Antibody having high affinity to methamphetamine and immunogen for obtaining the same.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates to a method for producing highly effective antibodies which play the most important role in immunoassay in the fields of medical diagnosis, chemical analysis, etc., and in particular to a method for producing antibodies which have a high affinity for methamphetamine, that of is not capable of eliciting immuno-responses and which has a central stimulating effect and is one of the analeptics.

RELATED ART

When detecting a trace component in blood or a specific component of the atmosphere, it is necessary to definitely detect a component present in a trace amount from a large number of contaminants. In recent years, the immunoassay based on the reaction of an antibody with an antigen has been extensively studied for this purpose.

An immunoassay is roughly divided into the radioimmunoassay using a radioactive isotope and the enzyme immunoassay (EIA) using an enzyme. In terms of security and simplicity, the EIA is more advantageous.

The enzyme-linked immunosorbent assay (ELISA), which is a typical EIA, is described below with reference to FIG. 1. In Fig. 1, reference numeral 1 denotes a microplate made of plastic such as polystyrene, which non-specifically adsorbs protein. Reference numeral 2 denotes a solid phase antigen adsorbed on the microplate, this antigen being produced by introducing a suitable functional group into an antigen as an analyte and chemically binding the antigen to protein via the functional group. Reference numeral 3 denotes an antibody capable of binding to the analyte, and the antibody binds to the solid phase antigen 2 in an equilibrium state. This means that the antibody 3 is immobilized on the microplate 1 by means of the solid phase antigen 2. When an antigen 4 is introduced into the system as an analyte, solid-phase antigen 2 and antigen 4 as analyte bind competitively to the antibody 3. Therefore, part of the antibody 3 binds to the antigen 4 as the analyte and, as a result, part of the antibody remains 3 not bound on the microplate 1. The larger the amount of the antigen 4 as the analyte, the more the amount of the antibody 3 not bound to the microplate 1 naturally increases. At this stage, the unbound antibody 3 can be removed by washing. After removal of the unbound antibody 3, antibody 5 which is capable of binding to antibody 3 is added to the system. Antibody 5 chemically binds to enzyme 6 in a defined ratio. After antibody 5 not bound to antibody 3 has been removed by washing, the activity of enzyme 6 is determined. There is a positive relationship between the activity and the amount of enzyme 6 so that the amount of enzyme 6 can be quantified by examining the reactivity. If the amount of enzyme 6 is known, it is possible to determine the amount of the antibody 3 bound to the microplate 1 via the solid phase antigen 2 and then to quantify the antigen 4 as analyte. In this case, an affinity of the antibody 3 for antigen 4 as the analyte is a primary factor in determining the sensitivity of detection.

An antibody is a protein formed by an organism and has the property of selective binding only to a specific substance, ie antigen. Antigens are roughly divided into two types because of the differences in the formation of an antibody binding to the antigen. One is an antigen which can induce the formation of an antibody by direct injection into an animal. In general, macromolecular substances with a molecular weight of tens of thousands or higher correspond to such an antigen. The other type has a relatively low molecular weight and, by chemically binding to a suitable protein, can act as an immunogen that induces the formation of an antibody. In particular, the latter antigen is called hapten. A method of forming an antibody to a hapten as an antigen is in "Haptens and Carriers", O. Makela and IJT Seppala, Handbook of Experimental Immunology, 4th Edition, Volume 1, Chapter 3, published by DM Weir, Blackwell Scientific Publications, Oxford 1986. That is, it is described that if hapten contains a functional group capable of chemical bonding, hapten is bound to protein via the functional group to form an immunogen. If any functional group is missing from the hapten, an immunogen is generally synthesized by introducing a suitable functional group such as an amino group, a carboxyl group and a hydroxyl group and a spacer consisting of an alkyl group having 1 to about 10 carbon atoms into the hapten to synthesize hapten derivatives and Obtain binding of the derivatives to a suitable protein using a crosslinking agent. More specifically, Takami et al. on antibody formation using methamphetamine (MA) with a central excitatory effect, which is one of the analeptics, as a hapten [Takami, Fukuda and Takahashi, Japan, J. Legal Med., 37 (4), 417, 1983]. That is, the aminobutyl group is reported to be introduced into the secondary amino group of MA to form aminobutyl methamphetamine (ABMA). The derivative is bound to protein and the product obtained is used as an immunogen.

