biotinylated peroxidase complex at 4°C for 2 hours. After thorough washing with PBS, color development was performed in PBS containing 0.05% 3.3'-diaminopentidine tetrahydrochloride and 0.01% hydrogen peroxide, followed by microscopic examination.

The results are shown in Figure 1.

Example 3 10.5.2.5G, 1.25.0 of sheep brain crude extract
．． 63.0.32.0.15 and Q, 07mg/
ml! Using the solution as an antigen, BLISA was performed by adding 50 μl each of a 2000-fold dilution of the monoclonal antibody obtained in Example 1. As a result, as shown in FIG. 2, it was revealed that the amount of antigen-antibody binding increased in proportion to the amount of antigen.

[Brief explanation of the drawing]

FIG. 1 is a photograph of a human spinal cord section stained with 3,3'-diaminobenzidine using a monoclonal antibody against sheep brain-derived AMP deaminase. Figure 2 shows that when the amount of monoclonal antibody was kept constant,
It is a drawing showing the relationship between the absorbance and the antigen amount obtained when the protein amount in a sheep brain crude extract is changed. Above Figure 1 Figure 2 O Protein amount (η/-)

Claims (1)

[Claims] 1. Monoclonal antibody against sheep brain-derived AMP deaminase. 2. The following properties: (1) antibody class IgM, (2) antibody titer 2,000 times, (3) human brain, human spinal cord, human heart,
2. The monoclonal antibody according to claim 1, which has cross-reactivity with AMP deaminase in human liver and rat brain. 3. A method for measuring AMP deaminase, which comprises adding a monoclonal antibody against sheep brain-derived AMP deaminase to a subject and performing an immune reaction.