KR20030080782A - The mass preparation method of monoclonal antibodies to methamphetamine - Google Patents

Abstract

PURPOSE: A mass preparation method of monoclonal antibodies to methamphetamine (MAP) is provided, thereby accurately detecting methamphetamine. CONSTITUTION: A mass preparation method of monoclonal antibodies to methamphetamine (MAP) comprises the steps of: binding MAP to BSA(bovine serum albumin) and OA(ovalbumin) using DSS(disuccinimidyl suberate) as a coupling agent to prepare MAP-BSA conjugate and MAP-OA conjugate; injecting the MAP-BSA conjugate to the abdominal cavity and under the skin of a mouse(Balb/c); collecting spleen cells of an immunized mouse and fusing the spleen cells with myeloma cells; culturing the fusion cell in HAT(hypoxanthine aminopterin thymidine) medium and selecting a monoclonal antibody producing cell line using the MAP-OA conjugate; injecting the monoclonal antibody producing cell line to the abdominal cavity of a mouse(Balb/c); collecting ascite produced; and purifying the ascite to obtain the monoclonal antibodies to MAP.

Description

Mass production method of single cell antibody against methamphetamine {THM MASS PREPARATION METHOD OF MONOCLONAL ANTIBODIES TO METHAMPHETAMINE}

The present invention relates to a mass production method of unicellular antibody against methamphetamine (MAP: Methamphetamine).

Recently, various social problems are caused by the abuse and abuse of psychotropic drugs, and the resultant addiction, and therefore, the development of fast and accurate detection method is urgently needed.

Mesamphetamine represented by the following formula (1) has been abused as a stimulant as a powerful sympathetic excitatory amine that stimulates the central nervous system.

<Formula 1>

F.W. 149 Da

As a method for detecting the intake of psychotropic drugs, trace chemicals contained in blood or urine are detected by gas chromatography or infrared spectroscopy.

However, the application of this method requires a number of cumbersome processes, such as preparation and concentration of the sample through solvent extraction.

In order to obtain objective results in a simple manner in a short time, immunoassay using an antigenic antibody response has been used.

For the detection of methamphetamine by immunoassay, a structural analog that specifically reacts with body fluids, especially methamphetamine or its metabolites present in urine, but has a similar structure, such as epinephrine used as a cough medicine, etc. need.

However, since methamphetamine has a low molecular weight and low immunogenicity, it is difficult to obtain a desired antibody by a widely used immunization method.

Korean patent application (Japanese Patent Application No. 1988-0013161) relates to a cell line producing a monoclonal antibody against methamphetamine.

In Korean patent application (Special 1996-045086), methamphetamine is added to the coupling carrier, EDAC (1- (3-dimethylaminopropyl-3-ethylcarbodiimide), to the BSA, a large transport protein, to make an immunogen, and using cell fusion technology. It is known to generate a specific antibody secreting cell line against methamphetamine.

However, these methods had a problem in that the yield of specific antibodies was low and the yield was low.

An object of the present invention is to efficiently and efficiently prepare a single cell group antibody against methamphetamine required for detection of methamphetamine by immunoassay.

It is also an object of the present invention to provide a kit for diagnosing a methamphetamine doser with a single cell antibody against the methamphetamine obtained.

Figure 1 is a mass production process diagram of the methamphetamine unicellular antibody of the present invention.

Figure 2 is a methamphetamine and the carrier protein BSA and OA conjugate manufacturing process diagram.

Figure 3 is a result confirming the production of the MAP-BSA conjugate that is an immunogen by RP-HPLC.

4 is a result of confirming the production of a detection MAP-OA conjugate by RP-HPLC.

5 is a result of measuring the cross-reactivity of the MAP-monocyte group antibody and the carrier protein by western blotting (westhern blotting).

The present invention relates to a mass production method of a single cell antibody against methamphetamine (MAP), which is a kind of drug.