However, studies by the present inventors have shown that the antibody produced using the immunogen has a higher affinity for the antigen derivative, ie ABMA, than for the antigen sought, ie MA. This means that the antibodies which have been formed by methods of the prior art are antibodies not for the antigen but for the antigen derivatives. The antigen derivatives have a structure different from that of the antigen because functional groups, spacers, etc. are introduced. It is therefore made clear that an antibody with a high affinity for the desired antigen is difficult to form. When an antigen is detected as an analyte by means of - for example - the previously described ELISA using the antibody formed by the methods of the prior art, even when the antigen is introduced into the system in large quantities as the analyte, the unbound antibody does not increase, so that it is impossible to detect the antigen as an analyte with high sensitivity.

SUMMARY OF THE INVENTION

An object of the present invention is to produce an antibody with high affinity for methamphetamine compared to the antibody produced using a conventional immunogen, which is produced by chemically binding a methamphetamine derivative to protein.

According to the invention, this object is achieved by an immunogen according to claim 1. Preferred embodiments are claimed in the subclaims.

It is the object of the present invention to provide an immunogen obtained by introducing an alkyl chain spacer and a functional group capable of binding to protein into the amino group of amphetamine, and to bind the antigen derivative thus prepared to protein via the functional group , The immunogen of the present invention is one with a chemical structure highly similar to that of methamphetamine compared to conventional immunogens using methamphetamine as a starting material, introducing a functional group and spacer into the starting material and binding to protein via the functional group were manufactured. As a result, an antibody with high affinity for methamphetamine can be produced.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows an explanatory diagram of the ELISA, reference numeral 1 designating a microplate, reference numeral 2 designating a solid phase antigen formed by inserting a suitable functional group into an antigen and chemical binding of the antigen to protein as an analyte, reference numeral 3 designating one for binding denotes the antigen as an analyte-capable antibody, reference numeral 4 denotes an antigen as an analyte, the reference number 5 denotes an antibody capable of binding to antibody 3, and the reference number 6 denotes an enzyme chemically bound to antibody 5.

2 shows a chemical structure of methamphetamine (MA). Figure 3 shows a chemical structure of aminobutyl methamphetamine (ABMA). 4 shows a chemical structure of aminobutylamphetamine (ABAP). 5 shows a chemical structure of amphetamine (AP).

Figure 6 is a graph comparing the detection sensitivity on MA to that on ABMA in the ELISA with antisera obtained according to the prior art and the method of the present invention, the abscissa logarithmic concentrations of added MA and ABMA and the ordinate indicates enzyme activity in relative absorbency values. Where a relative absorbency decreases at a certain concentration of MA, MA can be determined with this concentration. The horizontal, dashed line indicates 50% relative absorption. Curves a, b, c and d show: a curve for the detection of MA using an antibody produced according to the prior art method, a curve for the detection of ABMA using an antibody produced according to the prior art method, a curve for the detection of MA using an antibody produced by the method of the present invention, or a curve for the detection of ABMA using an antibody produced by the method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

As an embodiment of the invention, the production of an antibody with a high affinity for the antigen sought is described below, using methamphetamine (MA), which has a central stimulating effect and is one of the analeptics, as the antigen sought. In this embodiment, an antigen derivative (aminobutylamphetamine, ABAP) was obtained by introducing the aminobutyl group as a functional group capable of binding to protein in amphetamine (AP) (see Fig. 5), which corresponds to a compound in which methyl is the N- Alkyl group in MA is replaced by a hydrogen atom. The structures of MA, ABMA, ABAP and AP are shown in Figures 2, 3, 4 and 5, respectively. A high similarity between MA and ABAP can be seen.

Other preferred examples of the antigen derivatives include amphetamine derivatives represented by the following general formula:

where n is an integer. n is preferably an integer from 1 to 10. If n = 4, the structural formula given above represents ABAP.

These amphetamine derivatives can be easily obtained by reacting AP with, for example, an N-haloalkyl phthalimide such as N-bromobutyl phthalimide and then treating with hydrazine.

