JPH09127109A - Anti-2'-deoxycytidine antibody, and manufacture and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an excellent antibody having high specificity for 2'- deoxycytidine(dCyd) by transforming the 5-th or 3rd position hydroxyl group of dcyd into succinyl, dehydration-linking it with the amino group of carrier polypeptide via the carboxyl group, and using the dCyd antigen expressed by a formula. SOLUTION: 2'-deoxycytidine(dCyd) is transformed into succinyl by succinic anhydride, succinylated derivative at the fifth or third position is separated by HPCL, for example, and it is linked with carrier polypeptide or protein to prepare the dCyd antigen expressed by the formula I or II. An animal (mouse, cattle, horse) is immunized against this antigen, antibody forming cells are collected from the immunized animal and cell-fused with myeloma cells to form hybridoma, and an anti-dCyd monoclonal antibody is obtained. The dCyd in a specimen is reacted to the anti-dCyd antibody serving as a reagent, and the existence or abundance of dCyd in the specimen is detected from the reaction result.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibody against 2'-deoxycytidine (sometimes abbreviated as dCyd) and its production and use. More specifically, the present invention relates to an antibody against dCyd (which may be abbreviated as anti-2'-deoxycytidine antibody or anti-dCyd antibody), an antigen for producing the antibody, a method for producing the antibody, and the production of the antibody. Hybridoma, a method for measuring dCyd using the antibody, and a dCyd measurement kit containing the antibody.

[0002]

2. Description of the Related Art dCyd is a constituent of genetic DNA, and free dCyd was considered to be an intermediate in DNA biosynthesis or metabolism. Yoshioka et al. (Biol. Pharm Bull., 17
(2), 169-172, 1994] have shown that patients whose dCyd is increased in blood during chemotherapy after surgery in Egyptian breast cancer patients had a poor prognosis and died. On the contrary, it was found that the prognosis was good for patients who showed normal values without increase. Therefore, measurement of the concentration of dCyd in body fluids has become important for the prognosis and pathological condition of cancer.

Conventionally, dCyd has been separated and measured by thin layer chromatography, high performance liquid chromatography, etc., but these methods have low sensitivity and are complicated to operate, and many samples cannot be measured at one time. There was a drawback. On the other hand, the immunoassay method is generally excellent as a method capable of easily and quickly measuring a large number of samples. However, in order to perform this method, it is necessary to prepare the antigen and antibody. However, no antigen and antibody of dCyd have been prepared so far.

[0004]

Therefore, the present invention is based on dC
It is intended to provide an immunoassay method for yd and a kit therefor, and as a premise thereof, dCyd antigen, anti-
It is intended to provide a dCyd antibody, a hybridoma producing the antibody, and the like.

[0005]

In such circumstances, the present inventor has succinylated the hydroxyl group at the 5'-position or 3'-position of dCyd and dehydrated and condensed to the amino group of the carrier polypeptide via these carboxyl groups. It was found that an antibody against dCyd with high specificity can be obtained by using the dCyd antigen represented by the formula (I) or (II).

Therefore, the present invention provides the following formula (I) or (I
I):

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There is provided an antigen represented by the formula: wherein R is a carrier polypeptide or protein. This antigen has the following formula (III):

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It can be prepared by O-succinylation of 2'-deoxycytidine (dCyd) represented by and then binding it to a carrier polypeptide, for example in the presence of carbodiimide. The invention further provides antibodies to dCyd, in particular anti-dCyd antibodies raised against formula (I) or (II) above. This antibody is a polyclonal antibody, that is, an antiserum, or a monoclonal antibody. Affinity constant K with dCyd
Those in which a is 1 × 10 ⁴ M ⁻¹ or more are particularly preferable. Particularly, 10 ⁴ M ^-1 or more, particularly 10 ⁵ M ^-1 or more for a polyclonal antibody (antiserum), and 10 or more for a monoclonal antibody.
It is preferably ⁸ M ^-1 or more, particularly preferably 10 ⁹ M ^-1 or more.