The method of the present invention synthesizes methamphetamine with carrier proteins BSA (bovine serum albumin) and OA (ovalbumin) using disuccinimidyl suberate (DSS) as a coupling reagent, and MAP-BSA conjugate (immunogen) and MAP for detection. After the -OA conjugate was prepared and immunized by injection of the prepared immunogen into mice, splenocytes were harvested and fused with myeloma cells, followed by culturing in hypoxanthine aminopterin thymidine (HAT) medium to obtain monoclonal antibody against MAP. The cell lines to be produced are selected using a MAP-OA conjugate for detection, followed by injection of antibody-producing cell lines into the abdominal cavity of mice to obtain ascites, which are purified to obtain a large amount of single-cell antibody against methamphetamine. have.

Mass-produced monocellular antibodies have high titers of antibodies, do not cross-react with transport proteins, and competitive inhibition occurs with increasing concentrations of methamphetamine, and does not cross-react with other proteins contained in urine, It is used in diagnostic kits for detecting methamphetamine.

Referring to the mass production method of a single-cell antibody against methamphetamine of the present invention in more detail.

The methamphetamine and the carrier protein BSA were made using the coupling reagent DSS to make an MAP-BSA conjugate as an immunogen, and in the same manner as the MAP-OA conjugate for detection.

MAP-BSA conjugates, which are immunogens, are immunized by intraperitoneal and subcutaneous injection of mice (Balb / c species).

Splenocytes are obtained from mice and fused with myeloma cells.

The myeloma cells do not need to be specific cell lines, and do not produce immunoglobulins and may be myeloma cells that lack HGPRT (Hypoxanthine guanine phosphoribosyl transferase).

The cell-fused cells are cultured in HAT medium, and the methamphetamine unicellular antibody secreting cell line is selected using MAP-OA for detection.

Mice (Balb / c) who want to produce unicellular antibody are pretreated with Pristane (2,6,10,14-tetramethyldecanoic acid), an intraperitoneal cancer-promoting agent, to facilitate cancer. do.

Selected antibody secreting cell lines are injected intraperitoneally of the pretreated mice.

Get revenge.

From this ascites, methamphetamine unicellular antibody is purified.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

Example 1 Preparation of MAP-BSA Conjugate as an Immunogen and MAP-OA Conjugate for Detection

MAP-BSA conjugate immunogen was prepared using methamphetamine and carrier protein BSA (bovine serum albumin) using a cross-linker of Disuccinimidyl suberate (DSS) (see FIG. 2).

7.2 mg of methamphetamine and 15 mg of DSS (1: 1 mole ratio) were mixed with 6 mg of sodium bicarbonate and 330 µL DMSO solution, and reacted by stirring slowly at room temperature for 2 hours.

5.0 mg of BSA was dissolved in 1 ml of 10 mM PBS buffer (pH 7.4), and then the product reacted therein was dropped dropwise, and stirred at room temperature for 30 minutes to react again.

The reaction solution was centrifuged to remove the precipitate, and the supernatant was dialyzed three times with 10 mM PBS buffer (pH 7.4) to remove residual DSS.

Reverse phase high-speed column chromatography (RP-HPLC) using a Protein PAK 125 (Waters, USA, P / N 08406) column confirmed that the final product was a MAP-BSA conjugate (FIG. 3).

The manufacture of the detection MAP-OA conjugate produced the detection MAP-OA conjugate by the method similar to the manufacturing method of said MAP-BSA conjugate (FIG. 4).

Example 2 Immunization of Mice

Mice (Balb / c) were stabilized for 1 week.

70 μg of the MAP-BSA conjugate, an immunogen obtained in Example 1, was mixed 1: 1 with a complete Freund's adjuvant and injected into the abdominal cavity and subcutaneously of mice.

At two week intervals after the first immunization, the same amount of MAP-BSA conjugate immunogen was immunized three times by mixing with Incomplete Freund's adjuvant.

Experimental Example 1 Confirmation of Immunization of Mice

Using anti-serum of mice not immunized and mice immunized by the method of Example 2, antibody titers were measured using a MAP-OA conjugate by the indirect ELISA method and the results are shown in Table 1.