Next, ABAP is bound to keyhole limpet hemocyanin (KLH) via the aminobutyl group to form an immunogen. Alternatively, proteins such as gamma globulins and bovine serum albumin derived from chickens etc. can also be used. Other amphetamine derivatives can also be bound to protein such as KLH via the aminobutyl group in its molecule. Binding between amphetamine derivatives such as ABAP and protein can be accomplished in a conventional manner. That is, binding can be accomplished using common binding reagents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), tolylene-2,4-diisocyanate, glutaraldehyde, periodic acid, 1,5-difluoro-2,4- dinitrobenzene (DFDNB), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N- (τ-maleimidobutyryloxy) succinimide (GMBS) and N- (∊-maleimidocaproyloxy) succinimide (EMCS) become.

By injecting the immunogen thus obtained into an animal, an antibody with a high affinity for MA, namely an anti-MA antibody, is generated in the animal serum. For example, collecting sera from animals sensitized with the immunogen results in an antibody with a high affinity for MA. The sera obtained in this way can be used without further purification as an agent for an immunoassay, ie ELISA. Any animal can be used for immunization if it is a mammal. To simplify the experiment, mice were used in one embodiment of the present invention. A / J strain mice are beneficial because the strain has the highest immune response.

By fusion of the spleen cells collected from the animal sensitized with the aforementioned immunogen with myeloma cells, hybridoma cells which can produce a monoclonal antibody with high affinity for MA can be obtained. The hybridoma cells can be generated by the method known as the Kohler and Millstein method [Kohler et al., Nature, 256, 495 (1975)]. For example, the hybridoma cells using X63-Ag8.653, which is one of 8-azaguanine-resistant strains, as mouse myeloma cells, fusion of the cells with spleen cells in the presence of a fusion accelerator such as polyethylene glycol and Sendai virus , Culturing the fused cells obtained in HAT medium and collecting the hybridoma, which produces antibodies with a high affinity for MA, can be harvested.

The hybridoma is cultured in a suitable medium and a monoclonal antibody with a high affinity for MA can be obtained from the supernatant.

Using the antibody and monoclonal antibody with a high affinity for MA, which can be obtained according to the present invention, MA in the blood can be determined quantitatively with high sensitivity, for example by means of ELISA. Therefore, these antibodies are extremely useful in the field of diagnosis, chemical analysis, etc.

In the following, the invention will be described in more detail with reference to the following examples.

example 1

First, a method for producing antisera is described below.

(1) Synthesis of ABAP

A mixture of 2.00 g of amphetamine (AP), 4.17 g of N-bromobutyl phthalimide (manufactured by Aldrich Chemical Company Inc.) and 3.14 g of sodium hydrogen carbonate was refluxed in 10 ml of benzene for 16 hours. After removing the solvent from the reaction solution, the residue was purified by preparative thin layer chromatography (TLC, manufactured by Merck and Co. Inc.) using silica gel as a carrier. A mixture of methanol and chloroform in a volume ratio of 5:95, which had previously been saturated with ammonia, was used as the elution solvent. The Rf value of the product was 0.7. Extraction of the product on silica gel with ethanol gave 1.59 g of N- (4-phthalimidylbutyl) amphetamine (PIBAP).

In 5 ml of 95% ethanol, 1.50 g of PIBAP was dissolved and 0.25 g of hydrazine monohydrate was added to the solution. The mixture was refluxed for 1.5 hours to form a small amount of white precipitate. After the mixture was filtered again, the filtrate was acidified with 1N hydrochloric acid and the white precipitates formed were removed. The solution was then made alkaline with sodium hydroxide, whereby the oil phase was separated. The oil phase was extracted with diethyl ether. After removing the solvent by distillation, the residue was purified by TLC under the same conditions as described above. The product reacts with ninhydrin to form a pure, bluish color. Using the reaction, the product was determined on silica gel and 0.75 g of N- (4-aminobutyl) amphetamine (ABAP) was finally obtained.

(2) Preparation of immunogen

19.1 mg ABAP was dissolved in 200 µl 0.5 N hydrochloric acid. The solution was diluted with 4.8 ml of 0.1 M phosphate buffer solution (pH 7.5). A solution of 12.5 mg of thiol group-introducing N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP, manufactured by Pharmacia LKB Biotechnology) in 1 ml of ethanol was added to the solution, followed by its reaction for 30 minutes with stirring. To determine the progress of the reaction, the solution was developed on TLC. The same solvent mixture described above was used as the elution solvent. SPDP is identified on TLC as a black spot that absorbs UV light at 245 nm. After stirring for 30 min, the black spot on TLC in the reaction solution disappeared, indicating that SPDP fully reacted with ABAP. In this way, the conjugate of SPDP and ABAP (AP-SPDP) was obtained.