The present invention further relates to a hybridoma producing the above anti-dCyd. The present invention also provides dCyd and the anti-dCy expected to be present in a sample.
There is provided a method for measuring 2'-deoxycytidine in a sample, which comprises reacting with a d antibody and measuring whether or not a reaction has occurred or the amount of the reaction directly or indirectly.
According to one aspect of this method, dCyd in the sample and
A labeled dCyd reagent is allowed to compete with the anti-dCyd antibody, preferably immobilized on a solid support, and the labeled dCyd reacted with the antibody is then measured. . According to yet another embodiment, dCyd immobilized on the solid phase (preferably in the form of the antigen) and dCyd in the sample are allowed to compete with the antibody, and the dCyd immobilized on the solid phase Bound d-C
The yd antibody is measured.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the antigen of the formula (I) or (II) is a derivative obtained by succinylating dCyd with succinic anhydride and succinylated at the 5'-position and / or the 3'-position. For example, high performance liquid chromatography (HP
LC), which is then prepared by binding it to a carrier polypeptide or protein. Examples of the carrier polypeptide or protein include human serum albumin (H
SA), bovine serum albumin (BSA), globulin,
Bovine thyroglobulin, bovine globulin, hemocyanin (K
Proteins such as LH) and Ascaris, and synthetic polypeptides such as polylysine and polylysyl glutamic acid are used.

For succinylation of dCyd, succinic anhydride is reacted with dCyd at room temperature for several minutes, and the reaction solution is dried under reduced pressure to give a 5'-position derivative (5'-OS-dCyd) and a 3'-position derivative (5'-OS-dCyd). 3'-OS-dCyd). Each separation is carried out, for example, by using ODS-silica gel column as a mobile phase and 0.1% trifluoroacetic acid (TFA).
Performed by high performance liquid chromatography (HPLC) with a content of 0-30% acetonitrile. Confirmation of the 5′-position and / or 3′-position derivative separated by HPLC is as follows.
FAB-mass spectrometry (FAB-MS) and NMR.

The reaction solution prepared by reacting the carrier polypeptide or protein, the succinylated dCyd and the condensing agent dicyclohexylcarbodiimide (DCC) for one day is dialyzed against water to remove excess condensing agent and succinylated d.
By removing Cyd and lyophilizing the content liquid, dCyd antigen I or II can be obtained. The thus obtained dCyd antigen had an increase in absorbance at 270 nm. Therefore, when the carrier polypeptide was KLH, it was 336 in the formula I against 1 mol of KLH.
It has been confirmed that 5.6 moles of the compound were bonded, in formula II. Also, when the carrier polypeptide is BSA, BSA1
It was confirmed that 9 moles of the formula I and 8 moles of the formula II were bonded to the moles.

By immunizing an animal with dCyd antigen (I) or (II), an antibody against dCyd can be obtained. The antibody is prepared, for example, as follows. That is, the antigen (I) or (II) is used to prepare a w / o emulsion using Freund's complete adjuvant and administered to animals such as mice, rats, cows, rabbits, sheep and horses,
Approximately every two weeks using Freund's incomplete adjuvant
/ O Emulsion is prepared and additional dose is given. An increase in serum antibody titer is seen after the first immunization. The antibody titer can be measured by the EIA method, and by collecting this serum, an antibody against dCyd can be obtained.

Alternatively, monoclonal antibodies can be obtained by collecting antibody-producing cells from the animals thus immunized and fusing the cells with myeloma cells to form hybridomas. In the present invention, the antibody and dCyd
It is preferable that the affinity constant Ka with is 1 × 10 ⁷ M ⁻¹ or more. The Ka value can be measured by the method of Yagisawa (Aki Yagisawa, Protein Nucleic Acid and Enzyme Supplement, No. 31, P219-226, 1987). The present invention also provides a method for measuring dCyd in a sample using the anti-dCyd antibody.
This method is generally a method in which dCyd in a sample is reacted with an anti-dCyd antibody as a reagent, and the presence or amount of dCyd in the sample is determined from the reaction result.

