CN115825455A

CN115825455A - Application of valproic acid derivative in immunoassay

Info

Publication number: CN115825455A
Application number: CN202211396766.7A
Authority: CN
Inventors: 龚俊; 王贵利; 张启飞; 李垚艳; 刘希
Original assignee: Beijing Strong Biotechnologies Inc
Current assignee: Beijing Strong Biotechnologies Inc
Priority date: 2019-07-04
Filing date: 2019-10-30
Publication date: 2023-03-21
Also published as: CN110954707B; CN115684613A; CN110954707A; CN115616231A

Abstract

The application discloses the use of valproic acid derivatives in immunoassays. Specifically, the application relates to a valproic acid derivative and a preparation method thereof, and a kit containing the valproic acid derivative and a preparation method thereof. The technical scheme of the application uses a novel valproic acid derivative and a maleimide-sulfydryl coupling method with high selectivity, so that the derivative and enzyme are subjected to 1:1, the batch difference formed in the conventional coupling process is greatly reduced. The valproic acid detection kit is simple, convenient, rapid and low in cost, and can be used for carrying out automatic detection on various mainstream machine types.

Description

Application of valproic acid derivative in immunoassay

The application is a divisional application of a Chinese patent application No. 201911042028.0 'valproic acid derivative and application thereof in immunoassay', which is filed in 2019, 10, month and 30.

Technical Field

The application relates to a valproic acid derivative and a preparation method thereof, and a valproic acid homogeneous enzyme immunoassay kit and a preparation method thereof.

Background

Valproic acid (Valproic acid) has the following structural formula:

valproic acid is a drug for the treatment of epilepsy. The medicine components are sodium valproate and valproic acid, and can be used for single medicine treatment or additive treatment. Valproic acid is used for the treatment of generalized epilepsy: including absence seizures, myoclonus seizures, tonic clonus seizures, and the like. Treatment of partial epilepsy is indicated: simple partial attack, complex partial attack and partial secondary systemic attack. In addition to anti-epileptic, it can be used for treating febrile convulsion, dyskinesia, chorea, porphyria, schizophrenia, pain caused by herpes zoster, adrenal dysfunction, and preventing alcohol withdrawal syndrome.

Common adverse effects of valproic acid therapy are manifested as: digestive symptoms, menstrual cycle changes; less frequently, alopecia, constipation, drowsiness, vertigo, fatigue, headache, ataxia, slight tremor, abnormal excitement, restlessness and dysphoria; when taken for a long time, pancreatitis, liver injury, acute hepatic necrosis, and thrombocytopenia occasionally occur; allergy occasionally; there is an occasional hearing loss and reversible hearing impairment.

Therefore, attention needs to be paid to the monitoring of the adverse reaction of the medicine in the treatment process. Due to the difference of drug metabolism individuals, a reasonable administration scheme is formulated by combining blood concentration monitoring during clinical use, so that adverse reactions are avoided as much as possible.

Currently, there are many methods for clinically monitoring the plasma concentration of valproic acid, such as High Performance Liquid Chromatography (HPLC), chemiluminescence, homogeneous enzyme immunoassay, latex agglutination turbidimetry, etc. HPLC is time consuming and not easily automated; the direct chemiluminescence method has high sensitivity, but needs expensive chemiluminescence instruments, and has the disadvantages of complex operation, long detection time, low automation degree and poor repeatability. The existing homogeneous enzyme immunoassay and latex agglutination turbidimetry are limited in application due to poor repeatability and poor linearity.

Patent application CN102507917A discloses an enzyme-labeled conjugate of valproic acid, which is characterized in that tributylamine and isobutyl chloroformate are added into a valproic acid derivative solution to activate a valproic acid derivative; then coupling with glucose 6 phosphate dehydrogenase (G6 PDH) at-2 to-8 ℃, and purifying by using a gel chromatography column to obtain a valproate enzyme-labeled conjugate; finally diluting the valproate enzyme-labeled conjugate with a buffer solution.

Patent application CN103242445a discloses a preparation method for obtaining an enzyme-labeled conjugate: weighing glucose-6-phosphate dehydrogenase, and dissolving in phosphate buffer solution at room temperature to obtain a final concentration of 3-5mg/mL; dissolving the valproic acid derivative by using dimethylformamide, activating by a tributylamine method, carrying out a cross-linking reaction with a glucose-6-phosphate dehydrogenase enzyme solution, and purifying and dialyzing to obtain the glucose-6-phosphate dehydrogenase-valproic acid conjugate.

