JP2775445B2 - Anti-phenytoin monoclonal antibody and hybridoma producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an antiepileptic drug, phenytoin (PH
The present invention relates to a monoclonal antibody having high affinity and specificity for T) and a hybridoma producing the monoclonal antibody. The monoclonal antibody of the present invention can be used for accurately measuring the concentration of phenytoin in the blood of epileptic patients.

[Prior Art] At present, a large number of drugs including theophylline have been developed and used for treatment as antiepileptic drugs. However, regarding the treatment, evaluation of drug effects, confirmation of medication,
It is a well-known fact that blood drug concentration measurement is very important for diagnosis of drugs responsible for toxic symptoms, diagnosis of drug interaction, and the like.

In particular, phenytoin, which is used as effective for refractory partial seizure epilepsy, is absorbed in the duodenum, taken up in the blood, hydroxylated in the liver, and excreted in urine via normal metabolic pathways such as bile . Around 90% is a metabolite, and phenytoin excreted in urine as an unmetabolite is only 5% or less. In addition, the saturation phenomenon appears in the metabolic process, that is, the phenytoin dose does not compare with the blood concentration in the steady state, and when the dose reaches the saturation point, the blood phenytoin concentration sharply increases due to a slight increase in phenytoin administration. It is also known to rise. Accordingly, there is a need for treatment with a minimally effective amount without causing toxic symptoms. However, changing the dosage form of phenytoin may lead to differences in blood levels, etc., and further increase the therapeutic effect when used in combination with other drugs. It is known that it may increase blood levels and cause toxic symptoms. Therefore, it is always desirable to control the blood concentration upon administration.

Conventionally, most of anti-phenytoin antibodies used in various measurement systems are polyclonal antibodies produced using rabbits, sheep and the like. Conventionally used polyclonal antibodies generally have low antigen specificity,
The cross-reactivity to the main metabolites of phenytoin, p-hydroxydiphenylhydantoin (p-HPPH) and m-hydroxydiphenylhydantoin (m-HPPH), and related drugs, ethylphenylhydantoin (EPH) and phenobarbital (PB), was observed. large. That is, it shows 5-10% cross-reactivity with p-HPPH and EPH and nearly 100% cross-reactivity with m-HPPH.

In addition, anti-phenytoin monoclonal antibodies have been conventionally marketed, but their affinity and specificity for phenytoin are still unsatisfactory, and as shown in Examples below, the 50% binding concentration for phenytoin is about 1 μg / ml. And 5% with respect to p-HPPH, m-HPPH
Shows 100% cross-reactivity.

[Problems to be Solved by the Invention] Accordingly, an object of the present invention is to provide an anti-phenytoin monoclonal antibody having high affinity and specificity for phenytoin and a hybridoma producing the same.

[Means for Solving the Problems] As a result of intensive studies, the present inventors have produced a novel anti-phenytoin monoclonal antibody having much higher affinity and specificity for phenytoin than conventional monoclonal antibodies. And successfully completed the present invention.

That is, the present invention provides a 50% binding concentration of phenytoin.
10 ^-1 μg / ml or less, the cross-reactivity to p-hydroxydiphenylhydantoin is less than 2%, and the cross-reactivity to m-hydroxydiphenylhydantoin is
Less than 10%, less than 1% cross-reactivity to ethylphenylhydantoin, less than 1% cross-reactivity to phenobarbital, hybridoma PHT1
Produced by 6-17 (FERM P-10389). An anti-phenytoin monoclonal antibody is provided.

Furthermore, the present invention relates to a hybridoma PHT16-17 (FERM P-1) producing the above-described monoclonal antibody of the present invention.
0389).

[Effects of the Invention] According to the present invention, a novel anti-phenytoin monoclonal antibody having extremely high affinity and specificity for phenytoin and a hybridoma producing the same are provided. Since the anti-phenytoin antibody of the present invention has an extremely high affinity and specificity for phenytoin, phenytoin in a sample can be extremely reduced without interference from its metabolites and similar drugs by applying an immunoassay such as ELISA. Measurement can be performed with high accuracy. Further, since it has extremely high specificity, it can be used for quantification of phenytoin in blood using whole blood. Therefore, the present invention greatly contributes to the treatment and diagnosis of epilepsy using phenytoin.

