JPH02138992A - Anti-phenytoin monoclonal antibody and hybridoma producing same antibody - Google Patents

Abstract

PURPOSE:To raise affinity for and specificity to phenytoin by forming anti- phenytoin monoclonal antibody having a specific cross reaction value to p- hydroxydiphenylhydantoin. CONSTITUTION:An antibody-forming cell prepared by immunizing a mouse, etc., against phenytoin is fused with a tumor cell line such as myeloma cell, etc., to prepare a hybridoma. The hybridoma is singly cloned by using a selective medium such as HAT medium by limiting dilution, etc., and cultured to form an anti-phenytoin monoclonal antibody. The anti-phenytoin monoclonal antibody has <=10<-1>mug/ml 50% bond concentration of phenytoin, <2% cross reactivity to p-hydroxydiphenylhydantoin and <10% cross reactivity to m- hydroxydiphenylhydantoin.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a monoclonal antibody that has high affinity and specificity for phenytoin, an antiepileptic drug, and to a hyturitoma that produces the monoclonal antibody. The monoclonal antibody of this invention can be used to accurately measure the concentration of phenytoin in the blood of epileptic patients.

[Prior Art] Currently, a large number of antiepileptic drugs including thioferline have been developed and used for treatment. However, it is well known that blood drug concentration measurement is extremely important for such treatment, such as determining drug effectiveness, confirming medication compliance, diagnosing the drug responsible for toxic symptoms, and diagnosing drug interactions. This is a fact.

In particular, phenytoin, which is used to treat intractable partial-onset epilepsy, is absorbed in the duodenum, taken into the blood, hydroxylated in the liver, and excreted in the urine via normal metabolic routes such as bile. . Approximately 90% of phenytoin becomes a metabolite, and only 5% or less of phenytoin is excreted in the urine as an unmetabolized product. In addition, a saturation phenomenon occurs in the metabolic process, that is, the amount of phenytoin administered is not proportional to the blood concentration in the steady state, and when the dose reaches the saturation point, the blood phenytoin concentration rapidly increases due to a small amount of phenytoin administered. It is also known to rise. Therefore, it is required to treat the drug with the minimum effective dose without causing toxic symptoms. However, changes in the dosage form of phenytoin may result in differences in blood concentration, etc., and the metabolic pathway of phenytoin may be affected by the interaction between drugs, whether it is intended to increase the therapeutic effect by using it in combination with other drugs, and phenytoin It is known that it can increase blood concentration and cause poisoning symptoms. Therefore, it is desirable to always control the concentration during administration.

Most of the anti-phenytoin antibodies conventionally used in various measurement systems are polyclonal antibodies produced using rabbits, sheep, etc. Conventionally used polyclonal antibodies generally have low specificity for antigens, and are highly sensitive to p-human loxydiphenylhytantoin (p-HPPH) and m-human loxydiphenylhytantoin (m-HPPH), which are the main metabolites of phenytoin. ) and the related drugs ethyl phenylhydantoin (EPI+) and phenoharbital (PB). Against Sunawachi, p-FIPPll, and EPH 5~
10% cross-reactivity, and nearly 100% cross-reactivity for +1-HPPH.

Furthermore, although anti-phenytoin monoclonal antibodies have been commercially available, their affinity and specificity for phenytoin are still not satisfactory. It is about I, and 5 for p-HPPH.
%, +*-HPPH shows 100% cross-reactivity.

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

[Means for Solving the Problems] As a result of intensive research, the present inventors have produced a novel anti-phenytoin monoclonal antibody that has much higher affinity and specificity for phenytoin than conventional monoclonal antibodies. We succeeded in producing the resulting hybridoma and completed this invention.

That is, in this invention, the 50% binding concentration of phenytoin is less than 1Lg/l, and the cross-reactivity with p-hydroxydiphenylhytantoin is less than 2%,
An anti-phenytoin monoclonal antibody having less than 10% cross-reactivity with m-hydroxydiphenylhytantoin is provided.

Furthermore, the present invention provides a hyturidoma that produces the monoclonal antibody of the present invention.

