JPH06261782A - Production of monoclonal antibody producing cell line, monoclonal antibody producing cell line and monoclonal antibody - Google Patents

Abstract

PURPOSE:To provide a new monoclonal antibody producing cell line which produces monoclonal antibody useful for the detection of cocaine or its derivative with high sensitivity by immunoassay. CONSTITUTION:The monoclonal antibody producing cell line is obtained by fusing a mouse spleen cell sensitized with an antigen of a conjugate composed of a compound of formula (n is an integer of Z >=1) and a protein to a cell line derived from a myeloma and screening the fused cells. The immunoglobulin produced in the supernatant of the cell line culture specifically combines to cocaine or its derivative.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel monoclonal antibody-producing cell line and a monoclonal antibody obtained from this cell line. The monoclonal antibody produced by this cell line is useful for highly sensitively detecting cocaine or a cocaine derivative in blood or air by an immunoassay.

[0002]

2. Description of the Related Art Antibodies to cocaine and cocaine derivatives have been reported, for example, by Bulkrichena Cowl, Stefan J. Milian and Bernard Devidou et al. (Journal of Pharmacology and Experimental Therapeutics Vol. 171-178).

However, the reported antibody is a polyclonal antibody obtained by purifying blood of immunized sheep or rabbits. Further, ecgonine shown in (Chemical Formula 2) is used as the immunogen.

[0004]

[Chemical 2]

[0005]

Polyclonal antibodies, which are conventional techniques, have the advantage that the entire production process until obtaining them is simple, but the characteristics of the obtained antibodies depend on the individual immunized animals. Therefore, it is difficult to provide a reproducible antibody.

Further, since a polyclonal antibody is a mixture of antibodies having various affinities for antigens, it has a low average affinity and cannot be used for high-sensitivity measurement.

Furthermore, according to our follow-up test, when ecgonine was used as an immunogen, there was a large difference in structure between cocaine and ecgonine, so that a monoclonal antibody having high affinity for cocaine could not be obtained.

[0008] An object of the present invention is to provide a monoclonal antibody having a high affinity for cocaine or a cocaine derivative, and a cell line producing the same, which has been made in view of the conventional problems.

[0009]

In order to solve the above problems, the present invention provides a spleen of a mouse sensitized with an immunogen comprising a conjugate of a compound having the structure shown in (Chemical formula 1) and a protein. The cells were fused with a myeloma-derived cell line and then cloned to obtain a monoclonal antibody-producing cell line.

[0010]

[Function] Immunoglobulin-producing cells are accumulated in the spleen. Spleen cells have no proliferative capacity by themselves,
By hybridizing with a myeloma cell line, a hybridoma cell line that produces antibodies while proliferating can be prepared. When one hybridoma cell producing an antibody having the highest affinity and having a high proliferative ability is selected and cultured (cloning), a high-affinity monoclonal antibody is produced.

Since the monoclonal antibodies thus obtained are antibodies of the same species, high affinity can be obtained. Further, by culturing the hybridoma cell line, it is possible to permanently provide a monoclonal antibody having certain characteristics.

Further, as compared with the conventionally used immunogen ecgonine, the immunogen of the present invention has remarkably high structural commonality with cocaine itself, and as a result, a monoclonal antibody having a high affinity for cocaine is obtained. It became possible to make.

[0013]

[Examples] The present inventors fused spleen cells of an immunized mouse with a mouse myeloma-derived cell line and cloned the cells to produce a monoclonal antibody having a high affinity for cocaine or a cocaine derivative. We succeeded in producing the line.

In the present invention, the target antigen is cocaine or a derivative of cocaine, and since all of these substances are low molecular weight, they act as an immunogen for the first time by binding to a high molecular carrier such as protein.

The optimal protein as a carrier depends on the sensitized animal and the antigen. The inventors conducted experiments using various proteins, and as a result, found that keyhole limpet hemocyanin (hereinafter referred to as KLH) was the most suitable as the immunogen carrier in the present invention.

In order to obtain high affinity monoclonal antibody-producing cells, it is necessary to exert a high antibody titer in the antiserum stage. Such an immune response
It depends on the type and system of the moving object. The inventors sensitized mice of various strains (A / J, BALB / c, DBA / 2, C57BL / 6, C3H / He, etc.), and as a result, A / J mice had the highest titers of anti-antibody. It was found to produce serum.