[Description of the Invention] The anti-phenytoin antibody of the present invention is 50% of phenytoin.
Binding concentration (the concentration of phenytoin that binds to 50% of the antibody,
The smaller this value, the higher the affinity for phenytoin) is 10 ^-1 μg / ml or less. This is in comparison with conventional commercially available anti-phenytoin monoclonal antibodies.
10 times higher affinity. In addition, the cross-reactivity to phenytoin metabolites p-HPPH and m-HPPH is less than 2% and less than 10%, respectively. These values are also significantly lower (ie, higher specificity) compared to conventional anti-phenytoin monoclonal antibodies. In addition, the cross-reactivity of phenytoin to the related drugs EPH and PB is less than 1% each.

The monoclonal antibody of the present invention can be obtained as follows. That is, first, an animal such as a mouse is immunized with phenytoin, and an antibody-producing cell such as a spleen cell thereof is fused with a tumor cell such as a myeloma cell to prepare a hybridoma. Since phenytoin has low immunogenicity, it has immunogenicity such as KLH (keyhole).
It is preferable to use those bound to a carrier protein such as Limpet Hemocyanin) or BSA for immunization. Next, the hybridomas are selected using a selection medium such as a HAT medium, cloned and cultured by an appropriate method such as limiting dilution, and the culture supernatant is subjected to an appropriate immunoassay such as enzyme immunoassay. To check if an anti-phenytoin antibody is produced. Each of these steps can be performed by a known method, for example, Kohler and
Milstein Nature 256,495 (1975) or Secher et al. Nat
285, 446 (1980). Furthermore, p-HPPH, m-HPPH, EPH and PB
The cross-reactivity with respect to is examined, and those with low cross-reactivity falling within the scope of the present invention are selected. Next, the desired anti-phenytoin antibody is isolated from the culture supernatant by a known method, for example, a combination of separation and purification means such as affinity chromatography and electrophoresis.

The anti-phenytoin monoclonal antibody of the present invention can be used as a reagent for detecting and quantifying phenytoin. In particular, it can be used as a reagent for homogeneous enzyme immunoassay (EIA) as described in JP-A-61-80049. The anti-phenytoin antibody of the present invention,
Since the affinity and specificity are extremely high, in such a homogeneous EIA, phenytoin in blood can be quantified using whole blood as it is, that is, without dilution, as a specimen.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the following Examples.

[Examples] 1. Preparation of hybridoma and selection of monoclonal antibody 1-1 Preparation of immunological substance An immunological substance complex comprising phenytoin as a hapten and KLH as a carrier molecule was prepared by the method of Cook et al.
h Communication in Chemical Pathology and Pharmaco
logy.vol.25.767,1973).

1-2 Immunization of Mice as Donors Only mice of the Balb / c strain were used for immunization, and a group of 10 mice was immunized. An emulsion of phenytoin-KLH and complete Freund's adjuvant (CFA, manufactured by Difco) at a ratio of 1: 1 was administered intraperitoneally to each mouse. The dose was 20-50 μg of immunogen per mouse.

After 4 weeks, phenytoin-KLH in 0.9% NaCl was added for 20-50.
μg was boosted.

The immune response of the mice was monitored periodically by taking blood samples.

Culture of 1-3 mouse cancer cells P3X63Ag8UP (P3U1) which is a commercially available mouse myeloma cell
Dainippon Pharmaceutical Co., Ltd.) was used for the fusion. The cells were stored at -196 ° C in liquid nitrogen in freezing medium. Fusion 1
One week ago, a part was taken out, thawed, and placed in a Petri dish (10 cm in diameter) containing RPMI-1640 medium and cultured. Exponentially growing cells were used for fusion.