On the other hand, KLH was dissolved in 0.1 M phosphate buffer (pH 7.0) containing 0.1 M NaCl to prepare 2.2 mg / ml KLH solution. A solution of 51.6 mg of SPDP in 4.2 ml of ethanol was added dropwise to the KLH solution and the mixture was allowed to react for 12 hours with stirring. Thereafter, unreacted SPDP in the reaction solution was separated by gel filtration using Sephadex G-25 (manufactured by Pharmacia LKB Biotechnology) to obtain the conjugate of KLH and SPDP (KLH-SPDP). Sephadex G-25 was packed in a 4 cm diameter and 50 cm long column and used at a flow rate of 6.9 ml / min. Dithioerythritol (DTT) was dissolved in 0.1 M phosphate buffer and 1.8 ml of the obtained solution was added to KLH-SPDP to carry out the reduction. After the mixed solution was again subjected to gel filtration under the same conditions as described above using the Sephadex G-25 column, the AP-SPDP solution was gradually added to 106 ml of the solution. 30 Minutes later, gel filtration was carried out under the same conditions previously described using the Sephadex G-25 column, introducing 14.3 molecules of AP per 1 molecule of KLH. The conjugate of KLH and AP (AP-KLH) thus obtained was used as an immunogen.

(3) Methods of immunization

The AP-KLH solution was diluted with 0.1 M phosphate buffer (pH 7.0) to adjust the concentration of the KLH to 1 mg / ml. The AP-KLH solution was mixed with Freund's complete adjuvant in an equivalent amount and the mixture was homogenized in a homogenizer. The emulsion was injected intraperitoneally in mice 8 weeks of age. An injection dose was 100 µl per mouse. The mouse strain used was A / J, as previously described.

(4) Evaluation of antisera by ELISA

After 18 weeks after the AP-KLH immunization, the sera of the mice were collected. The ELISA was performed using the sera and the sensitivity of detection was determined using MA and ABMA as analytes. The ELISA was carried out under the following conditions.

Chemically bound conjugate of BSA and ABMA (MA-BSA), in which 0.4 molecule of ABMA as the antigen derivative was introduced in 1 molecule of bovine serum albumin (BSA), was used as the solid phase antigen. MA-BSA was immobilized on a 96-hole polystyrene plate for the ELISA (manufactured by Coaster Corporation). The antisera collected from mice were diluted 10,000-fold with phosphate buffered saline (PBS) and the diluted antisera were mixed 1: 1 with MA or ABMA solutions at various concentrations. The solution mixture was added separately to each well of the 96-well plate for the ELISA, 100 µL each. After 3 hours the solution mixture was removed and the 96-well plate for the ELISA was washed with PBS. This operation removed the sera antibody that was unbound to MA-BSA on the plate. Then, antibody capable of binding to the mouse antibody contained in the antisera was added separately in the same amount as described above. The antibody is a so-called PODF-labeled antibody, which is an antibody chemically linked to peroxidase (POD) is. After 30 min, the POD-labeled antibody solution was removed and the 96-well plate for the ELISA was washed again with PBS. This procedure removed the POD-labeled antibody that was not bound to the mouse antibody attached to the solid phase antigen on the plate. On the other hand, 40 mg of o-phenylenediamine (OPD) was dissolved in 10 ml of phosphate citrate buffer (pH 5.0) and 4 µl of 30% H 2 O 2 was added. mixed with the solution. The mixed solution was applied separately to the 96-well plate for the ELISA, with 100 µl per well, which had been washed with PBS buffer after removal of the unbound, POD-labeled antibody. After 1 to 2 min, OPD was oxidized by the action of POD of the POD-labeled antibody bound to the plate, whereby the formation of a color with maximum absorption at 492 nm was observed. After completion of the enzyme reaction by separately adding 25 µl of 4N sulfuric acid to each hole, the absorbance of the solution in each hole was determined at 492 nm by means of a spectrophotometer. The results are shown by solid lines in FIG. 6. For comparative purposes regarding the sera collected from mice 18 weeks after immunization, the ELISA was performed under the same conditions described above on mice synthesized with the chemically bound conjugate (MA-KLH) of ABMA and KLH in a manner comparable to that previously described conjugate, had been sensitized. The results are shown by broken lines in FIG. 6. In Fig. 6 the ordinate represents a relative absorbance at 492 nm and indicates the relative amount of mouse antibody in the antisera bound to the solid phase antigen. The abscissa represents a concentration of MA or ABMA in the solution mixture of the antisera used for the assay with MA or ABMA.