TABLE 1 Antibody titers measured in antisera of mice

Dilution 1/100 1 / 1,000 1 / 10,000 1 / 100,000 Antiserum 1st - 1.760 0.925 0.372 (1/100) 2nd 2.015 1.830 1.330 0.436 Normal serum (1/100) 0.178 0.067 - - ※ PBS 0.062

As shown in Table 1, the antibody titer of the mice not immunized was 0.067, and in the mice immunized by the method of Example 2, the average antibody titers were immunized to 1.830 when diluted 1,000-fold.

Example 3 Cell Fusion

Antibody titers were measured using MAP-OA conjugates by indirect ELISA method using antiserum of mice immunized in Example 2.

Mice with the highest antibody titers were selected and 100 μg of MAP-BSA conjugate dissolved in PBS was injected through the tail vein 3 days prior to cell fusion.

On the day of cell fusion, the spleens of the mice were extracted, washed lightly with 10 ml of RPMI 1640 medium, and lymphocyte cells were extracted using a cell dissociation sieve tissue grinder kit in 30 ml of RPMI medium.

After centrifugation at 400 g , the leukocyte components were separated by hemolysis with a buffer. After washing three times with 30 ml of RPMI 1640 medium, the cells precipitated in 10 ml of fresh RPMI 1640 medium were freed and stored in an incubator at 37 ° C.

Myeloma cells were used to receive SP2 / 0 plasmacytoma cells from LG Life Science Research Institute. This cell line does not produce immunoglobulins and lacks HGPRT (Hypoxanthine guanine phosphoribosyl transferase).

SP2 / 0 plasmacytoma cells were cultured using RPMI 1640 medium containing 10% FBS, and the cells were dispensed in the same amount of fresh medium the day before cell fusion to increase cell health.

Spleen lymphocyte cells and plasmacytoma cells of the prepared mice were stained with trypan-blue solution (0.04%, Sigma, St. Louis, USA, T-8154) and the viable cell number was measured using a hemocytometer.

10 ⁸ spleen lymphocyte cells and 10 ⁷ plasma cell tumor cells were mixed and the mixed cells were washed with RPMI 1640 medium.

After the last wash, the supernatant was completely removed, and the tube containing the precipitated cells was lightly struck with a finger and mixed well. The 50% (w / v) PEG / DMSO solution was maintained at 37 ° C. at a rate of 1 ml / min. The ml was fused with dropwise addition.

RPMI 1640 medium was added dropwise by 1 ml for 1 minute, followed by 30 ml at a rate of 3 ml / min.

Centrifuge the fused cells at 400 g for 10 min to collect and float in HAT RPMI medium (FBS added at 20%) to select only the fused cells, and then 50 in five 96 well culture plates containing feeder cells. Aliquots were aliquoted per μl / well.

50 μl / well of HAT RPMI medium added with 20% FBS was added thereto, followed by incubation for 24 hours at 37 ° C. in the presence of 5% CO ₂ , and then 50 μl / well of HAT medium was added thereto.

Thereafter, 100 μl / well of the same medium was exchanged at intervals of 3 to 4 days, and the proliferation of fused cells was observed with an inverted microscope (Olmympus, Japan, IMT2-21-W-PM20-35DX2).

Example 4 Selection of Monoclonal Antibody Secreting Cell Lines against MAP

After cell fusion, the cells were grown under reversed phase microscope, and when grown to the extent of 10-20% at the bottom of the well, the culture medium of the upper layer of the well was taken and the antibody titer was measured using the MAP-OA conjugate by the indirect ELISA method.

Antibody titers were negative control and O.D. The wells with a difference of more than 0.5 were selected, and when the cells grew about 50% in the wells, they were transferred to a 24 well culture plate and cultured.

Cells grown 10-20% in the culture plate was again measured in antibody titers by indirect ELISA method is shown in Table 2 below for the five well titers of high. Normal serum and PBS were used as negative controls.