According to one aspect of this method, dCyd in a sample and a predetermined amount of labeled dCyd as a reagent are allowed to react competitively with an anti-dCyd antibody.
In this case, as the amount of dCyd in the sample increases, the binding amount of dCyd to the anti-dCyd in the sample increases, and conversely, the binding of the labeled dCyd to the anti-dCyd antibody decreases, and the anti-dCyd of the labeled dCyd decreases. The amount of dCyd in the sample can be determined by measuring the amount of bound dCyd or the amount of unlabeled labeled dCyd. In this aspect, if the anti-dCyd antibody is immobilized on the solid-phase carrier, the solid-phase carrier is washed after the reaction so that the dCyd bound to the anti-dCyd antibody immobilized on the solid carrier is not bound to dCyd. Can be separated into a simple substance.

As the solid phase carrier in the above case, a solid phase carrier commonly used in immunoassay such as polystyrene beads, well plate for immunoassay and the like can be used. As the label of the anti-dCyd antibody, a label which is commonly used in immunoassays such as a radioisotope, a fluorescent substance and an enzyme can be used, and a radioisotope is particularly preferable. Examples of radioisotopes include ¹²⁵ I and ¹³¹
I, ³² P, ¹⁴ C, ³ H and the like which are commonly used in immunoassay can be used.

As the diluent for the sample and the reagent and the reaction medium, an aqueous buffer solution, for example, a conventional buffer solution such as a phosphate buffer solution having a pH of 6 to 8, an acetate buffer solution and a Tris buffer solution is used. The reaction is preferably carried out at room temperature for 10 to 60 minutes. According to another aspect for measuring dCyd in a sample, dCyd immobilized on a solid phase carrier and dCy in a sample are analyzed.
d is reacted with anti-dCyd as a reagent in a competitive manner. As the amount of dCyd in the sample increases,
The amount of anti-dCyd that binds to dCyd in the sample increases, and conversely, the amount of anti-dCyd that binds to dCyd immobilized on the solid phase carrier decreases, and d that is immobilized on the solid phase carrier d decreases.
By measuring the amount of anti-dCyd bound to Cyd, the amount of dCyd in the sample can be determined.

In this case, the above-mentioned solid phase carrier can be used. Also, dC immobilized on a solid phase carrier
yd is in the form of the above-mentioned antigen, namely dCyd
It is preferably used in the form of a conjugate of the protein with a carrier polypeptide or protein. Immobilization of dCyd on a solid phase carrier is
A buffer containing, for example, dCyd is brought into contact with the solid phase carrier by a conventional method, and then these are allowed to stand for a predetermined time, for example, 60 to
It may be incubated for 180 minutes at room temperature or overnight at 4 ° C., and then unfixed dCyd may be washed away with a washing solution.

The measurement of the anti-dCyd and the antibody bound to the solid phase carrier is carried out by labeling the anti-dCyd antibody itself or by labeling with the second antibody which binds to the anti-dCyd antibody. It can be done using things. As the second antibody, an antibody against an immunoglobulin produced by the animal species used for producing the anti-dCyd antibody, for example, if the anti-dCyd antibody is a mouse antibody, an antibody against a mouse antibody of an animal species other than mouse , Eg goat antibodies can be used.

As mentioned above, as a label for directly labeling the anti-dCyd antibody, a radioisotope, a fluorescent substance, an enzyme or the like can be used, and preferably an enzyme label is used. As the label of the second antibody, an enzyme label, a fluorescent substance, a radioisotope or the like can be used, and preferably an enzyme label is used. As the enzyme in this case, a commonly used enzyme label such as alkaline phosphatase, peroxidase, β-galactosidase, uricase or the like can be used, and a commonly used detection system can be used depending on the kind of the enzyme. For example, when alkaline phosphatase is used as the enzyme label of the second antibody, p-nitrophenyl phosphate is used as the substrate, and p- produced by the enzymatic reaction is used.
Nitrophenol can be measured by its absorbance at 405 nm.