[Effects of the Invention] The present invention provides a novel anti-phenytoin monoclonal antibody having extremely high affinity and specificity for phenytoin, and a hybridoma producing the same. The anti-phenytoin antibody of the present invention has extremely high affinity and specificity for phenytoin, so it can be used to detect phenytoin in a sample using immunoassay methods such as ELISA without being interfered with by its metabolites or similar drugs. It becomes possible to measure with extremely high precision. Moreover, since its specificity is extremely high, it can be used for quantifying phenytoin in blood using whole blood. Therefore, this invention greatly contributes to the treatment and diagnosis of epilepsy using phenytoin.

[Detailed Description of the Invention] The anti-phenytoin antibody of the present invention has 50% of phenytoin
The binding concentration (the concentration of phenytoin that binds to 50% of the antibodies (the smaller this value is, the higher the affinity for phenytoin) is 10-' pLg/ml or less. The affinity is more than 10 times higher than that of antibodies.Also, p-11PPH and m-HP, metabolites of phenytoin,
Cross-reactivity to PH is less than 2% and 10%, respectively
less than These values are also much lower (ie, highly specific) compared to conventional anti-phenytoin monoclonal antibodies. Furthermore, preferably, the cross-reactivity of phenytoin with 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 the antibody-producing cells such as tile cells are fused with a tumor cell line such as myeloma cells to produce a hybridoma. Phenytoin has low immunogenicity, so it does not have immunogenicity, such as KLH (
keyhole limpet hemocyanin
) or BSA is preferably used for immunization. Hybridomas are then selected using a selective medium such as HAT medium,
The monoclonal cells are cloned and cultured using an appropriate method such as limiting dilution, and the culture supernatant is checked for production of anti-phenytoin antibodies using an appropriate immunoanalytical means such as enzyme immunoassay. Each of these steps can be performed by a known method, for example, as described by Kohler and
Milstein's Nature 256°495 (
19751 or Nature 285 of 5echer et al.
．． 446 (19801).Furthermore, p-HPPH, m-HPPH,
The cross-reactivity with EPH and PB is investigated, and those with low cross-reactivity that fall 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. Since the anti-phenytoin antibody of the present invention has extremely high affinity and specificity, phenytoin in blood can be quantified using whole blood as a sample in such homogeneous EIA, that is, without dilution. .

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to the following Examples.

[Example] l. Hybridoma production and monoclonal antibody selection method 11 Preparation of immune substance An immune substance complex consisting of phenytoin as a hapten and KLH as a carrier molecule was prepared by the method of Cook et al. (R
esearch (:communication in
Chemical Pathology and Ph
armacology, vol, 25.767゜1
9731.

1-2 Immunization of mice as blood donors Only Balb/c strain mice were used for immunization, and groups of 10 mice were immunized. Phenytoin-KLF (and complete Freund's adjuvant (CFA, manufactured by Difco) l:l
The emulsion was administered intraperitoneally to each mouse. The dose was 20-50 μg of immunogen per mouse.

After 4 weeks, phenytoin in 0.9% NaCl 20~
A booster dose of 50 μg was given. Note that this booster immunization can be repeated every 4 weeks if necessary.

The mice's immune responses were tracked by taking regular blood samples.

1-3 Culture of mouse cancer cell line P3X63Ag8UP (obtained from P3U11), a commercially available mouse myeloma cell line, was used for fusion. The cells were frozen in liquid nitrogen in freezing medium.
It was stored at 6°C. One week before fusion, a portion was thawed, placed in a petri dish (diameter 10 cm) containing RPMI-1640 medium, and cultured. Cells in logarithmic growth phase were used for fusion.

1-4 somatic cell fusion 3 to 4 days after the last booster, under sterile conditions,
The lungs of immunized mice were removed.

