JP3091974B2 - 8-Hydroxy-2'-deoxyguanosine monoclonal antibody, method for producing the same, and hybrid cell producing monoclonal antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to an oxidative degradation product of DNA generated from a living body,
Hydroxy-2'-deoxyguanosine (hereinafter referred to as 8-
OHdG) as an index,
It is possible to evaluate oxidative stress by microquantification, and 8-OHdG monoclonal antibody and extremely useful in the fields of medicine, pharmacy, hygiene related to adult diseases, aging, stress, etc. The present invention relates to a production method thereof, a hybrid cell producing a monoclonal antibody, an 8-OHdG quantification kit, and a column for affinity chromatography.

(Prior art) Until now, quantification of oxidative degradation products of DNA constituent bases due to oxidative stress caused by radiation exposure, energy metabolism, drug metabolism, etc. has been performed using thin-layer chromatography, Separation and quantification were performed by high performance liquid chromatography and gas chromatography mass spectrometer.

(Problems to be Solved by the Invention) However, in each of the above methods, in the qualitative / quantitative determination of a natural product sample, coexistence of similar substances is obstructed, the target substance is a trace amount below the detection limit of chromatography, or separation using a column. It has been pointed out that it requires a long time for renewal, a relatively expensive equipment and a large amount of cost for renewal due to contamination of columns and the like, and is not economical.

That is, the first requirement is that the method be specific for quantitative evaluation of naturally occurring low-molecular-weight trace substances. Immunochemical methods are good for that purpose,
When considering coexisting interfering substances, it is advisable to use monoclonal antibodies to ensure specificity.
However, production of monoclonal antibodies requires low cross-reactivity of analogous substances, which requires a great deal of labor.

(Means for Solving the Problems) The present invention solves the above-mentioned problem, and
It was possible to create stable hybrid cells producing a highly specific monoclonal antibody (hereinafter referred to as hybridoma). Further, by constructing a kit for quantifying 8-OHdG using the anti- 8-OHdG antibody, the present invention can be put to practical use. Further, by constructing a kit for quantifying 8-OHdG using the anti- 8-OHdG antibody, the present invention can be put to practical use. Also, this anti-8
Using the -OHdG antibody, a column for affinity chromatography for enriching 8-OHdG in a natural biological component could be produced.

That is, a conjugate was prepared by binding 8-OHdG and KLH (Limpet hemocyanin), and a mouse was immunized using the conjugate as an antigen, and a monoclonal antibody against 8-OHdG was prepared by a cell fusion method. To this end, a very small number of antibody-producing hybridomas were obtained as a result of a large number of cell fusions, and antibody-producing clones were sorted out by limiting dilution from these, and clones with extremely high specificity for 8-OHdG were obtained. Obtained. Then, this clone was inoculated into the abdominal cavity of a nude mouse, and antibodies could be produced in ascites. In addition, the same monoclonal antibody was successfully produced by cell culture of this clone.

The hybrid cell of the present invention that produces a monoclonal antibody having high specificity for 8-OHdG can
Hydroxy-2'-deoxyguanosine (8-OHdG)
Spleen cells from mice immunized with a hapten antigen of the same type as myeloma and bovine serum albumin (BS
The sensitization value to A) is 40% or less of the sensitization value to 8-OHdG / BSA, and the concentration showing 50% inhibition by deoxynucleoside is 100 times the concentration showing 50% inhibition by 8-OHdG. This is an 8-OHdG hybrid cell producing the monoclonal antibody described above.

Further, the anti-8-OHdG monoclonal antibody proposed by the present invention is obtained by synthesizing mouse spleen cells immunized with a hapten antigen of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and syngeneic myeloma. The sensitization value to bovine serum albumin (BSA) produced by the proliferation of the produced hybrid cells is 40% or less of the sensitization value to 8-OHdG / BSA, and shows 50% inhibition by deoxynucleoside. The concentration is at least 100 times higher than the concentration showing 50% inhibition by 8-OHdG.

The present invention further provides an immunoassay kit for quantitatively quantifying 8-OHdG constructed using the anti-8-OHdG monoclonal antibody, and an immunoassay kit for enriching 8-OHdG produced using the anti-8-OHdG monoclonal antibody. It proposes an adsorption column for affinity chromatography.