Patent application CN108956971A discloses a valproic acid immunodetection reagent and a preparation method and a detection method thereof, wherein valproic acid, 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide and N-hydroxy thiosuccinimide are dissolved in morpholine ethanesulfonic acid (MES) solution for activation and then coupled with enzyme.

All three routes rely on activation of the reactive group originally carried by valproic acid prior to reaction with an enzyme. Such strategies have difficulty ensuring a directed reaction and achieving a 1:1, resulting in batch-to-batch instability. Most preferably, reagents produced by the above strategy have reported intra-and inter-batch precision CVs at levels of only about 4.7%.

Thus, there remains a need in the art for improved valproic acid derivatives, and for improved coupling methods.

Disclosure of Invention

According to some embodiments, there is provided a valproic acid derivative having the structure shown in formula I:

wherein

n is an integer from 1 to 10, preferably an integer from 2 to 6;

m is an integer of 1 to 10, preferably 1 to 5.

In some particular embodiments, the valproic acid derivative has the structure shown in formula II:

according to some embodiments, there is provided an enzyme label comprising a valproic acid derivative according to the present application and an enzyme. In some specific embodiments, the enzyme is covalently bound to said valproic acid derivative. In some specific embodiments, the enzyme is glucose hexaphosphate dehydrogenase.

According to some embodiments, there is provided a glucose hexaphosphate dehydrogenase variant having a substitution of the proline residue at position 254 to a cysteine residue, as compared to wild-type glucose hexaphosphate dehydrogenase. It is understood that the proline residue at position 254 and its equivalent are included within the scope, depending on the species to which the glucose hexaphosphate dehydrogenase belongs.

In particular embodiments, the species to which the glucose hexaphosphate dehydrogenase belongs may be selected from: leuconostoc mesenteroides (Leuconostoc mesenteroides).

According to some embodiments, there is provided an agent comprising a valproic acid derivative according to the present application.

According to other embodiments, a reagent is provided comprising an enzyme label according to the present application.

According to some embodiments, there is provided a method of preparing an enzyme label, comprising the steps of:

1) Providing a valproic acid derivative according to the present application, for example in an aprotic solvent;

2) Providing an enzyme, e.g., in a buffer;

3) At 18 ℃ to 28 ℃, according to the valproic acid derivative: enzyme =1:1 to 1:200 (preferably 1.50), contacting the valproic acid derivative and the enzyme for 1 hour to 4 hours (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4 hours, or any value in between) such that the valproic acid derivative and the enzyme are conjugated to provide an enzyme label.

In some embodiments, the enzyme label produced is purified as desired, such as, but not limited to, a molecular sieve chromatography process.

In the context of this application, step numbering should not be understood as an order of operation of the steps.

In some specific embodiments, steps 1) and 2) may be interchanged in order.

In some embodiments, the enzyme is provided in a buffer selected from the group consisting of: PBS, tris, TAPS, TAPSO, pH of the buffer is 6.0 to 8.0.

In some embodiments, the aprotic solvent provides a reaction medium environment selected from one or a combination of: acetonitrile, dimethylformamide, dimethylsulfoxide.

In some embodiments, the enzyme is glucose hexaphosphate dehydrogenase; in particular, the enzyme comprises at least one free thiol group prior to conjugation to allow for directed and controlled conjugation reactions.

In a specific embodiment, there is provided a method for preparing an enzyme label, comprising the steps of:

1) Dissolving a valproic acid derivative in N, N-dimethylformamide;

2) Dissolving glucose hexaphosphate dehydrogenase containing one free sulfhydryl group in a buffer solution;

3) Mixing the solutions of step 1) and step 2), contacting (allowing for sufficient mixing, e.g., moderate shaking) at 18 ℃ to 25 ℃ for 2 to 3 hours;

4) Performing molecular sieve chromatography treatment on the reaction product obtained in the step 3) to obtain the purified glucose hexaphosphate dehydrogenase labeled valproic acid derivative.

According to some embodiments, there is provided an enzyme label prepared according to the method of the present application.

According to some embodiments, there is provided a valproic acid detection kit comprising:

-a first agent comprising an anti-valproic acid antibody derived from: mouse, rat, primate, sheep, fowl, human, rabbit, horse, cow, camel; the anti-valproate antibody is selected from the group consisting of: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, antigen-binding fragments;

-a second reagent comprising an enzymatic label according to the present application;

-optionally, a quality control; and/or

-optionally, a calibrator.

In some embodiments, the quality control comprises from 30 μ g/ml to 120 μ g/ml valproic acid, e.g., 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 μ g/ml (or any value in between) valproic acid.

In some embodiments, the calibrator comprises 0 μ g/ml to 150 μ g/ml valproic acid, e.g., 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 μ g/ml (or any value in between) valproic acid.