After carefully grinding on a stainless steel mesh (pore size 100 μm), the connective tissue was separated from the tile cells and washed twice with RPMI-1640 medium (1000 μm).
rpm, centrifugation for 5 minutes). Next, RPMI-164
It floated to 0. In this mixed solution, the tile cells and the cancer cell line cells were mixed at a ratio of 2:1. Polyethylene glycol-RPMI-1640 (PEG4000 and RPMI16
Cell fusion was started by adding a 1:1 mixed solution of 1:40 to 1:1. After 1 minute, RPMI-1640 was added to dilute the PEG. The cell suspension was washed until PEG was completely removed and placed in hypoxanthine/aminopterin/thymidine selection medium (Littlefield's HAT medium) in water at approximately 10 6 cells/ml. Only fused cells can survive in this selective medium, and unfused tile cells and cell line cells in the suspension cannot survive.

1-5 After culturing and fusion of hybridoma cells, pipette the cell suspension (in HAT medium) for 20 minutes.
A sample of 0 μm was taken and placed into 96 wells of a microtiter (registered trademark) plate. 37℃, relative humidity 95%
Incubate in an incubator with ventilation in an atmosphere of 95% air and 5% carbon dioxide without changing the medium for 7 to 7 days.
Incubated for 10 days. Cell proliferation was monitored periodically during this time.

After 2-3 weeks, positive cultures (ie, cultures producing immunoglobulin with the desired specificity) were identified using the screening method described in 1-9 below. At the same time, the culture medium was transferred from HAT medium to HT medium, then R
The medium was sequentially changed to PMI-1640.

A cloning-positive growth culture of 1-6 hybridoma cultured cells was grown in a large-capacity culture vessel (manufactured by Costa, cell culture plate type 3524 or 3506). The following cloning was performed by the limiting dilution cloning method. In this method, the positive culture solution was diluted so that when a new culture was started, the number of cells transplanted was statistically 10 or 1, respectively. After 8-12 days, large colonies starting from single cells were already visible. Cloning operations were performed at least twice to ensure monoclonal cell proliferation.

1-7 Preparation of ascites in mice (mineral oil pristane (registered trademark, manufactured by Ross) to prepare in vivo peritoneum)
0.5 ml was administered intraperitoneally to Balb/c mice.

Within 7 to 60 days, the thus pretreated mice were intraperitoneally administered with a suspension of 104 to 107 hybridoma cells (in PES) per mouse. After 8-10 days, a cannula was inserted into the peritoneal cavity to collect ascitic fluid containing cells.

Cellular components were separated from the ascites by centrifugation (1000 rpm, 10 minutes). The supernatant fraction containing the monoclonal antibody was also aliquoted (diluted as necessary), and the following procedure was carried out in 1-1.
It was purified by the method described in 8.

1-8 Purification of ascites The method of Black et al. [J, Immunol, Math
, 53°313-319 +1982)].

1-8-1 Pretreatment of ascites The ascites was centrifuged at 1000 x g for 5 minutes to precipitate cellular components. Ultracentrifugation (10 (1,000 x g,
Cell fragments and fibrin clots were separated and removed by washing for 30 minutes). Next, the supernatant fraction was added to 100 times the volume of Tris-HCl buffer (0
, 02mglI2, pH 7,2) overnight. The mixture was then centrifuged at t'l(+,000 x g"cl) for 5 minutes.

1-8-2 Chromatography Ion exchange chromatography and gel filtration chromatography were performed under the following conditions. Ion exchange chromatography is performed using 0.0% as the starting buffer. Old M Tris
0-0.5 M NaC using HCI pH 8.0
1 was used for gradient elution. The carrier is DEAE-Sepharose (manufactured by Pharmacia), and the gel filtration is Sephacryl S.
-200 (manufactured by Pharmacia). The buffer solution was 0. Old M phosphate buffer pH 7.4 containing 0.15 M NaCl was used.

1-9 Screening test for antibody detection Monoclonal antibodies in culture soil were screened using an ELISA method using homologous antigens, and the specificity of the antibody was determined by 3H-
It was investigated by RIA method using phenytoin.

1-9-1 In-phase immunoassay (EL I 5A) ELIS
Preparation of A plate Perform ELIS using a phenytoin-BSA conjugate with a different carrier moiety from the antigen (phenytoin-KLH) used in immunization.
20 μg/m1 to cover the holes of the 96-well plate for A.
．． 50 μl, 4° C. - incubated overnight in a humid box. After removing the reaction solution, add PB containing 0.05% Tween 20.
Washed with S and prepared.