In addition, the method for producing an anti-8-OHdG monoclonal antibody proposed by the present invention comprises a method of synthesizing spleen cells of a mouse immunized with a hapten antigen of synthesized 8-hydroxy-2'-deoxyguanosine (8-OHdG). The hybrid cells produced by the myeloma are grown and the sensitization value to bovine serum albumin (BSA) is 40% or less of the sensitization value to 8-OHdG / BSA, and the deoxynucleoside is used.
After increasing the amount of the anti-8-OHdG monoclonal antibody at which the concentration exhibiting 50% inhibition is 100 times or more the concentration exhibiting 50% inhibition by 8-OHdG, the antibody is isolated by separating the anti-8-OHdG monoclonal antibody. This is for producing an 8-OHdG monoclonal antibody.

(Example 1) To prepare a hybridoma for producing an anti- 8-OHdG monoclonal antibody based on the 8-OHdG hapten antigen, succinyl-8-hydroxyideoxyguanosine (Suc-8) was used.
-OHdG) was dissolved in dimethylsulfoxide and N, N-carbonyl- ヂ -imidazole (CD
I) 126.8 μmol (manufactured by Tokyo Kasei Kogyo) was added and reacted at room temperature for 2 hours. To the bovine serum albumin BSA (manufactured by Seikagaku Corporation) aqueous solution (22.2 mg / ml) or limpet hemocyanin (KLH) (Sigma) aqueous solution (5.8 mg / ml), add each of the reactants, and stir at room temperature overnight. To complete the reaction of the conjugate. After the reaction, the distilled water was dialyzed to remove free CDI and Suc-8-OHdG. The Suc-
The binding ratio between 8-OHdG and protein was determined by reverse-phase chromatography using an ODS column (10% methanol solvent).
And quantified from the peak area ratio.

KLH was insolubilized by dialysis after reaction with 8-OHdG, but was solubilized again when the pH of the solvent was adjusted to 2.5. When this was hydrolyzed with NaOH and measured by HPLC,
It was estimated that 8-OHdG / KLH = 3.2 (molar ratio). Also, 8-O
HdG / BSA = 4.2 μg / ml.

(Example 2) Using 8-OHdG / KLH as an antigen for immunization, a single dose of 100 μg / mouse was intraperitoneally administered to a mouse (Balb / clb, 6 weeks old).
Mice were administered with Freund's complete adjuvant (FCA), the second and subsequent doses were administered with Freund's incomplete adjuvant (FIA), and cell fusion was performed after a total of 3 to 6 administrations.

P3UI cells (P3-X63-Ag8-UI) as myeloma cells
Using 10% FCS, 5 × 10 ^-5 M2-mercaptoethanol, 1
The cells were cultured in RPMI-1640 medium (manufactured by Nissui Pharmaceutical) supplemented with mM pyruvate.

Fusion of spleen cells and P3UI cells was performed as usual using 96 well
Hybridomas were selectively grown in HAT medium using the plates. On average, 10 fusions per cell
00 colonies were obtained.
The next screening was performed. Those for which antibody production was confirmed were subjected to average subculture. Secondary screening was performed by EIA on cells that were only allowed to proliferate in the 24-well plate, and antibody-producing cells were confirmed again. As a result, about one antibody-producing cell line was obtained by one cell fusion.

Therefore, the total number of antibody-producing strains obtained by 20 cell fusions was 16 strains. These 16 strains were cryopreserved in liquid nitrogen and prepared for cloning.

(Example 3) Cloning of antibody-producing cells and confirmation of cross-reactivity, and 8
-Confirmation of reaction specificity for OHdG.

Of the 16 antibody-producing strains, two were cloned: N45 strain and N310 strain. As a result, cloned N45.1 cells and N310.1 cells producing monoclonal antibodies were obtained.

The culture supernatant containing the monoclonal antibody produced by each of the N45.1 cells and the N310.1 cells was first examined for the cross-reactivity with the protein in order to examine the reaction specificity. 96we
Each of the five proteins was coated on an II plate, and whether or not the antibody reacted was examined. As a result, it was found that the N45.1 antibody strongly reacted with 8-OHdG / BSA, but did not react with BSA. It was suggested that OVA and KLH might react very weakly. On the other hand, the N310.1 antibody is 8-OHdG
In addition to / BSA, BSA (bovine serum albumin), OVA (ovalbumin), and KLH were found to react strongly.
Neither antibody reacted with casein (first
Figure).