As the diluting solution of the reagent in the sample, the reaction medium, the washing solution and the like in this measurement, those described above can be used. In carrying out the method, for example, the above-mentioned dCyd antigen solution is put into a well of an immunoassay plate, incubated, and then washed to fix the dCyd antigen to the well, and then the sample and anti-dC are fixed in the well.
After reacting with yd antibody, the wells are washed to remove unbound reagents and the like, and then a second antibody against anti-dCyd antibody is added to react and then washed with a labeled enzyme such as alkaline phosphatase. Alternatively, a substrate for the enzyme, such as p-nitrophenyl phosphate, may be added, and color development may be measured after a predetermined time.

[0022]

INDUSTRIAL APPLICABILITY By using the dCyd antigen of the present invention,
An antibody against dCyd with extremely high specificity can be obtained. By using this antibody, dCyd in a sample such as body fluid can be obtained.
The amount of yd can be quantified accurately and easily.

[0023]

EXAMPLES Next, examples will be described. Embodiment 1 FIG. Preparation of succinylated derivative of dCyd In an aqueous solution of dCyd 0.05 mmol / 1 ml, 0.4 mmol of succinic anhydride / triethylamine-dioxane (1:
(9, V / V) 1 ml and mixed at room temperature for 10 minutes.
The reaction solution was dried under reduced pressure to obtain a derivative. This was separated and purified by HPLC. The column contains 10 CAPCELL PAKs.
Use a commercially available product filled in × 250 mm, and the temperature is 40 ° C.
And

Elution was carried out for 30 minutes by a linear gradient elution method of 0 to 30% acetonitrile containing 0.1% TFA. The flow rate of the eluent was 3 ml / min. Detection is 28
The absorbance at 0 nm revealed 4 peaks.
The result is shown in FIG. The second and third peaks were separated. The second peak contains 5'-OS-dCyd and the third peak contains 3'-OS-dCyd.
It was revealed by B-MS and ¹ H-NMR. FAB-M
The result of S is shown in FIG.

Embodiment 2 FIG . 5'-OS-dCyd or
Condensation of 3'-OS-dCyd and carrier polypeptide 5'-OS-dCyd or 3'-OS-dCyd1
To 1 ml of a solution of 5 μmol of 12.5 mM phosphate buffer (pH 5.25), 1-ethyl-3 (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDAC.HCl) 7 was added.
Add 5 μmol and add KLH 0.95 nmol or BSA 78 nm
ol 50 mM phosphate buffer (pH 7.2) and 0.1 M Na
The pH was adjusted to 6.4 by further adding 1 ml of a Cl solution, and the reaction was carried out overnight in the dark. The reaction solution was dialyzed against water for 48 hours.

Then, the dialyzed solution is freeze-dried to give each antigen, 5'-OS-dCyd carrier polypeptide and 3 '.
-OS-dCyd carrier polypeptide was obtained, which was
Saved in. A part of the dialysate was diluted and the absorbance at 270 nm was measured to measure the amount of hapten binding. As a result, 336 mol of 5'-OS-dCyd was bound to 1 mol of KLH. Also, 586 moles of 3'-O
-S-dCyd was bound to 1 mol KLH. On the other hand, for 1 mol of BSA, 9 mol of 5'-OS-
dCyd was bound. 8 mol of 3'-OS-dC
yd was also bound to 1 mol BSA.

Embodiment 3 FIG . Preparation of antibody 1 mg antigen using KLH as carrier polypeptide
1 ml of Freund's complete adjuvant and w /
o An emulsion was prepared. This was injected into the abdominal cavity of female BALB / c mice (6 weeks old) by 0.1 ml each, and then boosted every 2 weeks. From the second immunization, Freund's incomplete adjuvant was used and an emulsion was prepared and administered in the same manner. Then, 3 days after the administration, blood was collected by cutting the tail of the mouse. This serum was left overnight at 4 ° C. to obtain an antibody. It was stored at -20 ° C.