TABLE 2 Selected Antibody Producing Cell Lines

Control Conjugate (ovalbumin) ovalbumin Antiserum (1/100) 3.002 1.495 Normalmedia 0.083 0.093 Supernatant Conjugate (ovalbumin) ovalbumin 1B9 1.557 1.646 2D9 2.987 0.136 2D10 2.911 - 2F9 1.161 0.546 3E1 2.900 -

Of these, 2D9 was selected and cultured in a 96 well culture plate by limiting dilution to 1 cell / well, and antibody titers were measured by indirect ELISA to select 4 cell lines that secrete monoclonal antibodies as shown in Table 3 below. .

At this time, anti-MAP-BSA serum was used as a positive control and normal media was used as a negative control.

<Table 3> Result of 1st Limited Dilution Dispensing Culture

Control Cultured media Antiserum (1/100) 0.837 2D92D4 1.048 Noermalmedia 0.071 2D96F4 0.974 Antiserum (1/100) 0.656 2D911E2 1.048 Noermalmedia 0.091 2D911H1 0.974

Each cell line was cultured by diluting the cells by secondary limiting dilution to 0.5 cell / well, and measuring antibody titers by indirect ELISA.

At this time, anti-MAP-BSA serum was used as a negative control for the normal medium as a positive control.

TABLE 4 Secondary limiting dilution aliquot culture results

Control Cultured media Antiserum (1/100) 0.981 2D92D41B7 1.143 2D92D42G1 0.994 Noermalmedia 0.081 2D96F41C4 0.956 2D96F42F3 1.005 Antiserum (1/100) 0.893 2D911E21B5 1.055 2D911E22C5 1.033 Noermal media 0.110 2D911H12F7 0.993

Example 5 Preparation of Ascite Fluid

In order to facilitate the cancer, 0.5 ml of Pristane was injected into the abdominal cavity of three old mice (Balb / c) older than 8 weeks.

One week after the injection of freesten, at least 5 × 10 ⁷ cells of 2D92D41B7, 2D96F42F3, and 2D911E21B5 of the single cell group antibody secreting cell lines selected in Example 4 were suspended in 400 μl of RPMI 1640 medium, and then inoculated into the abdominal cavity of mice.

Inoculated mice were observed for abdominal swelling at intervals of 2 to 3 days.

One week after the inoculation, when the abdomen was expanded to a nearly pregnant size, a sterile 16 G needle was inserted into the abdomen of the mice to obtain ascites.

Each obtained ascites was centrifuged at 600 g at 4 ° C. for 10 minutes, the supernatant was collected, the fat layer was removed, and 1 ml each was dispensed into 1.5 ml tubes and stored at −20 ° C.

Example 6 Purification of Monoclonal Antibodies to MAP

To each of the plurality obtained in Example 5, 1 M Tris-HCl buffer (pH 8.0) was added to a plurality of volumes of 1/10 to prevent pH change, followed by 1: 1 with saturated ammonium sulfate solution. It was mixed with and left for 4 hours at 4 ℃.

The supernatant was completely removed by centrifugation at 10,000 g for 20 minutes, and then dissolved in the same volume as a plurality of initial volumes with 20 mM sodium phosphate buffer (pH 7.0).

Ammonium sulfate was completely removed by dialysis four times at 4 ° C. using 100-fold volume PBS (pH 7.0) buffer.

The Protein G column (volume: 1 ml, Supelco, Bellefonte, USA, 54840-U) was washed with 20 mM sodium phosphate buffer (pH 7.0) to equilibrate the column, and then 1 ml of the dialysis sample was added. It was.

After washing with the same buffer to wash off the protein G bound proteins, bound IgG was eluted with 0.1M Glycine-HCl buffer (pH 2.7).

The pH was restored to the original state by adding 1/10 of 1M Tris-HCl buffer (pH 9.0) of the eluate volume to the eluted sample.

The eluted sample was concentrated (200-500 μl) using a concentrator (2 ml, Vivascience, USA, 99VS0248) to prepare a single cell antibody against the MAP of the present invention.

Experimental Example 2 Isotype Determination of Single Cell Antibody

The subclass of unicellular antibody produced by the seven cell lines established in Example 4 was immunopure ^? Monoclonal Antibody Isotyping Kit I was used. As a result, as shown in Table 5, all cell lines secrete IgG _2b with κ light chain.