The reaction inhibition experiment of both antibodies was performed using 8-OHdG / BSA as an antigen.
Free 8-OHdG was added to the EIA system to determine whether the antigen-antibody reaction could be competitively inhibited. N45.1 antibody is 8
The reaction was inhibited in a concentration-dependent manner between 1 μM and 500 μM of —OHdG. However, the N310.1 antibody was not inhibited even by 8-OHdG at 500 μM (FIG. 2).

Therefore, it was found that the N45.1 antibody binds to 8-OHdG and has high reaction specificity, and the N310.1 antibody does not bind to 8-OHdG.

From the above, the monoclonal antibody produced by clone 45.1 cells strongly reacted with 8-OHdG / BSA, and the reaction was inhibited by free 8-OHdG in a concentration-dependent manner. Therefore, N4
The 5.1 antibody was found to be an antibody that specifically reacts with 8-OHdG.

Next, a competition inhibition test by EIA was performed in order to examine the reaction cross-reactivity of the anti-8-OHdG monoclonal antibody N45.1.

That is, four types of nucleosides (2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, and 2'-deoxyuridine) are added to a system in which the 45.1 antibody reacts with the 8-OHdG / BSA antigen. Dissolve in PBS and add 2
mM solution was prepared and coated with 8-OHdG / BSA antigen
The nucleoside solution was placed in the first row of a 96-well microplate, and diluted in two-fold culture steps toward the twelfth row. A certain amount of the N45.1 antibody was added thereto, and the mixture was incubated at 37 ° C. for 1 hour to react with N45. .1 The amount of antibody was detected by color development using a peroxidase-labeled anti-mouse IgG antibody and OPDA (orthophenylenediamine).

As a result, the reaction of the N45.1 antibody was from 1 μM to 1 mM 8-
Inhibited only by OHdG (FIG. 3). The reaction of the N45.1 antibody was not inhibited by 2'-deoxyguanosine or 2'-deoxyadenosine, 2'-deoxycytidine, or 2'-deoxyuridine (Fig. 3).
Therefore, the N45.1 antibody was considered to be a more specific antibody for 8-OHdG.

(Example 4) Preparation of anti-8-OHdG antibody derived from mouse ascites N45.1 antibody was purified from ascites obtained by transplanting anti-8-OHdG antibody-producing hybridoma N45.1 cells into the peritoneal cavity of a nude mouse. Using this antibody, a competitive inhibition test using an 8-OHdG standard solution was performed in an EIA system. As a result, 8-OHd
G detection limit concentration is 1 ng / ml, 50% competitive inhibition concentration is 15 ng / ml.
ml.

To make ascites, nude mice (8-10 weeks old)
Two weeks after intraperitoneal injection (1 × 10 ⁷ cells / mouse) of anti-8-OHdG antibody producing hybridoma N45.1 cells,
ml was collected.

Purification of the N45.1 antibody was performed using a protein A column.

Prior to purification, the subclass of the N45.1 antibody was determined using a mouse IgG subclass assay kit (manufactured by Amasiyam Japan). As a result, the antibody was found to be IgG1. Therefore, protein A suitable for this subclass
Column purification conditions were used.

That is, the protein A column was equilibrated with 3M NaCl-1.5M glycine buffer (pH 8.9), and ascites containing the N45.1 antibody was passed through the column to adsorb the N45.1 antibody to the protein A. Thereafter, the N45.1 antibody was recovered with a 100 mM citrate buffer at pH 6.0, and dialyzed against PBS (containing 0.1% NaN ₃ ). Repeat the purification process twice in the same manner and add ascites
From 2 ml (151.4 mg of total protein), 12.4 ml of N45.1 antibody solution (27.46 mg of protein) was obtained. In addition, N4 purified twice
It was confirmed that the specific activities of the 5.1 antibodies were the same.

 After dialysis, the N45.1 antibody was stored frozen at -20 ° C.

(Example 5) Preparation of an 8-OHdG assay kit using an anti- 8-OHdG antibody (construction of an EIA system).