The results shown in Fig. 6 are checked in the following. First, the antisera obtained by immunization with MA-KLH are considered, and the MA concentration showing a 50% reduction in relative absorbance was found to be 10-3.6 M based on curve a. With regard to ABMA, the relative absorbency decreased to 50% at the concentration of 10-5.2 M based on curve b. This means that it can be concluded that ABMA inhibits the binding of the mouse antibody in the antisera to the solid phase antigen by about 50 times more than MA. In other words, the antibody obtained by immunization with MA-KLH binds to ABMA about 50 times more than to MA. On the other hand, considering that the solid phase antigen used for the ELISA is the chemically bound conjugate of ABMA and BSA and contains the same structure as that of ABMA, the antibody obtained by immunization with MA-KLH is expected to be present the solid phase antigen would bind more than MA. As previously described, the solid phase antigen and the antigen compete with the antibody as an analyte; as a result, the antibody is prevented from binding to the solid phase antigen, so that the amount of the antibody bound to the solid phase antigen decreases. This phenomenon is used for the ELISA. If the antibody binds more strongly to the solid phase antigen, it is difficult to effect the inhibition of the binding of the antibody to the solid phase antigen by the antigen as the analyte, resulting in a reduction in the sensitivity.

On the other hand, in the AP-KLH immunized sera from curve a, the MA concentration showing 50% reduction in relative absorbance was found to be 10-6.5 M, and from curve d, the ABMA concentration became 10-5. 6 M found. This means that the antibody obtained by immunization with AP-KLH binds to MA by about 50 times more than to ABMA in contrast to the case where the antibody was obtained by immunization with MA-KLH. Therefore, there is no chance of causing a reduction in measurement sensitivity as described above. In fact, in the ELISA using the antisera obtained by immunization with AP-KLH, the MA concentration, 10-6.5 M at which the relative absorbance was reduced by 50%, was as low as 1/1000 compared to the case the use of the antisera obtained by immunization with MA-KLH.

The immunogen based on the concept described above is also generally applicable to the production of an antibody to a hapten with a molecular weight below 1000 with respect to substances other than the MA mentioned in this example. In addition, an antibody produced from hybridoma cells obtained by fusing spleen cells collected from animals sensitized in this example with myeloma cells, a so-called monoclonal antibody, can of course also have the same properties as the antisera. This means that a monoclonal antibody can be obtained which has a very high affinity for a hapten as an antigen. A method for producing the monoclonal antibody is described below.

Example 2

A monoclonal antibody was produced using mice in which the formation of the antibody having a high affinity for MA instead of ABMA was confirmed in Example 1. In the following the procedure is described step by step.

(1) Booster response in mice

At 7 weeks after immunization with AP-KLH, the mice were boosted to induce spleen hypertrophy. For this purpose, AP-KLH was diluted to 1 mg / ml and 100 μl of the dilution was injected intraperitoneally into each mouse.

(2) cell fusion

3 Days after the booster treatment, spleen cells were removed from the mice and fused with mouse myeloma cells X63-A8.653. The mode of operation is shown in detail below:

(i) After killing and pasteurizing the mice, the mice were transferred to a clean bench. The following procedure was performed on the clean bench.

(ii) The spleen was removed from each mouse and transferred to a stainless steel sieve previously immersed in Isove's Modified Dulbeco Medium (IMDM) (manufactured by Sigma Chemical Company). The spleen was cut with scissors to make about 10 sections and carefully squeezed out using a glass rod to remove cells. Through this process, the spleen cells were suspended in IMDM. After 5 ml of IMDM with suspended spleen cells was transferred to a centrifuge tube, the spleen cells remaining on the sieve were further washed with 5 ml of IMDM. The washed cells were also transferred to the centrifuge tube. In the subsequent steps up to (ix) the centrifuge was cooled and an ice bath was used if necessary to keep the temperature of the cells as low as 4 ° C as much as possible.