In some specific embodiments, a valproic acid detection kit comprises:

-a first reagent comprising:

30mM to 300mM buffer solution,

5mM to 20mM glucose-6-phosphate,

5mM to 20mM oxidized beta-nicotinamide adenine dinucleotide,

0.01 to 10 mu g/ml of anti-valproic acid monoclonal antibody,

0.1g/L to 5g/L stabilizer,

0.1g/L to 5g/L of surfactant,

0.1g/L to 5g/L preservative;

-a second reagent comprising:

30mM to 300mM buffer solution,

0.01 to 10. Mu.g/ml of the enzyme label of the present application,

0.1 to 5g/L stabilizer,

0.1g/L to 5g/L of surfactant,

0.1g/L to 5g/L preservative;

according to some embodiments, there is provided the use of a valproic acid derivative of the present application in the preparation of a detection device.

According to some embodiments, there is provided the use of an enzyme label of the present application in the preparation of a detection device.

According to some embodiments, there is provided a use of a glucose hexaphosphate dehydrogenase variant of the present application in the preparation of a test device.

In some embodiments, the detection device is embodied in a form selected from: reagent, kit, orifice plate, particle, chip and test paper.

Drawings

Figure 1 is a scheme of the synthesis of valproic acid derivatives.

FIG. 2 is a graph showing the correlation analysis of the kit of the present application.

FIG. 3 difference in absorbance change between the application reagent and the control reagent.

Detailed Description

The present application is described in detail below with reference to specific examples, which describe in detail only the synthesis of the valproic acid derivatives listed in the following figures, and the specific method of coupling glucose hexaphosphate dehydrogenase using the same.

Example 1 Synthesis of Valproic acid derivatives and confirmation of their Structure

According to the synthetic route of valproic acid derivatives shown in FIG. 1, compounds represented by formula II are synthesized:

1. synthesis of Compound 3

Compound 1 (100mg, 0.69mmol) and compound 2 (150mg, 0.69mmol) were dissolved in DCM (15 mL), to which triethylamine (209mg, 2.07mmol) and HATU (314mg, 0.83mmol) were added, and stirred at room temperature (18 to 25 ℃ C.) for 6h. The solvent was removed under reduced pressure and purified by column chromatography to give compound 3 as a white solid (150mg, 64% yield).

2. Synthesis of Compound 4

Compound 3 (150mg, 0.46mmol) was dissolved using methanol (10 mL), to which was added concentrated HCl (2 mL) and stirred at room temperature (18 to 25 ℃ C.) for 2h. Water was added to the reaction system, and extraction was performed using ethyl acetate. The aqueous phase was adjusted to pH 10 with NaOH (1N), extracted with ethyl acetate, and the solvent was removed under reduced pressure to give Compound 4 (128mg, 100%).

3. Synthesis of valproic acid derivatives

Compound 4 (50mg, 0.18mmol) and compound 5 (38mg, 0.18mmol) were dissolved in DCM (8 mL), and triethylamine (55mg, 0.54mmol) was added dropwise thereto, followed by HATU (82mg, 0.22mmol) and stirring at room temperature (18 to 25 ℃) for 16h. Preparative plates were purified to give valproic acid derivative as a white solid (55mg, 71% yield).

4. By mass spectrometry (measuring M) ⁺¹ 525.5，M ⁺²³ 574.6 And nuclear magnetism ( ¹ H-NMR) to identify that the valproic acid derivative has correct structure.

Example 2 enzyme marker preparation method (method of the present application)

1. The valproic acid derivative obtained in example 1 was dissolved in N, N-dimethylformamide (10 mg/ml);

2. mu.l of a solution (6.4 mg/ml, 0.2M phosphate buffer, pH 8.0) of glucose hexaphosphate dehydrogenase (derived from Leuconostoc mesenteroides) which had been artificially modified to contain a single free thiol, i.e., proline at position 254 was substituted with cysteine to obtain a free thiol) was added to 750. Mu.l of a buffer solution (0.05M Na ₂ HPO ₄ 、150mM NaCl、10mM EDTA、0.1％ NaN ₃ pH 7.2);

3. then 50. Mu.l of an N, N-dimethylformamide solution of a valproic acid derivative was added thereto;

4. fully shaking the mixed solution at room temperature (18-28 ℃) for 2-3 hours;

5. performing molecular sieve chromatography to obtain glucose hexaphosphate dehydrogenase (concentration of 0.1mg/mL-2.0 mg/mL) labeled with valproic acid.