Transfer 50 u of each cell culture supernatant fraction to a plate coated with ELISA antigen.
1 at a time with a pipette. After incubating at 37°C for 60 minutes, the contents in the wells were discarded and the plate was heated with Tween 20.
Washed three times with PBS containing water. Next, a second antibody labeled with peroxidase (anti-mouse immunoglobulin antibody manufactured by CACO) was added to each well, and the reaction was further carried out at 37° C. for 60 minutes. After washing three times, the substrate ABTS f2.2°-amino-bis (3-ethylbenzothiazoline-6-sulfuric acid, manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) was added to initiate the enzyme reaction. Green color development was observed in positive wells.

1-9-2 RIA method for affinity testing Binding affinity for phenytoin was determined using RIA method using 3H-labeled phenytoin.
It was investigated by IA method.

This operation is described in the method of Vincent P. Butler (Methodin Enzymology vol.
, 84.558.1982), and for comparison, commercially available anti-monoclonal antibodies, the monoclonal antibody in the Emitutophenytoin kit (manufactured by Shiba) and the monoclonal antibody manufactured by Inuno Research, and phenytoin were tested. We also investigated the affinity of

1-9-3 RIA method for specificity test Wells in which the presence of antibodies against phenytoin was observed were P-HPPH,
++In order to examine the cross-reactivity with rHPPH, EPH and PHB, competitive reactions were performed between 3H-phenytoin and the monoclonal antibody using various cross-reactive substances mentioned above at varying concentrations, and free and antibody-bound phenytoin were mixed with dextran-charcoal. Separated in a tube. Specifically, this operation is performed as follows. 0.5 ml (7) 0.25%
Add 50 ml of 3■-phenytoin at a constant concentration and 50 μm of anti-phenytoin antibody at varying concentrations to 5 ml of BSA/TBS O, react at 25°C for 2 hours, and after the reaction is complete, add 0.25 ml of dextran charcoal. Free 3H-phenytoin and antibody-bound 3H-phenytoin were separated by centrifugation. Using an antibody concentration that binds 70% of the total 3■-phenytoin, this antibody, 3■-phenytoin, and a cross-reacting substance at varying concentrations were reacted at 25°C for 2 hours, and free 3H-phenytoin was mixed with dextran charcoal. The bound 3H-phenytoin was separated. 3H-phenytoin bound to the antibody in the absence of a cross-reactive substance was taken as 100%, the concentration of the cross-reactive substance showing 50% was determined, and the cross-reactivity % was calculated by comparing it with phenytoin.

For comparison, the above-mentioned Emitt monoclonal antibody manufactured by Shiba Corporation was also tested in the same manner.

1-10 Homogeneous EIA of phenytoin JP-A-1982
The homogeneous EIA system described in No.-80049 was established using the monoclonal antibody of the present invention, and its sensitivity and specificity were investigated. That is, 50 μl of a solution of an enzyme-antibody conjugate in which α-amylase and a monoclonal antibody were bound, 50 μl of a solution of polymerized phenytoin in which phenytoin was bound to horse ferritin, and 50 ml of 5% BSA/PBS in which a known amount of phenytoin was dissolved were mixed. , 20 at 37℃
Allowed to react for minutes. Add 1.0 ml of the substrate suspension to the reaction solution and react at 37°C for an additional 30 minutes.
The reaction was stopped by adding 5+nl of aOHO.

After stirring, the mixture was centrifuged at 3000 rpm for 2 minutes, and the absorbance of the supernatant at 620 nm was measured to obtain a calibration curve for phenytoin. In order to examine the specificity of a monoclonal antibody, 50 μl of an enzyme-antibody conjugate solution, 50 μl of a polymerized phenytoin solution, and 50 μl of a solution containing a constant concentration of phenytoin and various concentrations of cross-reactive substances were added.
μm was added and reacted at 37° C. for 20 minutes. The subsequent operations were performed under the same conditions as when calculating the calibration curve. The % cross-reactivity was calculated by comparing the measured values of the solution to which the cross-reactive substance was added to the measured values of the solution to which no cross-reactive substance was added.