Further, in order to obtain a monoclonal antibody-producing cell having a high affinity for cocaine, it is preferable to use an immunogen that retains the basic skeleton of cocaine shown in (Chemical Formula 3) as accurately as possible.

[0018]

[Chemical 3]

The conjugates of ecgonine and protein used as conventional immunogens do not have the methoxycarbonyl group and benzoyl group, which are the characteristic functional groups of the cocaine molecule.

The present inventors used a conjugate of a compound and a protein having a methoxycarbonyl group and a benzoyl group and having a structural formula shown in (Chemical formula 1) having a more basic skeleton more similar to cocaine as an immunogen.

Finally, the proliferation ability of hybridoma cells largely depends on the type of mouse myeloma-derived cell line used for cell fusion. As a result of cell fusion using various mouse myeloma-derived cell lines, the inventors have found that hybridoma cells fused with P3X63-Ag8.653 have the fastest division.

The monoclonal antibody-producing cell line of the present invention and the experimental method for producing the monoclonal antibody are described below. Preparation of immunogen, 2. 2. Monoclonal antibody-producing cell line and method for producing monoclonal antibody Describe in order of detection method.

1. Preparation of Immunogen First, a compound containing the structural formula shown in (Chemical Formula 1) and a KLH conjugate (hereinafter referred to as CC-KLH) were prepared, and then this was emulsified with an adjuvant to obtain an immunogen.

(A) KLH and O-succinimyl-3- (2-polydithio) -1-propionate (hereinafter referred to as SPDP) (Pharmacia)
A conjugate with and was prepared, and then CC-KLH was prepared. However,
As a method for producing CC-KLH, the method described in Japanese Patent Application No. 5-43502 was used.

That is, norcocaine shown in Chemical formula 4 was prepared from cocaine to prepare an aminobutyl derivative of cocaine. Next, the butylphthalimidized product of norcocaine shown in (Chemical Formula 5) was produced, and the amino derivative shown in (Chemical Formula 6) was produced. From the amino derivative thus obtained, a 2-polybenzylthio derivative of cocaine shown in Chemical formula 7 was prepared to prepare CC-KLH. The synthetic process is shown below.

(B) Preparation of Norcocaine Norcokine having the structure of the following (Chemical Formula 4) was prepared by the method shown below.

[0027]

[Chemical 4]

500 mg (1.65 mmol) of cocaine was dissolved in 21 ml of a mixed solvent (acetonitrile / water = 1/2), and 0.5 ml of acetic acid was added to make it weakly acidic (pH≈5).

21 ml of an aqueous potassium permanganate solution (KMnO ₄ = 534 mg, 3.78 mmol, 2.05 eq.) Was added dropwise to this solution over 3 hours, and the mixture was stirred overnight at room temperature.

After removing the formed precipitate by filtration, potassium carbonate was added to the filtrate to make it weakly alkaline (pH≈8).

It was extracted with ether three times, and anhydrous sodium sulfate was added thereto, followed by drying overnight. After removing sodium sulfate by filtration, ether was distilled off under reduced pressure to obtain a colorless liquid.

The obtained liquid was purified using silica gel thin layer chromatography (hereinafter abbreviated as TLC, developing solvent: ammonia saturated chloroform) to obtain 269 mg of norcocaine.

The yield was 56%. Next, this was butylphthalimidated to prepare an amino derivative.

(B) Butylphthalimidization of norcocaine A butylphthalimidated product of norcocaine having the structure of (formula 5) was prepared by the following method.

[0035]

[Chemical 5]

268 mg (0.93 mmol) norcocaine, 314 mg
(1.11 mmol, 1.2 eq.) Bromobutylphthalimide and
10 ml of 147 mg (1.39 mmol, 1.5 eq.) Anhydrous sodium carbonate
Was dissolved in benzene and refluxed under a nitrogen stream for 72 hours.

The reaction solution was cooled to room temperature and filtered. The filtrate was extracted 3 times with 1N hydrochloric acid, and then the aqueous layer was extracted 3 times with chloroform.