Example 4. Determination of antibody titer Antigen or BSA using BSA as carrier polypeptide
The solution itself was dissolved in 0.1 M phosphate buffer (PB) (pH 7.0) so as to have a concentration of 0.6 μg / ml. 50 μl thereof was dispensed into the sample hole of the microplate. Furthermore, it is left to stand at 37 ° C for 2 hours, and PB (T
The sample hole was washed 5 times with PB). The antiserum obtained in Example 3 was diluted with TPB and dispensed in 50 μl aliquots into the sample wells.

This was shaken at 37 ° C. for one and a half hours. TPB
After washing 5 times with 50 ml of 3 μg / ml alkaline phosphatase-labeled goat anti-mouse IgG TPB solution,
Shake at 37 ° C. for one and a half hours. ₂ including 1 mM MgCl 2
2.4 mM-Nitrophenyl phosphate 100 mM
A NaHCO ₃ solution (pH 9.8) was dispensed in 50 μl portions. Absorbance of generated p-nitrophenol is 405 nm
The wavelength was measured with an EIA reader of the following wavelength. The results are shown in FIGS.

Embodiment 5 FIG . Specificity of antibody 0.6 μg of antigen using BSA as carrier polypeptide
50 μl of PB solution / ml was dispensed into the sample holes of the microplate. It was left at 37 ° C. for 2 hours and washed 5 times with TPB. Further, 50 μl each of the hapten TPB diluted solution was further dispensed and left at 37 ° C. for 10 minutes. To this 10000
50 μl of a double-diluted antiserum solution was dispensed and left at 37 ° C. for 1 hour and a half. Next, after washing 5 times with TPB, the following Example 4 was used.
The same operation was performed. The results are shown in FIGS. FIG.
And 6, the antibody has an affinity constant Ka for dCyd.
= 1 × 10 ⁵ M ⁻¹ . This result indicates that the polyclonal antibody of the present invention is dCy among other nucleotides.
It shows that it specifically binds to d.

Embodiment 6 FIG . Preparation of Monoclonal Antibody Three days after the fifth booster in Example 2,
Mouse splenocytes were taken out and fused with cultured mouse myeloma cells P3U1 by a conventional method using polyethylene glycol. The fused hybridoma was cultured in HAT medium, and the resulting colonies were further liquid-cultured in RPMI 1640 medium containing 10% fetal bovine serum and the like, and cloned by performing limiting dilution 3 times to obtain hybridoma DCYD-1.

The monoclonal antibody in the culture supernatant was stored at 4 ° C. These monoclonal antibodies are dCy
It had an affinity constant Ka of 10 ⁹ M ⁻¹ for d. The hybridoma DCYD-1 is a mouse-mouse
It was named hybridoma DCYD-1 and deposited on August 24, 1995 as FERM P-15132 at the Institute of Biotechnology, National Institute of Biotechnology.

Example 7. Specificity of Monoclonal Antibody The monoclonal antibody-containing medium obtained in Example 6 was used in the same manner as in Example 5 except that the diluted antiserum was used. The results are shown in FIG.
As is apparent from FIG. 7, the monoclonal antibody of the present invention specifically reacts with dCyd in various other nucleotides.

