JPS63301800A - Determination of cu,zn-superoxide-dismutase - Google Patents

Abstract

PURPOSE:To easily determine Cu,Zn-superoxide.dismutase (Cu,Zn-SOD) useful in the field of immunology, etc., in high uniformity of reaction, by using a specific monoclonal antibody. CONSTITUTION:An antibody-producing spleen cell of an animal immunized with Cu-Zn-SOD is fused with a myeloma cell to obtain a hybridoma and/or a cell strain originated from the hybridoma. The cell strain is cultured to produce a monoclonal antibody and obtain (A) the immobilized 1st antibody and (B) the 2nd antibody capable of recognizing an antigen site different from the antibody A and recognizing a Cu,Zn-SOD labeled with a labeling agent. A specimen such as serum containing human Cu,Zn-SOD is made to react with the antibodies A, B in a well and the concentration of Cu,Zn-SOD is calculated by a solid-phase enzymatic immunoassay using a sandwich process.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention provides a method for detecting the presence of oxidation in a sample using a monoclonal antibody that recognizes Cu, Zn-superoxide dismutase (hereinafter abbreviated as Cu, Zn-9OD). Cu
, relates to a method for measuring Zn-9OD.

[Prior Art] Cu and Zn-8OD are enzymes that have a catalytic effect on the disproportionation reaction of superoxide radicals (hereinafter abbreviated as 02D), and their measurement has recently attracted clinical attention. Cu, Zn in various tissues such as liver and lungs
-8OD trace measurement is desired. Also Cu.

Zn-8OD has come to be used as a variety of therapeutic agents, and measurement of its blood concentration has become desirable.

For conventional measurements, the Me Cord
) et al. (J, Blol, Chem, vol. 244, 604)
9-6055, 1989). This method required complicated operations and did not have sufficient measurement sensitivity. Furthermore, the method of McCord et al. is a method of measuring the amount of a substance that eliminates O7τ, and is not specific only to Cu and Zn-9OD.

Superoxide dismutase (SOD) depends on the type of chelate metal required for the expression of enzyme activity.
It is classified into Zn type, Mn type and Fe type.

The cytoplasm of eukaryotes contains Cu, Zn-8OD,
Mitochondria contain Mn-9OD. 0 more
2τ is also eliminated by biological substances such as cytochrome C, epinephrine, and ascorbic acid. Therefore, it is impossible to specifically quantify only Cu and Zn-9OD in a sample using the method of McCord et al.

On the other hand, immunoassays such as radioimmunoassay and enzyme immunoassay have been conventionally used as methods for measuring trace components in body fluids.

Several patents have been published regarding immunoassay methods for Cu and Zn-8OD. For example, JP-A-57-102195 discloses an enzyme-labeled SOD-containing solution, an antibody-containing solution that has specific binding to SOD, and an SO
SOD is measured by mixing a sample solution in which D is to be quantified, removing the immune complex as a precipitate from the aqueous solution, and measuring the activity of the labeled enzyme in the aqueous solution or precipitate. Furthermore, in JP-A-57-102198 and JP-A-58-7580, an enzyme-labeled anti-SOD polyclonal antibody and an anti-SOD polyclonal antibody bound to an insoluble carrier,
The SOD measurement is carried out using the Sand-Deutsch method.

[Problems to be Solved by the Invention] The present invention uses monoclonal antibodies that can always obtain the same quality in the immunological determination of Cu and Zn-3OD, and achieves high reproducibility and sensitivity. This provides an excellent measurement method.

[Means and effects for solving the problem] The present inventors prepared a monoclonal antibody specific to Cu and Zn-8OD to be measured, and investigated an immunoassay method using this. Ta. As a result, it was discovered that the monoclonal antibody related to the present invention is extremely useful as a reagent for measuring Cu and Zn-8 OD, and the present invention was achieved.

That is, the present invention provides a monoclonal antibody that recognizes Cu and Zn-3OD, A: an immobilized first antibody, and B: a monoclonal antibody that recognizes a different antigen site from the first antibody and is labeled with a labeling agent. This is a method for measuring Cu and Zn-8OD, which is characterized by using a second antibody that is

The monoclonal antibody used in the present invention is preferably produced by fusing antibody-producing lung cells and myeloma cells of an animal immunized with Cu, Zn-8OD to produce a monoclonal antibody that recognizes Cu, Zn-3OD. A producing hybridoma is obtained, and then the hybridoma and/or a cell line derived therefrom are cultured and harvested from the culture. Hybridomas that produce monoclonal antibodies that recognize Cu and Zn-3OD can be produced using the cell fusion method (G, Kohler (Ko)), which is a known method per se.
hler) and C. Milstein.
, Nature vol. 258, p. 495,
(1975).