1-4 Somatic cell fusion The spleen of the immunized mouse was removed under aseptic conditions 3 to 4 days after the last booster immunization. After careful grinding on a stainless steel mesh (pore size 100 μm), connective tissue was separated from splenocytes and washed twice with RPMI-1640 medium (100 rpm, centrifugation for 5 minutes). next
Floated on RPMI-1640. The spleen cells and the tumor cells were mixed at 2: 1 in this mixture. Polyethylene glycol-R
PMI-1640 (a 1: 1 mixture of PEG4000 and RPMI-1640) was added to initiate cell fusion. 1 minute later RPMI-16
40 was added to dilute the PEG. The cells were washed until the PEG completely disappeared, and suspended in a hypoxanthine / aminopterin / thymidine selection medium (Littlefield's HAT medium) to about 10 ⁶ cells / ml. Only the fused cells can survive in this selective medium, and the unfused splenocytes and cell line cells in the suspension cannot survive.

Culture of 1-5 hybridoma cells The cell suspension (in HAT medium) after fusion was pipetted to 200 μl.
One by one and dispensed into wells of a 96-well culture plate. Incubation was carried out for 7-10 days in an atmosphere of 95% air and 5% carbon dioxide at 37 ° C. and 100% relative humidity. During this time, cell growth was monitored periodically.

Two to three weeks later, a positive culture (i.e., a culture producing an immunoglobulin having the desired specificity) was identified using the screening method described in 1-9 below. At the same time, transfer the culture from HAT medium to HT medium, then RPMI
The medium was sequentially changed to -1640 medium.

Cloning of 1-6 hybridoma cultured cells The positive growth culture was grown in a large-capacity culture vessel (Costar, cell culture plate type 3524 or 3506). The following cloning was performed by the limiting dilution cloning method. In this method, the number of cells transplanted when a new culture was started by diluting the cells in the positive culture solution was set so that the number of cells transplanted was approximately one per well. 8
After ~ 12 days, large colonies starting from single cells were already visible. The cloning procedure was performed at least twice to ensure that the monoclonal cells were growing.

Preparation of ascites from 1-7 mice (in vivo) To adjust ascites, 0.5 ml of Pristane (registered trademark, manufactured by Ross) was intraperitoneally administered to Balb / c mice.

The mice thus pretreated were administered intraperitoneally with a suspension of 10 ⁶ to 10 ⁷ hybridoma cells per mouse (in PBS) within 7 to 60 days. Eight to ten days later, a 19G needle was inserted into the peritoneal cavity to collect ascites fluid containing cells.

Cell components were separated from the ascites fluid by centrifugation (1000 rpm, 10 minutes). The supernatant fraction containing the monoclonal antibody was diluted (if necessary) and purified by the method described in 1-8 below.

1-8 Purification of ascites Black et al.'S method [J. Immunol. Math. 53, 313-319 (19)
82)].

1-8-1 Ascites pretreatment Ascites was centrifuged at 1000 × g for 5 minutes to precipitate cell components. By ultracentrifugation (100,000 × g, 30 minutes), the fibrin clot of the cell fragment was separated and removed. Next, the supernatant fraction was
It was dialyzed overnight against twice the volume of Tris-HCl buffer (0.02 M / pH 7.2). It was then centrifuged at 10,000 xg for 15 minutes.

1-8-2 Chromatography Ion exchange chromatography and gel filtration chromatography were performed under the following conditions. Ion exchange chromatography was performed using 0.01 M Tris HCl p as starting buffer.
Using H8.0, gradient elution was performed with 0 to 0.5 M NaCl.
The carrier was DEAE-Sepharose (Pharmacia), and the gel filtration was Sephacryl S-200 (Pharmacia). Buffer is 0.15 in 0.01M phosphate buffer pH 7.4
Those containing M NaCl were used.

1-9 Screening Test for Antibody Detection Monoclonal antibodies in the culture supernatant were screened by ELISA using immobilized antigen, and the antibody specificity was ³ H
-Investigated by RIA method using phenytoin.

1-9-1 Solid-Phase Immunoassay (ELISA) Preparation of ELISA Plate The wells of a 96-well ELISA plate were coated with a phenytoin-BSA conjugate having a different carrier part from the antigen (phenytoin-KLH) used for immunization ( 20 μg / ml, 50 μl, 4
° C, overnight). After removing the reaction solution, PB containing 0.05% Tween 20
Prepared by washing with S.