A confirmation test of reproducibility as an EIA system was performed by the same competitive inhibition test method as in Example 4 above. That is, a competition inhibition test was performed at n = 6, and the standard deviation (SD value) was shown on the prepared standard curve (FIG. 5). Judging from this SD value, it can be said that there is little variation in the measured values within the sample. Therefore, it was found that the reproducibility of antigen detection by the present EIA system was stable. In order to determine the optimal concentration of N45.1 antibody, the concentration of the added antibody was 0.2 μg / m
8-OHdG to set 2 points of l and 0.1μg / ml
The amount of / BSA antigen was 4.2 μg / ml (FIG. 4).

A comparison was made for each antigen detection limit. As a result, free 8-O when the amount of added antibody was 0.2 μg / ml
The minimum detection limit of HdG antigen was 5 ng / ml, but when the added antibody amount was 0.1 μg / ml, the antigen detection limit was 1 ng / ml.
And the measurement sensitivity increased. On the other hand, the maximum color value obtained when no free 8-OHdG antigen is added (zero antigen on the standard curve) is OD = 0.84 when the added antibody is 0.2 μg / ml, whereas the OD = 0.84 when the added antibody is 0.1 μg / ml. In this case, OD = 0.69. For this purpose, the slope of the standard curve is
The use of μg / ml resulted in a steeper gradient. Therefore, in order to obtain measurement accuracy, it can be said that an antibody concentration of 0.2 μg / ml is good. In this EIA kit, the concentration of the added antibody was 0.1 μg / ml in order to improve the sensitivity of the measurement.

(Example 6) Preparation of a column for affinity chromatography using an anti-8-OHdG monoclonal antibody.

The anti-8-OHdG antibody purified as described above is adsorbed on CNBr-activated Sepharose (or tosyl-activated agarose), packed into a column of an appropriate size, and a sample containing 8-OHdG is slowly passed and adsorbed. Elution is easily performed with an aqueous solution containing a chaotropic salt such as thiocyanate. Take an appropriate amount of CNBr Sepharose, swell with 1 mM HCl, then wash several times with 1 mM HCl on a glass filter. Then, the plate is washed with a coupling buffer and immediately mixed with a monoclonal antibody solution diluted with the coupling buffer (monoclonal antibody 5 mg / ml gel or less). Room temperature 2
The reaction is allowed to stir for 4 hours or overnight at 4 ° C. Next, the supernatant is removed, a blocking solution is added, and the mixture is left at room temperature for 2 hours or at 4 ° C. overnight to block active groups that have not reacted with the antibody. Next, the carrier is washed five times alternately with a coupling buffer and a 0.1 M acetate buffer (pH = 5.0), and the monoclonal antibody that has not reacted with the carrier is washed away. Furthermore, PBS
Wash and store at 4 ° C with 0.05% NaN ₃ .

The carrier prepared as described above is packed in a column, and a solution containing 8-OHdG is flowed under appropriate conditions. PBS overnight at room temperature or 4 ° C
In good eluate After washing column (SCN ^-, pH7~8.3M) eluting with. After elution, wash the column as soon as possible with a buffer containing 10% dioxane, then wash alternately with 0.1 M Tris-HCl buffer and 0.1 M acetate buffer, and finally with PBS.
Add 0.05% NaN ₃ and store at 4 ° C.

(Example 7) About quantification of 8-OHdG in urine by an anti- 8-OHdG monoclonal antibody. (EIA method) Dilute to a concentration of 4.2 μg / ml of 8-OHdG / BSA antigen and 100 μl to coat a microplate (96 well)
/ well, and allowed to stand at 4 ° C. overnight.

Next, remove the supernatant and add PBS (0.1% NaN ₃ ) at 200 μl / weld.
l Dispensed and washed.

300 μl of blocking solution (0.1% BSA-PBS 0.1% NaN ₃ )
Dispense 1 / well and allow to stand at room temperature for 2 hours or at 4 ° C. overnight to block the adsorption of the antibody to the plate.

After blocking, 200 µl / wel of PBS (0.1% NaN ₃ )
l Dispensed and washed.

8-OHdG standard solution is 0 (PBS only), 65 pg / ml, 320 pg / m
l, 1.6ng / ml, 8ng / ml, 40ng / ml, 200ng / ml (final concentration)
Were taken at 50 μl / well into a plate, and a calibration curve was prepared at n = 3.

For the sample, use the first urine after waking up, 50 μl / well
And dispensed into plates.