In the present invention, Cu,
A monoclonal antibody that recognizes Zn-5OD was obtained.

These were monoclonal antibodies that recognized mutually different antigenic sites of the Cu and Zn-9OD.

Therefore, solid-phase enzyme-linked immunosorbent assay (enzyme-11n) using two monoclonal antibodies based on the Sand-Deutsch method was used.
ked iou++uno-sorbent assa
y) became possible.

In the present invention, a monoclonal antibody that recognizes Cu and Zn-3OD is immobilized as the first antibody, and a known method can be used for the immobilization.

Examples of materials for immobilizing antibodies include glass.

Polystyrene, polyethylene, polyvinyl chloride.

Beads and microplates made of latex, agarose, cellulose, methacrylate, etc. are preferably used.

Also, labeling methods and means for preparing the second antibody,
The method or means of detecting it is not limited in any way,
It can be measured by known methods and means. As a labeling agent, in the method using an enzyme (EIA), an enzyme such as peroxidase, β-D-galactosidase, or alkaline phosphatase is used, and in the method using a radioactive substance (RIA), a fluorescent substance such as 125I' 3H is used. In the method using (FIA), fluorescein isothiocyanate and the like are usually used, but other materials may also be used.

When the labeling agent is an enzyme, a substrate is used to measure its activity. For example, substrates for horseradish peroxidase include 2.2''-azinodi[3-ethylbenzthiazolinesulfonic acid] ammonium salt (hereinafter referred to as ABTS)-1l?O?, 5-aminosalicylic acid-t1202.o- Examples of substrates for β-D-galactosidase include 0-nitrophenol-β-D-galactopyranoside, and substrates for alkaline phosphatase include p-nitrophenylphosphate. 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.

In the method of the present invention, Cu and Zn-8OD in the sample are particularly 1.
This is a preferred method for measuring in the range of 25-320 ng/ml. This is because it becomes difficult to measure anything beyond this range using current detection methods.

[Effects of the Invention] The method of the present invention is a specific measurement method for Cu and Zn-8OD, and is not affected in any way by the presence of substances that eliminate 02D other than Cu and Zn-3OD. Also,
The monoclonal antibodies used in the present invention can be obtained in large quantities and have high uniformity of reaction. And these can also be produced industrially.

[Examples] Hereinafter, the present invention will be explained in detail with reference to Examples. However, the present invention is not limited to these examples.

Example 1 a) Preparation of anti-human Cu, Zn-9OD monoclonal antibodies Anti-human Cu, Zn-9OD monoclonal antibodies were prepared according to the method of G. Köhler and C. Milstein. That is, an emulsion prepared by mixing P[3S (phosphate buffered saline) in which human Cu and Zn-3OD were dissolved and Freund's complete adjuvant was intraperitoneally administered to BALB/c mice twice at 4-week intervals. After confirming the increase in blood antibody titer by solid-phase enzyme immunoassay using a 96-well plate, PBS in which human Cu and Zn-8OD were dissolved was added.
was injected into the tail vein of the mouse. Four days later, the mouse lung cells were removed and treated with polyethylene glycol.
Cell fusion was performed with azaguanine-resistant myeloma cells 5P210. Cells were cultured in 96-well plates and subjected to conventional HAT selection. Is hybridoma screening using solid-phase enzyme immunotherapy using 96-well plates? The 1Pl standard method was used, and hybridomas producing anti-HiI-SOD antibodies were cloned by the limiting dilution method. The hybridoma obtained as described above was cultured in a flask, then transplanted into the peritoneal cavity of a BALB/C mouse and cultured to obtain ascites containing monoclonal antibodies. This is precipitated by ammonium sulfate.
Anti-human C by ion exchange column chromatography
u, Zn-8OD monoclonal antibody was obtained.