ELISA method 50 μl of each cell culture supernatant fraction on a plate coated with antigen
One by one was inserted with a micropipette. After reaction at 37 ° C. for 60 minutes, the contents in the wells were discarded, and the plate was washed three times with PBS containing Tween 20. Next, a second antibody (anti-mouse immunoglobulin antibody manufactured by DACO) labeled with peroxidase was added to each well, and further reacted at 37 ° C. for 60 minutes. After washing three times, the substrate ABTS (2,2'-amino-bis (3
-Ethylbenzothiazoline-6-sulfonic acid), manufactured by Wako Pure Chemical Industries, Ltd.), and the enzyme reaction was started. Green color was seen in positive wells.

1-9-2 RIA Method for Affinity Test The affinity of binding to phenytoin was examined by an RIA method using ³ H-phenytoin. This operation is
P. Butler's method (Method in Enzymology vol.84,558,19
82). Further, for comparison, the affinity of phenytoin with the monoclonal antibody in the commercially available anti-monoclonal antibody Emit phenytoin kit (manufactured by Shiva) and the monoclonal antibody manufactured by ImmunoResearch Inc. were also examined by the same method.

1-9-3 well seen the presence of antibodies to RIA method phenytoin for specificity tests to investigate P-HPPH, m-HPPH, a cross-reactivity with EPH and PB, ³ H- and phenytoin The reaction with the monoclonal antibody was allowed to compete with the above various cross-reactive substances having different concentrations, and free and antibody-bound phenytoin were separated by dextran-charcoal. This operation was specifically performed as follows. 50 μl of a fixed concentration of ³ H-phenytoin and 50 μl of an anti-phenytoin antibody having a different concentration in 0.5 ml of 0.25% BSA / TBS were added, and reacted at 25 ° C. for 2 hours.
After the reaction was completed, 0.25 ml of dextran charcoal was added and centrifuged to separate free ³ H-phenytoin and antibody-bound ³ H-phenytoin. Using an antibody concentration that binds 70% of the total ³ H-phenytoin, the antibody was reacted with ³ H-phenytoin, a cross-reactant of varying concentration, at 25 ° C. for 2 hours, and free ³ H-phenytoin was added with dextran charcoal. And join
³ H- phenytoin was separated. The amount of ³ H-phenytoin bound to the antibody in the absence of cross-reactive substances was taken as 100%,
The concentration of the cross-reactive substance showing% inhibition was determined, and the cross-reactivity% was calculated in comparison with phenytoin. For comparison, the above-mentioned Emitt manufactured by Shiva and the monoclonal antibody manufactured by ImmunoResearch were also tested in the same manner.

Homogeneous EIA of 1-10 phenytoin A homogeneous EIA system described in JP-A-61-80049 was established using the monoclonal antibody of the present invention, and its sensitivity and specificity were examined. That is, 50 μl of a solution of an enzyme-antibody conjugate in which α-amylase and a monoclonal antibody are bound, 50 μl of a solution of polymerized phenytoin in which phenytoin is bound to horse ferritin, and 50 μl of 5% BSA / PBS in which a known amount of phenytoin is dissolved are mixed. And reacted at 37 ° C. for 20 minutes. Add 1.0 ml of the substrate suspension to the reaction solution, and add
After reacting for 0.5 min, the reaction was stopped by adding 0.5 ml of 0.5N NaOH. After stirring, the mixture was centrifuged at 3000 rpm for 2 minutes,
The absorbance at nm was measured to obtain a calibration curve for phenytoin. To examine the specificity of the monoclonal antibody, 50 μl of an enzyme-antibody conjugate solution and 50 μl of a polymerized phenytoin solution, 50 μl of a solution in which a cross-reactive substance was added at various concentrations to phenytoin at a fixed concentration, were added at 37 ° C.
Allowed to react for minutes. Subsequent operations were performed under the same conditions as when the calibration curve was obtained. The cross-reaction% was calculated by comparing the measured value of the solution to which the cross-reactive substance was added with the measured value of the solution to which no cross-reactive substance was added.

