JPS5888367A - Compound for immunochemical assay of phenobarbital or primidone - Google Patents

Abstract

NEW MATERIAL:The compound of formula (X is O or H; the substituent groups of the benzene ring are m- or p-positions with each other). EXAMPLE:m-(Succinimidoxyadipoylamino)phenobarbital. USE:Raw material for the preparation of enzyme-labeled hapten and hapten antigen of phenobarbital or primidone is the body fluid. It has constant activity, and excellent storage stability and reactivity. CONSTITUTION:The compound of formula can be prepared by reacting an adipic acid monoester with amino-substituted derivative of phenobarbital or primidone, and hydrolyzing the product with NaOH.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for treating phenobarbital (hereinafter referred to as "PB") in biological fluids.
It is abbreviated as ) or brimidone (hereinafter abbreviated as "PM").

As with other antispasmodic agents, PB and PM have a small difference between effective and toxic doses, and the blood concentration in each patient is measured before administration to determine the desired level (PB H) ~25py/1nl.
! It is necessary to maintain PM 5-10)'f/me) and to reduce the occurrence of side effects as much as possible.

Blood concentration can be measured using gas chromatography, high performance liquid chromatography, ultraviolet absorption measurement, radioimmunoassay, and enzyme immunoassay (hereinafter referred to as "EIA").
However, methods other than EIA have drawbacks such as requiring a large amount of sample and special and expensive equipment.

The present inventors have already succeeded in quantifying FB or PM by IA (Japanese Patent Application Laid-open No. 52-117419, No. 53-658).
86 and No. 54-119026).

These publications describe the use of a diazonium salt of iso-amino PB or tome-amino PM as a raw material for the production of enzyme-labeled no-butene and no-butene antigen. Salts have drawbacks such as difficulty in long-term storage and slight differences in reaction temperature or reaction time can significantly vary the activity of the target product in EIA.

The present inventors conducted extensive research to overcome this drawback and completed the present invention.

The present invention relates to PB represented by the following formula (when X is an oxygen atom)
or PM (when X is a hydrogen atom) enzyme-labeled hapten and a novel compound used for producing a hapten antigen.

(In the formula, X means an oxygen atom or a hydrogen atom, and the substituents on the be/zene nucleus are in the book-position or up-position relationship with each other.) It can be synthesized by reacting with an acid monoester and then hydrolyzing with NaOH (see Examples 1 to 3), and is excellent in both storage stability and reactivity.

Furthermore, the enzyme-labeled hapten and hapten antigen synthesized from the compound of formula (1) have constant activity and are extremely useful as raw materials for EIA reagents. Note that the activity of these derivatives in terms of preservative 1 reactivity and EIA is almost the same for compounds in which the substituents on the benzene nucleus are located at the opposite positions and those at the 2nd position.

A conjugate of a compound of formula (Natsu) and a polymeric protein can be prepared according to the reaction formula below. Suitable polymeric proteins include albumin, globulin, thyroglobulin, shellfish hemocyanin, and edestin.

The conjugate thus prepared is mixed with a suitable adjuvant and used in rabbits, guinea pigs, and goats.

Antibodies against PB or PM (hereinafter abbreviated as "antibodies") can be obtained by subcutaneously administering to animals such as sheep, collecting serum, and performing known treatments.

A conjugate of the compound of formula (1) and an enzyme (hereinafter abbreviated as "enzyme-labeled hapten") can also be prepared by the same reaction as in the above-mentioned conjugation with a polymeric protein, and suitable enzymes include:
β-galactosidase, peroxidase, lipase,
Examples include alkaline phosphatase and glucose oxidase.

The above-mentioned antibodies and enzyme-labeled haptens are PR or PM EI.
Used for measurements by A.

It is convenient to combine the antibody and enzyme-labeled hapten into a quantitative kit. The kit also includes a standard PB or PM solution for creating a calibration curve, and reagents for activity measurement (
(substrate, substrate solution, enzyme reaction stopper, etc.).

The method for quantifying PR or PM using this kit is to mix the above-mentioned antibody, enzyme-labeled hapten, and a sample containing PB or PM, perform an antigen-antibody reaction, and then quantitate the "antigen bound to the antibody" and ""Antigen that is not bound to an antibody" is B.
/F separation, measuring the labeling activity of either of them,
It consists of quantifying PR or PM in a subject.

In carrying out quantification, FB or PM in the sample and enzyme-labeled hapten are subjected to a competitive antigen-antibody reaction with the antibody, and then ``antigen bound to the antibody'' and ``antigen not bound to the antibody'' are determined. It is necessary to separate the "antigen" into B/F.

There are three methods for this B/F separation. One is a method in which the antibody is made insolubilized in advance and an antigen-antibody reaction is performed.