Add 50 μl / N45.1 antibody (protein amount 0.2 μl / ml)
After incubating at 37 ° C. for 1 hour, removing the supernatant after the reaction, removing 200 μl of 0.05% Tween 20-PBS (NaN ₃ free) 200 μl.
l / well was dispensed and washed.

Next, a peroxidase-labeled anti-mouse 1gG antibody (1/100
0 dilution) was dispensed at 100 μl / well into a plate and incubated at 37 ° C. for 1 hour. Then, the supernatant was removed and 0.05% Tween 20
Washing was performed with 200 μl / well of PBS (NaN ₃ ).

To detect by color development by o-phenylendiamin (OPDA), prepared 50mM citrate -100mMNa ₂ HPC ₄ · 12H ₂ O buffer pH 4.5, and dissolved while shielding at a concentration of 10 mg / 25 ml of OPDA,
Immediately before use, 10 μl of a 35% H ₂ O ₂ solution was added. 100μl / wel
Then, the mixture was incubated at room temperature for 30 minutes under a subcutaneous shield, and a 2NH ₂ SO ₄ solution was added to the plate at 100 μl / well to stop the reaction.

After the reaction was stopped, the absorbance at 492 to 620 nm was measured, and a calibration curve was prepared. Then, the amount of 8-OHdG in the sample was determined. As a result, it became possible to quantify 8-OHdG in urine.

(Effects of the Invention) According to the present invention, an 8-OHdG antibody can be efficiently produced, and by using this, a very small amount of 8-OHdG (1 ng / ml) can be reproduced much more efficiently than in the conventional method. There is an effect that can be quantitatively determined. For the evaluation of genotoxicity, the 8-OHdG antibody has the effect of being efficiently concentrated by an affinity column and traceable even at a trace level.

[Brief description of the drawings]

FIG. 1 is a graph showing the detection of cross-reactivity of N45.1 antibody and N310.1 antibody against various proteins, and FIG.
FIG. 3 is a graph showing a competition inhibition test for 0.1 antibody, FIG. 3 is a graph showing a competition test of N45.1 antibody with nucleoside,
FIG. 4 is a standard curve diagram of the N45.1 antibody concentrations of 0.1 μg / ml and 0.2 μg / ml, and FIG. 5 is a graph showing the simultaneous reproducibility of the standard curve in the 8-OHdG competitive inhibition test.

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁷ Identification code FI G01N 33/577 C12N 15/00 C

Claims (5)

(57) [Claims] The present invention comprises spleen cells of a mouse immunized with a hapten antigen of synthesized 8-hydroxy-2'-deoxyguanosine (8-OHdG), and syngeneic myeloma. Sensitization value is 8-OHdG /
The concentration which is less than 40% of the sensitization value to BSA and shows 50% inhibition by deoxynucleoside is 50% by 8-OHdG.
8-OHdG hybrid cells producing a monoclonal antibody that is at least 100 times the concentration showing% inhibition. 2. A hybrid cell produced by a spleen cell of a mouse immunized with a hapten antigen of synthesized 8-hydroxy-2'-deoxyguanosine (8-OHdG) and a syngeneic myeloma. The sensitization value to bovine serum albumin (BSA) was 40% or less of the sensitization value to 8-OHdG / BSA, and the concentration showing 50% inhibition by deoxynucleoside was 50% inhibition by 8-OHdG. An anti- 8-OHdG monoclonal antibody that is at least 100 times the indicated concentration. 3. An immunoassay kit for quantifying 8-OHdG constructed using the anti- 8-OHdG monoclonal antibody according to claim 2. 4. An adsorption column for affinity chromatography for concentrating 8-OHdG prepared using the anti- 8-OHdG monoclonal antibody according to claim 2. 5. A hybrid cell produced from a spleen cell of a mouse immunized with a hapten antigen of synthesized 8-hydroxy-2'-deoxyguanosine (8-OHdG) and a syngeneic myeloma, and The sensitization value to serum albumin (BSA) was 4 times the sensitization value to 8-OHdG / BSA.
0% or less and 50% by deoxynucleosides
After increasing the amount of the anti-8-OHdG monoclonal antibody whose concentration showing% inhibition is 100 times or more that of the concentration showing 50% inhibition by 8-OHdG, the anti-8-OHdG monoclonal antibody is separated. A method for producing an anti- 8-OHdG monoclonal antibody.