Example 8. Measurement of dCyd 0.6 μg of antigen using BSA as carrier polypeptide
50 μl of PB solution / ml was dispensed into the sample holes of the microplate. It was left at 37 ° C. for 2 hours and washed 5 times with TPB. The standard substance of dCyd (0.1 to 100 μM) and the sample are diluted with TPB and dispensed in 50 μl portions.
It was left for 30 minutes at ℃. Next, the monoclonal antibody DCYD
-1 was added and the mixture was left at 37 ° C. for 1 hour and a half. 5 more with TPB
After washing twice, the same operation as in Example 4 was performed. FIG. 8 shows the result. This curve revealed that 0.1 μM dCyd can be measured. Embodiment 9 FIG. On a 2'deoxycytidine BSA-coated plate prepared in the same manner as in Example 8, 20 μl of the sample, and the monoclonal antibody DCYD-1 culture supernatant diluted solution 200
μl was added and left at room temperature for 1 hour. TP the plate
After washing 5 times with B, peroxidase-labeled goat anti-mouse I
The gG antibody (Jackson Immuno Research) was diluted 10,000 times with 1% BSA-containing T-PBS, 100 μl thereof was added, and the mixture was allowed to stand at room temperature for 30 minutes. After washing 5 times with TPB, 100 μl of OPD substrate solution was added and left for 20 minutes, 100 μl of 2N sulfuric acid was added as a reaction terminator, and the absorbance at 492 nm was measured by an EIA plate reader. The results are shown in Tables 1 and 2. Specimens were measured by diluting 16 healthy subjects and 19 cancer patients 100-fold. Specimens: 16 healthy subjects Average 5.1 μg / ml Cancer patients 19 average 35.0 μg / ml Each serum was measured at 100-fold dilution.

[Table 1]

[Table 2]

[Brief description of the drawings]

FIG. 1 is an HPLC elution diagram of succinylated dCyd, which is a synthetic intermediate of the antigen of the present invention.

FIG. 2 is a synthetic intermediate 5 ′ of the antigen of the present invention.
FIG. 3 is a mass spectrum diagram of —O-succinylated dCyd.

FIG. 3 shows that the antiserum of the present invention is 5′-OS-dCy.
It is a figure at the time of measuring an antibody titer by making it react with d-KLH antigen or bovine serum albumin (BSA).

FIG. 4 shows that the antiserum of the present invention was treated with 3′-OS-dC.
It is a figure at the time of reacting with yd-KLH antigen or BSA, and measuring an antibody titer.

FIG. 5 is an anti-5′-OS-dCyd of the present invention.
FIG. 3 shows the binding specificity when KLH antiserum was reacted with various haptens. In the figure, the abbreviation of hapten is cytosine (Cyt), cytidine (Cyd), 2'-deoxycytidine (dCyd), cytidine-5'-phosphate (C
MP), 2'-deoxycytidine-5'-phosphate (dC
MP), 2'-deoxyadenosine (dAdo), 2 '
-Deoxygua (dGuo), thymidine (Thd).

FIG. 6 is an anti-3′-OS-dCyd of the present invention.
FIG. 6 is a diagram showing the binding specificity when the KLH antiserum is reacted with various haptens, and the abbreviations of the haptens in the diagram are as shown in FIG.

FIG. 7 is a diagram showing the binding specificity when the monoclonal antibody of the present invention is reacted with various haptens,
Abbreviations of haptens in the figure are as shown in FIG.

FIG. 8 is an example of a standard curve when dCyd is measured using the monoclonal antibody DCYD-1.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location G01N 33/577 9162-4B C12N 15/00 C

Claims (6)

[Claims] 1. The following formula (I) or (II): A 2'-deoxycytidine antigen represented by the formula: wherein R is a carrier polypeptide or protein. 2. An antibody against 2'-deoxycytidine. 3. A hybridoma producing the antibody according to claim 2. 4. 2'- expected to be present in a sample
A method for measuring 2'-deoxycytidine in a specimen, which comprises reacting deoxycytidine with the antibody according to claim 2 and measuring whether or not the reaction has occurred or the amount of the reaction directly or indirectly. . 5. The method according to claim 2, wherein 2′-deoxycytidine immobilized on a solid phase and 2′-deoxycytidine in a sample are contained.
Determination of 2'-deoxycytidine in a sample, characterized in that the amount of the antibody bound to 2'-deoxycytidine immobilized on a solid phase is determined by competing with the antibody described in 1. Method. 6. An antibody according to claim 2, comprising
A kit for measuring 2'-deoxycytidine.