Antibodies are produced using bacterial cell walls, natural insoluble polysaccharides, chemically treated dextran gels, agar gels, plastic beads,
Acrylamide gel, glass beads.

It can be insolubilized using fine metal powder or the like.

Another method is to produce an antibody before, during, or at the same time as the antigen-antibody reaction, and an antibody (secondary antibody) against r-globulin of the animal that produced the antibody.
This method involves reacting with antibodies (antibodies) to make them insoluble. Second
Antibodies are usually used in a solution state, but if the second antibody is insolubilized in advance with bacterial cell walls or the like, there is an advantage that the amount used is small and the reaction time is short.

Measure.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Preparation of succinimidoxyadipoylamino PB Tetrahydrofuran (hereinafter referred to as "THE")
It is abbreviated as ) solution (2,1'/20mt') and a THF solution of adipic acid monomethyl ester (1,6y-), and while cooling, a THF solution of dicyclohexylcarbodiimide (hereinafter abbreviated as "DCC") ( 2,06f/2
After stirring for 1 hour, the mixture was further stirred overnight at room temperature, and the precipitated dicyclohexylurea was removed by filtration. Concentrate the F' solution under reduced pressure and add 10% to the residue.
After the methyl ester is thawed by adding an aqueous NaOH solution, it is immediately acidified with hydrochloric acid, extracted with ethyl acetate, and an aqueous solution of sodium carbonate is added and shaken to separate the organic layer. Acidified with hydrochloric acid again, extracted with ethyl acetate and concentrated under reduced pressure.
Obtain crystals of (hemiadipoyl)PB (420QF
) o m, 9.197℃・Mass spectrum rrv/e3
75 (M+) Two-element analysis (actual value) C: 58.1% H
: 5.60% N: 11.32% (Cl8H2106N
The calculated values for 3 are 57.59.5.64.1 respectively.
1.20%) T)IF bath solution of (hemiadipoyl)PB (100my/1.5mt') and N-hydroxysuccinimide THF solution (30.61ngArn) were mixed, and under cooling, DCC THF solution (100my/1.5mt') was mixed. 54,8 width'
d) was added and stirred for 2 hours. After further stirring overnight at room temperature, the precipitated dicyclohexylurea was removed by filtration, concentrated under reduced pressure, and a small amount of ethanol was added to obtain the scaly form of Asahi-(succinimidoxyaziboyl)amino PB. Obtain crystals (1
02~).

m, p, 154℃ single element analysis (actual value) C: 58.1%
H: 5.3 inches N: 12.1 inches (C22N24011
The calculated values for N4 are 57.91.5.26, respectively.
12.28 h) Example 2 Preparation list of F-(succinimidoxyadipoylamino)PB - p- (hemiadipoyl)FB crystals were obtained (620■).

m, p, 261℃, mass spectrum rrV/e375
(M”), elemental analysis (actual value) C: 58.2%
H: 5.5% N: 11.2 - Using p- (hemiadipoyl) PR instead of chicken (hemiadipoyl) PR,
By operating in the same manner as in Example 1, crystals of p-(succinimidoxyadipoylamino)PB were obtained (80
■).

m, p, 189-192℃ single element analysis (actual value) C
: 55.36'%H: 5.84ch N: 10.82
%(C22N24N408・C2)150H Calculated values are 55.59.5.83.10.81 respectively) Example 3 Preparation of PM - Amino PM dimethylformamide (hereinafter referred to as "DMF
”. ) solution (1,87y/10mt') was added with a DMF solution of adipic acid monomethyl ester (1,28y/10mt').
151n was added, and while cooling, DCCODMF solution (1,
65y15-) was added with stirring, and after stirring for 2 hours, the mixture was further stirred at room temperature overnight. The precipitated dicyclohexyl urea is removed by filtration, water (30 me) is added to the r solution to remove the remaining dicyclohexyl urea, excess ethyl acetate is added, shaken and extracted, and concentrated under reduced pressure. 10%NaOH
Hydrolysis is carried out by adding a water bath solution (10rnI), immediately neutralized with hydrochloric acid, and ethyl acetate and water are added and shaken to separate the insoluble mucilaginous substance. When diluted with a mixture of
11#g).

m, p, 231℃, mass spectrum m7'e 361 (
M"), elemental analysis (actual value) C: 59.7% H
:, 6.4chi N: 11.3% (Css HB 05
The calculated values for N3 are 59.82° and 6.42°, respectively.
11.62%) DMF solution of (hemiadipoylamino)PM (13
14E) and N-human d-xysuccinimide in DMF solution (42fng10.5mt') under cooling.
A DMF solution of CC (75.21W/0.5*e) was added under stirring, and after stirring for 2 hours, the mixture was further stirred at room temperature overnight.