2. Results 2-1 Establishment of hybrid monomer cell line Table 1 shows the number of wells in which colonies are growing, fusion frequency (%), number of wells producing anti-phenytoin antibody, and 10% in m-HPPH obtained in the above procedure. The number of wells producing the following antibodies is indicated. As shown in Table 1, after fusion, a fusion frequency (ratio of wells in which colonies of fused cells were present in HAT selection medium) of almost 100% was regularly obtained. As a result of performing approximately 20 fusion operations and screening 7680 wells, anti-phenytoin antibodies were produced and the cross-reactivity to m-HPPH was 10%.
Only one hybridoma producing the following antibody was obtained and was named hybridoma PHT16-17.
It was named 6-17. Hybridoma PHT16-17
has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.
Its accession number is Microtechnical Research Institute No. 10389.

2-2 Monoclonal antibody against phenytoin pHT
Properties of 15-17 2-2-1 Immunoglobulin subclass PHT16-17 is an IgG, immunoglobulin, and the L chain is of the type.

2-2-2 Affinity for phenytoin 1-9-2 above
The figure shows the affinity for phenytoin determined by the method described above. IJI also shows results for the commercially available Emitt monoclonal antibody as a control. As is clear from Figure 1, PT816-17 has a 50% bound concentration of phenytoin or approximately 0.5 x 10-' u g/m
1, which is more than 10 times the affinity of the commercially available Emitt monoclonal antibody. The results for a commercially available anti-phenytoin monoclonal antibody manufactured by ImmunoResearch were almost the same as for the Emitt antibody.

2-2-3 Specificity PHT1 investigated by the method described in [above]-9-3
Table 2 shows the cross-reactivity of the 6-17 and Emitt monoclonal antibodies to various substances. As shown in Table 2, the monoclonal antibody of the present invention, PHT16-17
The cross-reactivity of P is low, i.e. the specificity is high, especially P
-HPP)l and -HPPII, especially rs -H
The cross-reactivity to PPH is much lower than that of conventional anti-fetomonoclonal antibodies.

Table 2 2-2-4 Homogeneous EIA The results of the homogeneous 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, and it can be seen that immunoassay is possible using this method. Further, the results shown in Table 3 are almost the same as the results of the RIA method shown in Table 2. Also, Figure 3
Emmitt Fenit. A correlation diagram of the results of InKit and this homogeneous EIA method is shown, and
It corresponds to From these reasons, PHTI6-17
It can be seen that phenytoin can be immunoanalyzed with high accuracy by the homogeneous EIA method using the method.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the affinity of the monoclonal antibody of the present invention and conventional commercially available monoclonal antibodies for phenytoin, FIG. 2 is a diagram showing the calibration curve of homogeneous EIA using the monoclonal antibody of the present invention, and FIG. 3 is a diagram showing the affinity of the monoclonal antibody of the present invention for phenytoin. results of homogeneous EIA using a monoclonal antibody and EIA using a conventional anti-phenytoin monoclonal antibody kit.
A correlation diagram of IA results is shown. Anai River PHT t7L (P9/vnJL) Shima? Supplementary talent, L';' 21st 618 old stories

Claims (5)

[Claims] (1) 50% binding concentration of phenytoin is 10^-^1μ
g/ml or less, the cross-reactivity to p-hydroxydiphenylhydantoin is less than 2%, and the cross-reactivity to m-hydroxydiphenylhydantoin is less than 1%.
Anti-phenytoin monoclonal antibody that is less than 0%. (2) The monoclonal antibody according to claim 1, which has a cross-reactivity to ethyl phenylhydantoin of less than 1% and a cross-reactivity to phenobarbital of less than 1%. (3) The monoclonal antibody according to claim 2, which is monoclonal antibody PHT16-17. (4) A hybridoma producing the monoclonal antibody according to any one of claims 1 to 3. (5) Claim 4, which is the hybridoma PHT16-17
Hybridoma described.