Table 5 Subclass Analysis of Monoclonal Antibodies

IgG1 IgG2a IgG2b IgG3 IgA IgM Kappa Lamda Normal 2D92D41B7 0.122 0.490 1.252 0.099 0.125 0.095 1.094 0.184 0.133 2D92D42G1 0.098 0.513 1.195 0.089 0.130 0.091 1.072 0.168 0.116 2D96F41C4 0.099 0.324 1.059 0.086 0.133 0.088 0.854 0.148 0.102 2D96F42F3 0.110 0.497 1.188 0.087 0.134 0.094 1.055 0.182 0.107 2D911E21B5 0.117 0.342 1.017 0.109 0.141 0.102 0.963 0.176 0.125 2D911E22C5 0.119 0.307 1.003 0.095 0.127 0.091 0.939 0.161 0.111 2D911H12F7 0.109 0.403 1.025 0.091 0.137 0.093 0.950 0.160 0.098

Experimental Example 3 Measurement of Titer of Monoclonal Antibody to Prepared MAP

The titer of unicellular antibody against MAP obtained in Example 6 was measured by ELISA method and the results are shown in Table 6.

TABLE 6 Titers of unicellular antibody against MAP

Antibod conc. (ng / ml) One 10 100 1,000 10,000 2D92D41B7 0.257 0.853 1.521 1.691 1.968 2D96F42F3 0.208 0.568 1.360 1.613 1.913 2D911E21B7 0.323 1.142 1.724 1.965 2.059 PBS 0.167

The titers of these antibodies showed high titers of 0.56-1.14 ng / ml at concentrations of 10 ng / ml.

Experimental Example 4 Measurement of Cross-Reactivity between Monoclonal Antibody and Carrier Protein

The cross-reactivity of the unicellular antibody prepared in Example 6 of the present invention with the carrier protein was measured by an Indirect ELISA method, and the results are shown in Table 7.

Table 7 Cross-Reactivity Results with Mesamphetamine Monoclonal Antibodies and Carrier Proteins

Cell-line MAP-OA Conjugate OBA (ovalbumin) BSA 2D92D41B7 1.084 0.061 0.068 2D92D42G1 1.140 0.045 0.062 2D96F41C4 1.135 0.042 0.055 2D96F42F3 1.119 0.042 0.056 2D911E21B5 1.225 0.057 0.069 2D911E22C5 1.181 0.046 0.061 2D911H12F7 1.185 0.047 0.065 Antiserum (1/100) 1.363 0.460 0.125 Normal media 0.070 0.061 0.090

As shown in Table 7, all seven unicellular antibody antibodies did not cross react at all with the immunogen or the proteins used for analysis.

Experimental Example 5 Competitive Inhibition of Antibody Using ELISA

Competitive ELISAs were performed to determine whether established monocellular antibodies reacted with pure MAP, and the results are shown in Table 8.

TABLE 8 Competitive Inhibition Rate of Monocytic Antibody-secreting Cell Lines Against Mesamphetamine (%)

MAP conc. (Μg / ml) 10 100 1,000 2D92D41B7 9% 18.2% 72% 2D96F42F3 8.2% 1.7% 43.5% 2D911E21B5 - 2 % 41%

As a result, it was observed that the competitive inhibition of the antibody isolated from three cell lines (2D92D41B7, 2D96F42F3, and 2D911E21B5) increased with increasing MAP concentration.

In addition, 72% of antibodies isolated from the cell line 2D92D41B7, 41% of 2D96F42F3, and 43.5% of 2D911E21B5 were inhibited under the same conditions of MAP concentration of 1000 µg / ml and antibody concentration of 50 ng / ml.

As a result, the affinity of the antibody isolated from the cell line 2D92D41B7 with the highest inhibition rate showed the highest affinity.

Competitive ELISA of antibodies isolated from cell line 2D92D41B7 was carried out again with the same concentration of antibody in order to examine the wider range of competitive inhibition. 48.7% at MAP 400 ㎍ / ml, 84.2% at 2000 ㎍ / ml, 10000 At μg / ml, 90.1% inhibition was observed (see Table 9).