2. Results 2-1 Establishment of Hybridoma Cell Line Table 1 shows the number of wells in which colonies are growing, the fusion frequency (%), the number of anti-phenytoin antibody producing wells, and % Indicates the number of wells producing less than% antibody. As shown in Table 1, almost 100% of the fusion frequency (the ratio of the wells where the colonies of the fused cells were present in the HAT selection medium) was regularly obtained after the fusion.
As a result of performing about 20 fusion operations and screening 7680 wells, an anti-phenytoin antibody was produced, and m
-Only one hybridoma producing an antibody having a cross-reactivity to HPPH of 10% or less was obtained, named hybridoma PHT16-17, and the monoclonal antibody was named monoclonal antibody PHT16-17. The hybridoma PHT16-17 has been deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology, and its accession number is No. 10389 of Microbial Laboratory.

2-2 Monoclonal antibody PHT16 against phenytoin
-17 nature 2-2-1 immunoglobulin subclass PHT16-17 the L chain is IgG ₁ immunoglobulin is k type.

2-2-2 Affinity for phenytoin Affinity for phenytoin examined by the method of 1-9-2 above is shown in FIG. FIG. 1 also shows the results for a commercially available Emit monoclonal antibody as a control. As is clear from FIG. 1, PTH16-17 has a phenytoin 50% binding concentration of about 0.5 × 10 ⁻¹ μg / ml or less,
This is the affinity of commercial Emit monoclonal antibodies.
More than double. The results for the commercially available anti-phenytoin monoclonal antibody manufactured by ImmunoResearch were almost the same as those for the Emit antibody.

2-2-3 Specificity PHT16 examined by the method described in 1-9-3 above
Table 2 shows the cross-reactivity of -17 and Emit monoclonal antibodies to various substances. As shown in Table 2, the cross-reactivity of PHT16-17, which is the monoclonal antibody of the present invention, is low, that is, the specificity is high, and particularly the cross-reactivity with p-HPPH and m-HPPH, especially with m-HPPH. Much smaller than conventional anti-phenytoin monoclonal antibodies.

2-2-4 Homogeneous EIA The results of the homogenous EIA described in 1-10 are shown in Table 3 and FIGS. 2 and 3. FIG. 2 shows a calibration curve using PHT16-17. The absorbance changes depending on the concentration of phenytoin, indicating that immunoassay is possible by this method. The results shown in Table 3 are shown in Table 2.
It is almost the same as the result of the RIA method. FIG. 3 shows a correlation diagram between the results of the Emit phenytoin kit and the homogeneous EIA method, which almost correspond to 1: 1.
From these, the homogeneous EIA using PHT16-17
It can be seen that phenytoin can be immunoassayed with high accuracy by the method.

[Brief description of the drawings]

FIG. 1 is a diagram showing the affinity of the monoclonal antibody of the present invention and a conventional commercially available monoclonal antibody for phenytoin, FIG. 2 is a diagram showing a calibration curve of a homogeneous EIA using the monoclonal antibody of the present invention, and FIG. 3 shows a correlation diagram between the results of homogeneous EIA using the monoclonal antibody of Example 1 and the results of EIA using a conventional anti-phenytoin monoclonal antibody kit.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/53 G01N 33/577 B 33/577 C12N 5/00 B (C12P 21/08 C12R 1:91) (56) References FED. PROC. 46 [3] (1987) 709 Int. J. Immunopharm ac. 9 [3] (1987) 391- 399 (58) Field surveyed (Int. Cl. ⁶ , DB name) BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A phenytoin 50% binding concentration of 10 ^-1 μg / ml.
The following, the cross-reactivity to p-hydroxydiphenylhydantoin is less than 2%, the cross-reactivity to m-hydroxydiphenylhydantoin is less than 10%, the cross-reactivity to ethylphenylhydantoin is less than 1%, The cross-reactivity to phenobarbital is less than 1% and the hybridoma PHT16-17 (FER
MP-10389) produced by the anti-phenytoin monoclonal antibody. 2. A hybridoma PHT16-17 (FERM P-10389) which produces the monoclonal antibody according to claim 1.