Example 3 preparation of enzyme labels (control method, coupling of carboxyl group of activated valproic acid to amino group on G6 PDH)

1. Dissolving 15mg of glucose hexaphosphate dehydrogenase in 12mL of Tris buffer, and then sequentially adding 225mg of reduced nicotinamide adenine dinucleotide NADH,135mg of glucose-6-phosphate, 0.75mL of Carbitol (Carbitol) and 2.25mL of dimethyl sulfoxide; the pH value of the Tris buffer solution is 9.0, and the concentration of each component is as follows: 0.05mol/L Tris, 3.3mmol/L magnesium chloride, 145.4mmol/L sodium chloride.

2. Activation of valproic acid derivative: dissolving 10mg of valproic acid derivative in 420. Mu.L of dimethyl sulfoxide and 180. Mu.L of dimethylformamide, adding 6. Mu.L of tributylamine and 350. Mu.L of isobutyl chloroformate, and stirring at 2-8 ℃ for 30 minutes;

3. and (3) mixing the solutions obtained in the steps (1) and (2), stirring for 12 to 16 hours at the temperature of between 2 and 8 ℃, and purifying the coupled enzyme-labeled antigen by using a G-25 gel chromatography column to obtain the glucose-6-phosphate dehydrogenase-labeled valproic acid derivative.

EXAMPLE 4 preparation of reagents

1. Preparation of the first reagent:

2. preparation of the second reagent:

3. quality control product and calibrator:

the essential performance and effect of the reagent do not depend on quality control products and calibration products, and self-made or commercially available products can be adopted.

The quality control product is prepared by diluting pure valproic acid with buffer solution, and the concentrations are 30-40 μ g/ml, 70-80 μ g/ml and 115-125 μ g/ml respectively.

The calibrator is pure valproic acid and is obtained by diluting with buffer solution, and the concentrations are 0 mug/ml, 10 mug/ml, 25 mug/ml, 50 mug/ml, 100 mug/ml and 150 mug/ml respectively.

4. Assembling a kit:

and assembling the reagents (optionally containing quality control products and calibration products) into a valproic acid homogeneous enzyme immunoassay kit.

Example 5 Performance test of Valproic acid homogeneous enzyme immunoassay kit

1. The kit is based on competitive reaction, and the detection principle is as follows:

in a homogeneous reaction system, valproic acid and glucose hexaphosphate dehydrogenase-valproic acid conjugate in a sample compete for binding to an anti-valproic acid antibody site simultaneously, and as the enzyme activity is reduced after the antibody is bound to the conjugate, the more valproic acid is free in the sample, the more antibody sites are bound, the less antibody is bound to an enzyme marker, and the enzyme marker which is not bound to the antibody catalyzes beta-nicotinamide adenine dinucleotide oxidation type (NAD) ⁺ ) Converted into beta-nicotinamide adenine dinucleotide reduced form (NADH), and the concentration of valproic acid in the sample is formed by the generation amount of NADHAnd (4) obtaining the concentration of the valproic acid in the sample in a proportional way through the change of the absorbance.

2. Biochemical analyzer parameters

TABLE 1 Valproic acid kit Hitachi 7180 parameters of the present application

Analysis point	[Rate-A][10][19][24]
		WAVE(SUB/MAIN)	[410][340]
S.VIL.	[2.0]
		S.R1	[180]
S.R3	[60]
		ABS.LIMIT:	[32000][ increasing by one]
CALIB type	[Logit-Log4p]
		Point parameter	[6]SPAN POINT[6]
Calibration article	0.0、10.0、25.0、50.0、100.0、150.0μg/ml
		Sample(s)	Various physiological samples (such as serum, plasma, saliva, whole blood, urine, etc.)

3. Repeatability test

The calibration curve established above is used for repeated measurement, and the high-quality control product, the medium-quality control product and the low-quality control product are respectively detected for 20 times.

TABLE 2 repeatability test of valproic acid kit of the present application

Number of times of measurement	Quality control 1	Quality control 2	Quality control 3
				1	28.1	76.6	119.7
2	29.0	77.3	123.0
				3	28.2	76.7	119.6
4	28.0	78.3	131.0
				5	28.1	79.2	119.1
6	28.1	80.3	127.0
				7	27.9	75.1	124.2
8	27.5	76.6	123.9
				9	28.0	79.5	121.7
10	27.5	77.8	126.1
				11	27.9	77.5	127.0
12	26.7	78.4	122.9
				13	27.6	76.7	126.8
14	27.5	76.7	121.8
				15	27.4	77.8	124.3
16	26.5	78.7	119.5
				17	27.7	77.2	122.8
18	27.4	74.4	126.4
				19	26.9	78.1	121.8
20	27.1	78.0	119.6
				Mean value	27.7	77.5	123.4
Standard deviation of	0.580	1.40	3.23
				CV	2.10％	1.81％	2.62％

As shown in table 1, the sample test was repeated 20 times with CV less than 2.6%.