(iii) The suspension of the spleen cells was centrifuged at 800 g for 7 min and the supernatant was removed using an aspirator.

(iv) To destroy the red blood cells, which are unnecessary components, 10 ml of Tris buffer containing NH 4 Cl was added, and the cells adhering to the bottom were stirred with a pipette to detach them. After standing on ice for 5 to 10 minutes, centrifugation was carried out at 800 g for 7 minutes and the supernatant was removed by an aspirator.

(v) Hanks' equilibrium saline (manufactured by Research Institute for Microbiological Disease, Osaka University), 10 ml, was added to the system and the cells remaining at the bottom of the centrifuge were stirred to detach and placed on ice for 5 to Let stand for 10 min. When the tissue fragments were deposited, the cell suspension alone was transferred to another centrifuge tube so that the fragments were not taken away. The cell suspension was centrifuged at 800 g for 7 min and the supernatant was removed using an aspirator.

(vi) Again 10 ml of Hanks' equilibrated saline was added and the cells at the bottom of the vessel were stirred to detach. Centrifugation was carried out at 800 g for 7 min and the supernatant was removed using an aspirator.

(vii) Hanks' equilibrated saline, 13-14 ml, was added to the cells and the cells at the bottom of the vessel were stirred to detach.

(viii) To 50 µl of the spleen cell suspension obtained in (vii) was added 50 µl of nigrosine solution, which was a cell staining solution. About 1 min later, cell counting was done with a hemocytometer. The number of cells was 5.1 x 10? Cells / ml.

(ix) (The following operation is preferably performed alternatively, using the centrifugation time of the previous step (vi) to thereby count the spleen cells and myeloma cells at the same time. Furthermore, centrifuges at different temperatures are used in the following operation so that the cooling of the myeloma cells below normal temperature is avoided.) Myeloma cells, 45 ml, were added to 50 ml of a centrifuge tube during the cultivation process, followed by centrifugation at 800 g for 7 min. The supernatant liquid was aspirated using an aspirator.

(x) Hanks' equilibrated saline, 13 ml, was added to the cells and the cells at the bottom of the vessel were stirred to detach. Centrifugation was carried out at 800 g for 7 min and the supernatant was aspirated. This process was repeated twice.

(xi) After adding 13 ml of Hanks' equilibrated saline, the myeloma cells were stirred at the bottom of the vessel to detach them.

(xii) The cells were counted in a manner similar to that in step (viii). The cell count showed 1.5 x 10? Cells / ml.

(xiii) 9 ml of the spleen cell suspension obtained under (viii), which had been brought to normal temperature, were mixed well with 6 ml of the myeloma cell suspension obtained under (xii). After centrifugation at 2000 g for 5 min, the supernatant was removed using an aspirator.

(xiv) (The following operation was carried out on a water bath placed on the clean work bench at about 40 ° C.) With gentle stirring, about 0.5 ml of polyethylene glycol (PEG) with an average molecular weight of 1,500 was added to those obtained in (xiii) deposited cells were added over 1 min. Stirring was continued for an additional 1.5 min.

(xv) After further adding 5 ml of IMDM at a rate of 1 ml / min, 5 ml of IMDM was added over 1 min thereafter. After 10 ml of IMDM was finally added, centrifugation was carried out at 1000 g for 7 min, and the supernatant was removed by an aspirator.

(xvi) To the deposited cells obtained in (xvi), 3 ml of the spleen cell suspension of (viii) was added as additional cells, and HAT medium (manufactured by Sigma Chemical Company) containing 10% fetal cow serum ( FCS) was added to bring the total volume to 23 ml. After detaching the cells with a pipette, 100 µl was added separately to each well of a 96-well plate for incubation. The above operation was performed using 2 mice, and the cell suspension was individually placed in 6 plates in total.

(xvii) The plates were transferred to a CO 2 incubator and the incubation was initiated. The CO 2 concentration and temperature in the incubator were set at 5% and 37 ° C.

(xviii) One day later, 100 µl of HAT medium was added to each well. Incubation was continued for 1 week.