By removing the precipitated dicyclohexyl urea by filtration, door-(saguccinimidoxyaziboylamino)PM
A containing DMF solution is obtained. A small amount of this DMF solution was applied to a thin silica gel plate (Kiesel get 60 Fzs
After developing with a developing solvent chloroform: ethyl formate: formic acid (5:10:0.5), air-drying and examining ultraviolet absorption spots revealed the target object, Rt O, at around Rf O, 25. The presence of unreacted raw materials can be confirmed around 15.

A DMF solution containing sag-(sagsinimidoxyaziboylamino)PM is stable when stored at -20°C, and can be used as it is to introduce PM groups into enzymes and high-molecular proteins.

Example 4 Preparation of enzyme-labeled PB Escherichia coli-derived β-galactosidase solution (Boehringer) thoroughly dialyzed with 0.02M IJ acid buffer (pH 7)
(200) Lf! Arn! ), the p'-(saguccinimidoxyaziboylamino)PB-containing ethanol solution (250/'y/0.25 me
) was added under stirring, and after stirring at room temperature for 1 hour, 'Bio
Applied to gel P-4 column (2X30Cm), 0.9
Elute with Cl-0.02M phosphate buffer (pH 7) and fractionate into 5 rtte portions.

Fractions A7, 8.9 were collected and β-galactosidase-labeled P
get B.

In place of β-galactosidase, porcine pancreatic lipase (Sigma) and calf small intestine alkaline phosphatase (Sigma) are used to obtain lipase-labeled PB or alkaline phosphatase-labeled PB in the same manner.

Example 5 Preparation of enzyme-labeled PM A β-galactosidase solution (200/+y-10,8r) sufficiently dialyzed against 0.02 MIJ acid buffer (+) H7)
nI! ') was added to the DMF solution containing oligo-(saguccinimidoxyaziboylamino)PM prepared in Example 3 (100
/JP/1.61#L), reacted at room temperature for 1 hour, and applied to a Biogel P-4 column (2x30cm).
°0.94 NaCI-0.02M phosphate buffer (pH 7
) and fractionated in 5d increments. β-galactosidase-labeled PM is obtained by collecting A6,7.8.

Pig pancreatic lipase, calf small intestine alkaline phosph, atase was used instead of β-galactosidase, and lipase-labeled PM or alkaline phosphatase-labeled P was similarly used.
M is obtained.

Example 6 Preparation of PB hapten antigen and antibody Bovine serum albumin (Fraction V) (Armor Inc.)
0.04M phosphate buffer (pH 7) (15011F
/12me), the methanol solution containing F-(saguccinimidoxyaziboylamino)FB prepared in Example 2 (
454/6rnl) was added and stirred at room temperature for 2 hours, dialyzed overnight against 5 tons of the same buffer, poured into a 5ephadex G25 column, IA (3.8 x 30'cm), and passed through a 11 m
Fractionate by e.

Collect fractions &11-15 and lyophilize (approximately 150 WIg
).

It was confirmed from the ultraviolet absorption spectrum that a hapten antigen into which 1 molecule of sialupmin (67'c) and 15.4 moles of PB had been introduced had been prepared.

The hapten antigen of this PB was thoroughly emulsified at a ratio of 41 to 0.9 and then used for adult rabbits (2.5 to 3.5 Ky).
For the first time, 0.5% injections were given in 2 places on the foot and 0.25% in 4 places on the back, and then every 2 weeks, 0.2% - 0.2% was given in 10 places on the hind thigh or ventral side. Inject 0.4■. Two to four months after the start of immunization, an antiserum that can be used for immunological measurement of pH is obtained.According to conventional techniques, an immunoglobulin fraction is obtained from the antiserum by ammonium sulfate fractionation, and this fraction is further separated from the bacterial cell wall. By chemical conjugation, an anti-PB immunoglobulin lysate can be prepared. Example 7 Preparation of PM hapten antigen and antibody Instead of the FB active ester-containing methanol solution in Example 6, the DMF solution (saguccinimidoxyaziboylamino)PM-containing obtained in Example 3 (2rrlI) was used. Approximately 150 hapten antigens containing 13.1 moles of PM per molecule of albumin were obtained by dissolving the hapten antigen in 10 rIf! of methanol. An antiserum that can be used for immunological measurement of PM was obtained by immunizing with the immunoglobulin fraction of this antiserum.
By binding to bacterial cell walls, anti-PM immunoglobulin lysates can be prepared.

Patent applicant: Dainippon Pharmaceutical Co., Ltd. Agent Akira Kojima

Claims (1)

[Scope of Claims] A compound represented by the general formula (wherein, X means an oxygen atom or a hydrogen atom, and the substituents on the benzene nucleus are in an allegorical or p-position relationship with each other).