After drying with anhydrous sodium sulfate,
Chloroform was distilled off under reduced pressure to obtain colorless crystals.

The obtained crystals were purified by TLC (developing solvent: ammonia saturated chloroform / benzene = 1/3) to obtain 270 mg of butylphthalimidide.

The yield was 60%. (C) Preparation of Amino Derivative An amino derivative having the structure of the following (Chemical Formula 6) was prepared by the method shown below.

[0041]

[Chemical 6]

100 mg of butylphthalimidide (0.20 mmo
l) was dissolved in 3 ml of 95% ethanol, 10 mg of hydrazine monohydrate (0.20 mmol) was added thereto, and the mixture was refluxed for 2 hours.

The reaction solution was purified using TLC (developing solvent: ammonia saturated chloroform / methanol = 10/1) to obtain 57 mg of an amino derivative.

The yield was 78%. (D) Preparation of 2-pyridyldithio derivative of cocaine A 2-pyridyldithio derivative of cocaine having the structure shown below was prepared by the method shown below.

[0045]

[Chemical 7]

84 mg (0.23 mmol) of the amino derivative was dissolved in 1 ml of ethanol and added to 1 ml of O-succinimidyl-3.
-(2-Pyridyldithio) -1-propionate (hereinafter SPDP
Abbreviated) / ethanol solution (SPDP = 73 mg, 0.23 mmol)
Was dripped.

After stirring at room temperature for 1 hour, the product was purified using TLC (developing solvent: ammonia saturated chloroform).
Obtained mg of pyridyldithio derivative.

The yield was 100%. (E) Preparation of KLH-SPDP 200 mg of KLH was added to 30 ml of phosphate buffer sarin (hereinafter referred to as PB).
S) and 0.5 ml of SPDP / with stirring.
An ethanol solution (SPDP = 9.4 mg, 15 eq.) Was added dropwise.

After stirring at room temperature for 30 minutes, the formed precipitate was removed by centrifugation (10 min., 20000 rpm).

The obtained supernatant was subjected to gel filtration using a Sephadex G25 column (Pharmacia, diameter 2 cm × length 80 cm) to obtain 40 ml of KLH-SPDP / PBS solution.

The number of SPDP bound per KLH molecule was determined as follows. The obtained KLH-SPDP PBS
The absorbance A ₂₈₀ at ₂₈₀ nm was measured using 1 ml of the solution, and the measurement result was 3.6005.

To this, 50 μl of 100 mM dithiothreitol (hereinafter referred to as DTT) aqueous solution was added, and after standing for 5 minutes, 3
The absorbance A ₃₄₃ at 43 nm was measured, and the measurement result was 1.576.

Pyridine released by DTT reduction
Assuming that the molecular extinction coefficient of 2-thione at 343 nm is 1.12 × 10 ⁵ , its concentration can be calculated as in the following (Equation 1).

[0054]

[Equation 1]

This concentration is equal to the concentration of SPDP introduced into KLH. Further, since the 2-pyridyl sulfide group of SPDP contributes to the absorbance at 280 nm, the following correction is necessary for the calculation of the KLH concentration.

Absorbance A at 280 nm due to KLH
_{280, CGG} is as follows. _{A 280, CGG = 3.6005- (1.9505} × 10 -4 × 5.1 × 10 3) = 2.6057 However, a 2-pyridyl 5.1 × 10 ³ molar absorption coefficient at 280nm of disulfide groups.

Therefore, the concentration of KLH and the number of SPDP molecules introduced per molecule of KLH are as shown in the following (Equation 2).

[0058]

[Equation 2]

(F) Preparation of Cocaine / KLH Conjugate A cocaine / KLH conjugate was prepared by the following method.

40 ml of KLH-SPDP / PBS solution (2.
77.1 mg (50 eq.) Of DTT was added to 3265 × 10 ⁻⁵ M), and the mixture was stirred at room temperature for 30 minutes.

The resulting precipitate was centrifuged (10 min., 20000r
pm), and the resulting supernatant is Sephadex G2
Gel filtration through 5 columns (Pharmacia, diameter 2 cm x length 80 cm) and 48 ml of KLH-SPDP (SHfree) / PB
An S solution (1.7316 × 10 ^-5 M) was obtained.

4.63 mg (8.312 × 10 ⁻³ mmo) was added to the obtained solution.
l) Pyridyldithio derivative was added, and the mixture was stirred at 4 ° C. overnight.

The resulting precipitate was centrifuged (10 min., 20000r
The absorbance A ₃₄₃ at ₃₄₃ nm was measured, and the measurement result was 1.0352.

The obtained supernatant was subjected to gel filtration with a Sephadex G25 column (Pharmacia, diameter 2 cm × length 80 cm), and 48 ml of cocaine / KLH conjugate / PBS was obtained.
A solution was obtained.

The number of bonds of the pyridyldithio derivative per molecule of KLH was determined as follows. A ₃₄₃ immediately after the reaction is 1.0
Since it is 352, if the molecular extinction coefficient of the released pyridine-2-thione at 343 nm is 8.08 × 10 ³ , its concentration can be calculated as in the following (Equation 3).

[0066]

[Equation 3]

This concentration is equal to the concentration of the pyridyldithio derivative introduced into KLH. The concentration of KLH is 1.40 × 10
^Since it is ⁻⁵ M, the number of molecules of the pyridyldithio derivative introduced per one molecule of KLH is as shown in the following (Equation 4).

[0068]

[Equation 4]

As a result, CC-KLH having 7.4 cocaine bound per molecule of KLH was obtained. Although SPDP was used as the cross-linking agent in this example, it is naturally possible to easily prepare the conjugate using a cross-linking agent other than this.

(B) Preparation of Adjuvant Emulsion CC-KLH obtained by the above synthesis was diluted with phosphate buffered saline (hereinafter, PBS) to obtain 6 mL of 1 mg / mL solution.

6 mL of an adjuvant (complete Freund's adjuvant containing human tuberculosis killed, Wako Pure Chemical Industries, H37Rv) was stirred well, and 6 mL of CC-KLH solution was added in 3 portions, while stirring with a homogenizer. While emulsifying (10,000 rpm), a small amount was dropped onto the water until it did not spread.

In this example, KLH was used as the protein.
Although the immunogen was prepared by using, the immunization for preparing the monoclonal antibody-producing cell line was performed by using the same synthetic method even when other proteins such as bovine serum albumin and chicken-γ-globulin were used as carriers. Hara can be made.

2. Monoclonal antibody producing cell line
And a method for producing a monoclonal antibody Hereinafter, a method for producing a monoclonal antibody-producing cell and a monoclonal antibody will be shown step by step, and an example will be described.

(A) Immunization of Mice About 8 weeks after birth, 10 mice (A / J) were intraperitoneally injected with 1. 10% of the adjuvant emulsion containing the immunogen obtained in the preparation of the immunogen
Each injection was 0 μL.

(B) Check of antibody production For mice 70 days after the immunization injection, 50 to 100 μL of blood was collected from the ocular vein into a centrifuge tube.

When the serum was centrifuged and screened by the ELISA method (described later), production of anti-cocaine antibody was confirmed in all mice.

(C) Boosting of Mice Two mice, which had particularly high titers in the screening of (B), were boosted (injection of weak immunogen) to enlarge the spleen of the mice.

The immunogen was 1 mg / m 2 obtained by diluting CC-KLH with PBS.
The L solution was used as it was without adjuvant.

71 days after immunization, the immunogen was
100 μL was injected intraperitoneally. (D) Cell fusion The spleen cells of the mouse 3 days after the boost were removed,
A mouse myeloma-derived cell line (P3X63-Ag8.65) was prepared by a conventional method using polyethylene glycol having an average molecular weight of 1,500.
Fused with 3).

Using the same mouse spleen cells as feeders (cells supplying growth factors), 96-well plate 2
The plate was cultured on a HAT medium containing 15% fetal calf serum (hereinafter, FCS).

After 1 week, the medium was replaced with HT medium containing 15% FCS. (E) Cloning Screening was carried out by the ELISA method, and the top 12 wells were selected from those having high titers.

96 well microplate 1 was diluted to a concentration containing 1 cell per well (limit dilution)
Dispensed into 2 pieces.