(3) Formation of monoclonal antibody

The following operation was carried out on the plate on which 1 week after the initiation of the culture of the hybridoma cells in HAT medium:

(1) From each well, 100 µl of the supernatant culture liquid was taken out and diluted with PBS to make a 10,000-fold serial dilution. Using the serial dilution, the ELTSA was performed. Conjugate (MA-BSA) in which 0.4 molecule of MA was chemically bound to 1 molecule of BSA was used as the solid-phase antigen, but no analyte was added. The other conditions were identical to those of the ELISA described above. Such an ELISA enables the binding capabilities of the antibodies in the respective supernatant culture fluids to be compared to the solid phase antigen. That is, in the supernatant culture liquid showing high absorbency at a high degree of dilution, there is a high possibility that an antibody having a high affinity for the solid phase antigen is contained. Several supernatant culture fluids were selected from those that had a high absorbance, and incubation was continued only with the cells in the wells from which these supernatant culture fluids had been removed. A culture scale with a suitably larger increase was established.

(ii) for the supernatant culture liquids selected in (i), the ELISA was carried out in more detail. The supernatant culture fluids were diluted accordingly and MA-BSA was used as the solid phase antigen. MA was added as an analyte. The other conditions were identical to those of the ELISA described above. 19 Supernatant culture fluids were selected from those that showed high sensitivity in measuring MA, and the incubation was continued only with the cells in the wells from which these supernatant culture fluids were obtained.

(iii) The cells that formed the selected 19 supernatant culture fluids were each suspended in IMDM medium and the cells were counted using nigrosine.

(iv) Each cell suspension was diluted with HT medium (manufactured by Sigma Chemical Company) to adjust to 1 cell per 100 µl.

(v) The thymus was removed from mice 5 weeks after birth. The thymus cells were suspended in HT medium to make 2 x 10? Adjust thymus cells per 100 µl.

(vi) The thymus cell suspension of (v) was applied individually with 100 µl to each well in 10 plates of 96-well plates for incubation. Furthermore, the cell suspension of (iv) was added at 100 µl per well.

(vii) While continuing to incubate in a CO 2 incubator, the supernatant culture fluids were subjected to the ELISA. The conditions were the same as in (ii). In the cells where the sensitivities showed a high sensitivity to MA, the incubation was continued while gradually increasing the degree of incubation appropriately. As a result of the continuous selection by ELISA, 5 holes were finally selected. This means that 5 cell lines producing monoclonal antibodies were obtained. These cells were continuously cultured in 200 ml of IMDM medium until 5 x 10? Cells per ml were included.

Of the 5 cell lines, a cell line 2D55A, which produced a monoclonal antibody with the highest affinity for MA, was deposited with the Fermentation Research Institute Agency of Industrial Science and Technology (FRI, Ibaraki-ken, JAPAN) under the Budapest Treaty and received the accession number FERM BP-2564.

(4) Storage of cells

Each culture fluid from the cell lines finally selected was transferred to a centrifuge tube. After centrifugation at 800 g for 7 min, the supernatant culture liquids were removed using an aspirator. The cells on the bottom were suspended in a 9: 1 solution of FCS in dimethyl sulfoxide (DMSO). The suspension was made up to 5 x 10? Cells set per 1 ml. After freezing the suspension at -80 ° C, the frozen suspension was transferred to liquid nitrogen, resulting in a storage condition for a long period of time.

(5) Antibody purification

Monoclonal antibody was purified from the supernatant culture liquid by affinity chromatography using Protein A Sepharose 4B (manufactured by Pharmacia LKB Biotechnology). It was confirmed by SDS polyacrylamide gel electrophoresis that the purified monoclonal antibody was IgG consisting of H chain with a molecular weight of approximately 50,000 and L chain with a molecular weight of approximately 20,000.

(6) Comparison of antibody affinity for different haptens

ELISA was performed on the purified monoclonal antibody generated by cell line 2D55A. MA-BSA was used as the solid phase antigen and the assay was performed on 4 haptens from MA, ABMA, AP and ABAP. The other conditions for the assay were the same as those of the ELISA described above. These haptens were compared for the concentration required for a 50% reduction in relative absorbance at 492 nm. AP showed the highest concentration and the concentration then decreased in the order of ABMA, ABAP and MA. AP required concentration about 100 times that of MA to effect a 50% reduction in relative absorbance at 492 nm. This means that it was shown that by using AP-KLH as an immunogen, the antibody with the highest affinity for MA could be formed from the 4 haptens described above.