Table 9 Competitive Inhibition Rate of Mesamphetamine Monoclonal Antibody-secreting Cell Lines According to Mesamphetamine Concentration (%)

MAP conc. (Μg / ml) 0 3.2 16 80 150 400 2,000 10,000 PBS (control) OD 1.763 1.170 1.404 1.396 1.399 1.156 0.714 0.640 0.517 % inhibition 0 47.6 28.8 29.5 29.2 48.7 84.2 90.1 100

Experimental Example 6 Measurement of Cross-Reactivity with Urine Protein in Humans by Indirect ELISA

Nonspecific cross-reactivity of the prepared unicellular antibody with other proteins contained in urine was measured.

The BCA method (Smith at el., 1985) was used to measure the amount of protein. Antigens were attached at the concentration of 1 μg / ml in the same manner as the methamphetamine-BSA conjugate, and measured using the antibody purified from the methamphetamine mononuclear cell line at a concentration of 50 ng / ml.

Table 10: Nonspecific cross-reactivity of the prepared amphetamine monoclonal antibodies with other proteins contained in urine

Antibody conc. (ng / ml) 10 100 1,000 MAP-OA conjugate 0.378 0.829 1.071 Proteins in urine 0.084 0.095 0.259 PBS 0.086

The results showed that the titer for MAP-OA conjugate was 0.378 and 0.084 for urine protein when the concentration of antibody was 10 ng / ml.

When the antibody concentration was 100 ng / ml, the titer for the MAP-OA conjugate was 0.829, 0.095 for the urine protein, and 0.086 for the PBS.

These results indicate that there are no cross-reactions with unicellular antibody in urine proteins as well as carrier proteins.

The present invention provides a method for producing a large number of unicellular antibody against methamphetamine.

Monoclonal antibodies prepared by the present invention, the antibody titers are high, do not cross-react with the transport protein, the competitive inhibition occurs with increasing concentrations of methamphetamine, cross-react with other proteins contained in the urine Does not cause

Single-cell antibody produced in large quantities by the present invention is used in diagnostic kits for detecting methamphetamine.

Claims (4)

In the method for producing a single cell antibody against methamphetamine (MAP), Synthesis of methamphetamine (MAP) with carrier proteins BSA (bovine serum albumin) and OA (ovalbumin), respectively, using DSS (disuccinimidyl suberate) as a coupling reagent to prepare a MAP-BSA conjugate as an immunogen and a MAP-OA conjugate for detection. and, The prepared MAP-BSA conjugate was immunized by injection into the abdominal cavity and subcutaneously of mice (Balb / c), Splenocytes from immunized mice were harvested and fused with myeloma cells. While fusion cells were cultured in HAT (hypoxanthine aminpterin thymidine) medium, cell lines producing unicellular antibody were selected using a detection MAP-OA conjugate, Selected unicellular antibody-producing cell lines were injected intraperitoneally of mice (Balb / c), To obtain the resulting Ascite, Consisting of a method of purifying the ascites obtained to obtain a single cell antibody against methamphetamine, Mass production method of unicellular antibody against methamphetamine. The method of claim 1, In mice (Ab / c) to collect ascites 2,6,10,14-tetramethyldecanoic acid is treated in advance to create conditions favorable for inducing celiac cancer, Characterized in that ascites is generated by injecting the selected unicellular antibody-producing cell line into the abdominal cavity of a mouse (Balb / c), Mass production method of unicellular antibody against methamphetamine. The method according to claim 1 or 2, Myeloma cells do not produce immunoglobulins and are characterized by a lack of HGPRT (Hypoxanthine guanine phosphoribosyl transferase). Mass production method of unicellular antibody against methamphetamine. Prepared by the method of claim 1, High titers of antibodies, no cross-reaction with transport proteins, Higher concentrations of methamphetamine result in more competitive inhibition, Does not cross-react with other proteins contained in urine, Single cell antibody against methamphetamine used in a kit for detecting methamphetamine recipients.