Five-week-old mice (Balb
/ c) pleural cells were used to promote early proliferation.

The culture is advanced while increasing the size of the plate, and the supernatant is repeatedly screened by the ELISA method at appropriate times to finally select a cell line showing a high titer against cocaine and good growth. , 200m
The culture was advanced to a concentration of 5 × 10 ⁵ cells / mL in l.

(F) The finally selected cell line is
The supernatant was centrifuged and suspended in 1 mL of FCS: dimethylsulfoxide = 9: 1 solution at a concentration of 5 × 10 ⁶ cells / mL, frozen at -80 ° C, and then transferred to -135 ° C for long-term storage. I was in a state.

(G) A monoclonal antibody was purified from the cell culture supernatant by affinity chromatography using a protein A-binding gel (protein A Sepharose CL-4B, manufactured by Pharmacia).

This monoclonal antibody was confirmed by SDS polyacrylamide gel electrophoresis to be an IgG composed of a H chain having a molecular weight of about 50,000 and an L chain of about 20,000 by comparison with a standard protein.

In this example, KLH was used as a protein to bind to cocaine, but a monoclonal antibody-producing cell line was similarly prepared when other proteins such as bovine serum albumin and chicken-γ-globulin were used as carriers. it can.

3. Detection method (ELISA method) The ELISA method was used for evaluation of the antiserum and culture supernatant obtained in 2 and detection of cocaine and cocaine derivatives using a monoclonal antibody.

The operation method is described below. (A) Antigen coating A conjugate (CC-BSA) obtained by the same synthesis method as CC-KLH and having one molecule of cocaine bound to one molecule of bovine serum albumin (BSA) was added at 0.1 mg / ml and 0.04%. An antigen solution having a BSA concentration of 0.1 mg / mL was prepared by diluting with PBS containing sodium azide (BSA / PBS / Az).

100 μL / well of the antigen solution was injected into a microplate (vinyl chloride 96-well plate, Costar) and stored at 20 ° C. overnight.

After removing the antigen solution with an aspirator, PB
It was washed 3 times with S and the remaining PBS was removed with an aspirator.

(B) Blocking BSA / PBS / Az was injected at 250 μL / well and left at room temperature for 30 minutes.

After that, BSA / PBS / Az was removed with an aspirator. When no subsequent experiments were performed on the same day, they were stored in this state at 4 ° C. together with filter paper moistened with water.

(C) Antibody reaction Antibody solutions (serum, diluted with BSA / PBS / Az at various dilution ratios)
Culture supernatant, purified antibody, etc.) 100 μL / well was injected.

When carrying out the inhibition experiment, 50 μL / well of the inhibitor solution was injected, and 50 μL / well of the antibody solution was further added while shaking.

After storing at room temperature for 3 hours, the antibody solution was removed with an aspirator and washed three times with PBS to remove the remaining PBS with the aspirator.

(D) Reaction of secondary antibody 0.2 μg / mL peroxidase-labeled anti-mouse IgG antibody goat origin (KPL, Cat.141806 lot. HL10-5) was added to P of 1% BSA.
50 μL / well of a solution (second antibody solution) dissolved in a BS solution was injected, and the mixture was left at room temperature for 30 minutes.

The second antibody solution was removed with an aspirator, and PB
It was washed 3 times with S, and the remaining PBS was removed with an aspirator.

(E) Reaction and Termination of Substrate 40 mg of o-phenylenediamine (for biochemistry) was dissolved in 10 mL of citrate-phosphate buffer (pH 5), and immediately before use,
100 μL of a solution (substrate solution) containing 4 μL of 0% hydrogen peroxide solution
/ Well was injected and left at room temperature.

After 10 minutes, 25 μL / well of 4N sulfuric acid was injected to stop the reaction. (F) Measurement The absorbance at 492 nm or more was measured using a Toyo Soda Microplate Reader. Usually, in the first column, pure water was injected to serve as a reference value, and a blank value in which only the item (C) was appropriately omitted was used.

An example of the binding ability between the monoclonal antibody solution of each concentration and CC-BSA which is the solid phase antigen coated on the plate by the above method is shown in FIG. The vertical axis is the absorbance and the horizontal axis is the logarithmic value of the antibody concentration (mole / l, hereinafter, M).

As shown in FIG. 1, it can be seen that the bond is saturated at a concentration higher than 10 ⁻⁹ M. Next, FIG. 2 shows an example of detecting cocaine at each concentration with the antibody 10 ^-9 M. In FIG. 2, the vertical axis is the inhibition (%) and the horizontal axis is the logarithmic value of the cocaine concentration (M).

The inhibition (%) is a value obtained by the following calculation formula. Inhibition (%) = (1-ODx / ODmax) x 1
00 ODx: Absorbance at cocaine xM ODmax: Absorbance at cocaine 0M In this detection method, the higher the concentration of cocaine, the lower the absorbance. Therefore, the inhibition represented by the above formula increases as the concentration of cocaine increases.

Under these conditions, as shown in FIG.
^10-9.0 M of cocaine could be detected.

It is known that a monoclonal antibody usually shows affinity for a substance having a structure similar to that of an immunogen. Therefore, even if the monoclonal antibody immunized with CC-KLH as shown in the examples is used, the cocaine derivative can be detected although the sensitivity is slightly lowered.

The relative sensitivities of cocaine and derivatives of cocaine when the detection is carried out in the same manner as in this example are listed below.

Here, the relative sensitivity indicates the lowest detectable concentration of each substance when the lowest detectable concentration of cocaine is 1, and therefore the higher the number, the lower the sensitivity.

Norcocaine ------ 10 ecgonine ------ 13 methyl ecgonine ---- 20 benzoyl ecgonine -30

[0110]

INDUSTRIAL APPLICABILITY According to the present invention, mouse spleen cells sensitized with an immunogen comprising a conjugate of a compound having a specific structural formula and a protein and a myeloma-derived cell line are fused and cloned. The method for producing the obtained monoclonal antibody-producing cell line, the immunoglobulin produced in this production method, in which the immunoglobulin produced in the culture supernatant specifically binds to cocaine or a cocaine derivative, and a monoclonal antibody-producing cell line Since it is a nal antibody, it has become possible to provide a monoclonal antibody having a high affinity for cocaine or a derivative of cocaine and a cell line producing the same.

[Brief description of drawings]

FIG. 1 is a graph showing the binding ability of a monoclonal antibody to a solid phase antigen in an ELISA method using the monoclonal antibody in one example of the present invention.

FIG. 2 is a graph showing the results of detecting cocaine by the ELISA method using the monoclonal antibody in one example 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 A 8310-2J // (C12P 21/08 C12R 1: 91) (72) Inventor Tadayasu Kouji 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd.

Claims (6)

[Claims] 1. Spleen cells of a mouse sensitized with an immunogen consisting of a conjugate of a compound having the structural formula shown in (Chemical formula 1) and a protein are fused with a myeloma-derived cell line and then cloned. A method for producing a monoclonal antibody-producing cell line, which is obtained by [Chemical 1] 2. The method for producing a monoclonal antibody-producing cell line according to claim 1, wherein the protein is keyhole limpet hemocyanin. 3. The myeloma-derived cell line is P3X63-Ag8.65.
3. The method for producing a monoclonal antibody-producing cell line according to claim 1, which is 3. 4. The method for producing a monoclonal antibody-producing cell line according to claim 1, wherein the type of mouse is an A / J strain. 5. Spleen cells of a mouse sensitized with an immunogen comprising a conjugate of a compound containing the structural formula shown in (Chemical formula 1) and a protein are fused with a myeloma-derived cell line and then cloned. A monoclonal antibody-producing cell line obtained by the method for producing a monoclonal antibody-producing cell line obtained as above, wherein immunoglobulin produced in the culture supernatant specifically binds to cocaine or a cocaine derivative. 6. Spleen cells of a mouse sensitized with an immunogen comprising a conjugate of a compound containing the structural formula shown in (Chemical formula 1) and a protein are fused with a myeloma-derived cell line and then cloned. The monoclonal antibody-producing cell line obtained by the method for producing a monoclonal antibody-producing cell line, which is produced in a culture supernatant, is obtained by a monoclonal antibody-producing cell line that specifically binds to cocaine or a derivative of cocaine. Characterized monoclonal antibody.