Preparation of the first antibody 1 μg/ml dissolved in 0.1 M sodium carbonate buffer (pH 9,6) was added to each well of an untreated 96-well microtiter plate (trade name Nunc Immunoplate, manufactured by Intermed). Mouse anti-human Cu, Zn
The monoclonal antibody prepared with -8OD monoclonal antibody (a) is hereinafter referred to as 5I12A. IgG)250
μ was added and incubated overnight at 4°C. next,
The solution in each well was removed and washed three times with a PBS solution containing 0.04% Tveen-20 (hereinafter referred to as PBS-T solution), and then washed with PBS-T solution containing 1% BSA (bovine serum albumin). Add 300 μm of T solution and
A blocking treatment (a treatment in which BSA is adsorbed to non-specific binding sites for antigens or antibodies present in the carrier) was carried out at 4°C, and the mixture was stored at 4°C.

c) Preparation of second antibody Horseradish peroxidase (hereinafter referred to as HRPO) solution dissolved in 0.3M sodium bicarbonate buffer (pH 8,1) (5 ml 1 g/ml) 1% 1-fluoro-2, Ethanol solution of 4-dinitrobenzene 0
．． 1 ml was added, and the mixture was allowed to react at room temperature for 1 hour. then 0.0
8M Sodium Periodate 1. oml was added and reacted for 30 minutes. 0.18 of unreacted sodium periodate
After adding and removing 1.0 ml of M ethylene glycol,
Dialysis was performed against 0.01M sodium carbonate buffer (pH 9.5).

Next, 5+ng of a monoclonal antibody (1gG) prepared using a mouse anti-human Cu, Zn-9OD monoclonal antibody (a) which recognizes an antigen site different from 5H2A was added and allowed to react for 5 hours. 5 mg of sodium borohydride was added, and the mixture was left at 4°C overnight.

The reaction product obtained as above was added to TSK Gel G-30.
It was purified by high performance liquid chromatography using 00Sl/ (manufactured by Toyo Soda Kogyo, registered trademark) to obtain an HRPO-labeled monoclonal antibody.

2) Human Cu, Zn- in serum by enzyme immunoassay
Quantification of 8OD The microtiter plate on which the antibody was immobilized (in this example) was returned to room temperature, washed three times with PBS-T solution, and the purified human Cu, Zn-9OD was 1.25 to 320.
20 μm of normal human serum containing 1 g/ml was added to each well as a standard substance. Then in each well
4.5 mg/ml of the IIRPO-labeled antibody prepared in 1.
5% normal rabbit serum, 0.01% myoglobin, 0
．． A solution diluted 1000 times with PBS-T solution containing 0.075% refined white sugar and 0.05% ovalbumin was added to each well by 300 μm, and after incubation at room temperature for 3 hours, the solution was removed and then PBS-T was added. Washed three times with solution. Furthermore, 1.2%ABTS, 0.01%H2
0, 1M citrate buffer containing O2 (pH 4,1)
After adding 200 μl to each well and incubating for 30 minutes at room temperature, add lOOμ! of 1M citric acid solution. In addition, the reaction was stopped.

After stopping the reaction, the absorption intensity of each well at a wavelength of 415 ns and a reference wavelength of 492 nm was measured using an automatic microtiter plate reader (MPR-A4. Trademark manufactured by Toyo Soda Kogyo).
The concentration and absorption intensity of the standard substance were plotted to obtain the results shown in Table 1, and a calibration curve was created.

Using this calibration curve, human Cu and Zn-8 in each sample were determined.
OD concentration can be determined.

Table 1 Example 2 Under the same conditions as in Example 1 b), bovine Cu and Zn were used instead of human Cu and Zn-3OD.
Anti-bovine Cu, Zn-8OD monoclonal antibodies (IgG) were produced using -8OD. A first antibody and a second antibody were prepared in the same manner as in Example 1 (a) and c) for two anti-bovine Cu and Zn-8OD monoclonal antibodies that recognize different antigenic sites, and these were used to prepare a first antibody and a second antibody. Bovine Cu, Zn-8OD1.
Bovine Cu and Zn-8OD were measured using PBS containing 25 to 320 ng/ml and combined with 1% BSA as a standard substance, and a calibration curve was created. The results are shown in Table 2.

Using this calibration curve, determine the amount of bovine Cu in each measurement sample.

Zn-9OD concentration can be determined.

Table 2 From the above results, the present invention has demonstrated that Cu in various samples,
It can be seen that this is an excellent method for measuring Zn-8OD.

Claims (1)

[Scope of Claims] A monoclonal antibody that recognizes Cu, Zn-superoxide dismutase, comprising: A: an immobilized first antibody; and B: a monoclonal antibody that recognizes a different antigen site from the first antibody and that is a labeling agent. A method for measuring Cu, Zn-superoxide dismutase, which comprises using a labeled second antibody.