4. Accuracy test

The USP pure (USP) was dissolved in DMSO at different concentrations in stock solutions, diluted in serum by the same fold (minimum 20 fold dilution), prepared as different concentrations of VPA (variation differentiation analysis) solutions, reagents tested and calculated for deviation from the theoretical values.

TABLE 3 Valproic acid kit accuracy experiment of the present application

USP	Measured value 1	Measured value 2	Measured value 3	Mean value	Relative deviation of	Absolute deviation
							25	22.9	23.5	22.9	23.1	-7.6％	-1.9
30	27.4	27.5	28.0	27.6	-7.9％	-2.4
							50	49.7	50.3	50.2	50.1	0.1％	0.1
75	78.6	74.6	77.7	77.0	2.6％	2.0
							100	100.5	104.7	104.8	103.3	3.3％	3.3
125	123.1	122.4	125.3	123.6	-1.1％	-1.4

5. Drug interference experiments

13 compounds and drugs were selected and at valproic acid concentrations around 90 μ g/ml, the following concentrations of compounds did not interfere significantly in a statistical sense.

TABLE 4 anti-interference experiment

6. Linear experiment

The low value sample and the high value sample are selected to be diluted according to an equal difference dilution method, each sample is repeatedly tested for 3 times, and the measured linear data are shown in the table 5 and the figure 2.

TABLE 5 Valproic acid kit Linear data

	Measured value 1	Measured value 2	Measured value 3	Mean value	Theoretical value	Relative deviation of	Absolute deviation of
								1	10.0	10.2	10.1	10.1	9.8	3.1％	0.3
2	27.5	25.8	25.1	26.1	26.5	-1.4％	-0.4
								3	41.6	44.7	43.1	43.1	43.2	-0.2％	-0.1
4	58.0	62.7	65.3	62.0	59.9	3.6％	2.1
								5	75.5	75.9	79.0	76.8	76.6	0.3％	0.2
6	88.5	89.3	89.8	89.2	93.2	-4.3％	-4.0
								7	108.4	108.2	110.7	109.1	109.9	-0.8％	-0.8
8	130.5	129.1	130.8	130.1	126.6	2.7％	3.5
								9	139.8	144.9	147.7	144.1	143.3	0.5％	0.8
10	159.7	155.8	157.9	157.8	160.0	-1.4％	-2.2
								11	172.9	180.9	179.7	177.8	176.7	0.6％	1.1

7. Difference between batches

Three batches of the reagent of the present application (example 2) and the control reagent (example 3) were individually calibrated and the difference in absorbance change between the different batches was calculated, as shown in Table 6, FIG. 3.

TABLE 6 reagent run-to-run data

The application has the advantages that a novel valproic acid derivative is used, and a maleimide-sulfydryl coupling method with high selectivity is used, so that the derivative and enzyme can be coupled one by one, and the batch difference formed in the common coupling process is greatly reduced.

The valproic acid reagent prepared by the method has better specificity and has no obvious cross reaction with 13 common medicines; the accuracy and precision are very high, the detection CV is less than 2.6%, and the recovery deviation is less than 8%. The valproic acid detection kit is simple, convenient, rapid and low in cost, and can be used for carrying out automatic detection on various mainstream machine types.

Claims

1. Use of an enzyme label in the preparation of a detection device, wherein:

the enzyme label comprises or consists of: a valproic acid derivative and a glucose hexaphosphate dehydrogenase variant;

said glucose hexaphosphate dehydrogenase variant and said valproic acid derivative are covalently bound;

the valproic acid derivative has a structure shown in formula I:

wherein, the first and the second end of the pipe are connected with each other,

n is an integer from 1 to 10, preferably from 2 to 6;

m is an integer of 1 to 10, preferably 1 to 5;

(ii) the substitution of the proline residue at position 254 of the glucose hexaphosphate dehydrogenase variant with a cysteine residue, as compared to wild-type glucose hexaphosphate dehydrogenase of Leuconostoc mesenteroides;

preferably, the valproic acid derivative is covalently bound at amino acid residue 254 of the glucose hexaphosphate dehydrogenase variant.

2. The use according to claim 1, the detection device being prepared as a reagent or a kit.

3. The use according to claim 2, said reagent or kit being a homogeneous enzyme immunoassay reagent or kit.

4. The use of claim 1, wherein the valproic acid derivative is of formula II: