Valproic acid homogeneous enzyme immunoassay kit, preparation method and application

Abstract

The invention relates to a valproic acid homogeneous enzyme immunoassay kit and a preparation method and application thereof, the valproic acid homogeneous enzyme immunoassay kit comprises a reagent R1 and a reagent R2, the reagent R1 comprises a buffer solution, a mouse anti-valproic acid monoclonal antibody, L-malic acid, a stabilizer, a preservative and a surfactant; and the R2 reagent comprises a buffer solution, beta-NAD < + >, a malate dehydrogenase-valproic acid conjugate, a protective agent and a preservative. Aiming at the defect that an existing valproic acid homogeneous enzyme immunoassay kit is poor in stability, glucose 6-phosphate dehydrogenase in a system is replaced with malic dehydrogenase, so that the analysis sensitivity, precision, thermal stability and long-term stability of the kit are remarkably improved. Various performance indexes of the kit are further improved by adjusting the content of the malate dehydrogenase-valproic acid conjugate and beta-NAD < + > in the system, so that the valproic acid homogeneous enzyme immunoassay kit with high analysis sensitivity and excellent precision and stability is obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro diagnostic reagents for biochemical detection, and specifically relates to a valproic acid homogeneous enzyme immunoassay kit and a preparation method and application thereof. Background Art

[0002] Valproic acid is a broad-spectrum antiepileptic drug, which is mainly used clinically to treat systemic or partial types of epileptic seizures. During the clinical administration of valproic acid, due to the differences in individual pharmacokinetic metabolism in patients, the correlation between the dosage of valproic acid and the blood concentration is poor.

[0003] At present, there are many methods for monitoring the blood concentration of valproic acid in clinical practice, such as high performance liquid chromatography (HPLC), chemiluminescence, homogeneous enzyme immunoassay, latex agglutination turbidimetry, etc. HPLC is time-consuming and difficult to automate; direct chemiluminescence has high sensitivity, but requires expensive chemiluminescence instruments, cumbersome operation, long detection time, low degree of automation, and poor repeatability. Homogeneous enzyme immunoassay is a competitive binding analysis method. Compared with other methods of drug detection, homogeneous enzyme immunoassay uses enzyme markers to bind to corresponding antigens or antibodies, and the activity of the marker enzyme changes. Without separating the bound enzyme markers and free enzyme markers, the content of the substance to be tested is determined by measuring the change in the activity of the marker enzyme. And because it is a homogeneous enzymatic reaction system, it not only avoids the influence of antigen and antibody separation on the results, but also the reaction can reach equilibrium in a relatively short time, and the use of a fully automatic biochemical analyzer can achieve rapid high-throughput detection of samples.

[0004] The marker enzymes used in homogeneous enzyme immunoassay include lysozyme, malate dehydrogenase, β-D-galactosidase and glucose-6-phosphate dehydrogenase. Glucose-6-phosphate dehydrogenase is currently commonly used. However, glucose-6-phosphate dehydrogenase has many disadvantages: (1) It has a high content in the human body, a wide range of presence, and strong enzyme activity. During the detection process, glucose-6-phosphate dehydrogenase in the serum may cause false positive interference to the results; (2) glucose-6-phosphate dehydrogenase loses a large amount of enzyme activity during the coupling reaction, and the coupling efficiency is low; (3) The stability of glucose-6-phosphate dehydrogenase conjugates is poor, resulting in poor stability of the corresponding detection reagents, which affects the validity period of the kit. Summary of the invention

[0005] In order to overcome the defects of poor stability and specificity of the above-mentioned valproic acid homogeneous enzyme immunoassay kit due to the use of glucose-6-phosphate dehydrogenase conjugate, the present invention provides a valproic acid homogeneous enzyme immunoassay kit, which has the effects of strong stability and good specificity. At the same time, the present invention also provides a preparation method thereof.

[0006] The technical scheme of the valproic acid homogeneous enzyme immunoassay kit provided by the present invention is as follows:

[0007] A valproic acid homogeneous enzyme immunoassay kit comprises R1 reagent and R2 reagent, characterized in that the R1 reagent comprises a buffer, a mouse anti-valproic acid monoclonal antibody, L-malic acid, a stabilizer, a preservative and a surfactant; and the R2 reagent comprises a buffer, β-NAD+, a malate dehydrogenase-valproic acid conjugate, a protective agent and a preservative.

[0008] The specific reaction principle of the kit is as follows: the malate dehydrogenase-valproic acid conjugate contained in reagent R2 is bound to the mouse anti-valproic acid monoclonal antibody in reagent R1 through valproic acid, and the activity of the malate dehydrogenase of the conjugated species will be inhibited, and it will not be able to catalyze the conversion of β-NAD+ in the system. When testing the sample, since the sample contains small molecule valproic acid, it can compete with the malate dehydrogenase-valproic acid conjugate to bind to the mouse anti-valproic acid monoclonal antibody in the system. Small molecules are often more stable than relatively larger conjugates to bind to the mouse anti-valproic acid monoclonal antibody, thereby releasing the malate dehydrogenase-valproic acid conjugate, enhancing the activity of malate dehydrogenase, and then catalyzing L-malic acid and β-NAD+ to generate pyruvate and NADH, wherein NADH has an absorption peak at 340nm. The change in the content of NADH is detected by the analyzer, and the corresponding standard curve is drawn, so that the concentration of valproic acid in the sample can be calculated by detecting the change in the content of NADH.

[0009] The mouse anti-valproic acid monoclonal antibody (clone name: VP10-234.5, IgG1 type) was purchased from Shanghai Bio-Pac Biotechnology Co., Ltd. (Pac Bio).

[0010] Furthermore, the pH of the R1 reagent is 6.5-7.0, and specifically includes: (50-250) mM buffer, (1-5) g / L mouse anti-valproic acid monoclonal antibody, (1-5) g / L L-malic acid, (0.1-5) g / L stabilizer, (0.5-5) mL / L preservative, and mass concentration (0.1-10) g / L surfactant; the pH of the R2 reagent is 8.0-8.5, and specifically includes: (50-250) mM buffer, (5-10) g / L β-NAD+, (0.1-10) g / L malate dehydrogenase-valproic acid conjugate, (1-10) g / L stabilizer, and (0.1-5) g / L preservative.

[0011] Furthermore, the buffer is one or more of HEPES buffer, PBS buffer, acetic acid-sodium acetate buffer, Tris buffer, MOPSO buffer, TAPSO buffer, MES buffer, and PIPES buffer.

[0012] Furthermore, the valproic acid homogeneous enzyme immunoassay kit is characterized in that the stabilizer is one or more of casein, sorbitol, trehalose, mannitol, and sucrose.

[0013] Furthermore, the valproic acid homogeneous enzyme immunoassay kit is characterized in that the preservative is one or more of sodium azide and ProClin 300.

[0014] The present invention also provides a method for preparing the above-mentioned valproic acid homogeneous enzyme immunoassay kit, the method comprising:

[0015] [S1] Preparation of R1 reagent: L-malic acid, mouse anti-valproic acid monoclonal antibody, stabilizer, preservative and surfactant were sequentially added to phosphate buffer to obtain R1 reagent;

[0016] [S2] Preparation of malate dehydrogenase-valproic acid conjugate: After dissolving the valproic acid derivative with activation buffer, an activator is added for activation, and then malate dehydrogenase is added for coupling to obtain a mixture solution containing malate dehydrogenase-valproic acid conjugate.

[0017] [S3] Preparation of R2 reagent: Add β-NAD+, malate dehydrogenase-valproic acid conjugate, stabilizer and preservative to the buffer solution in sequence and adjust the volume to obtain R2 reagent.

[0018] Further, step [S2] is specifically as follows: ① firstly dissolve the valproic acid derivative in a dimethyl sulfoxide organic solvent, and then add 4-morpholineethanesulfonic acid to the activator 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to fully dissolve it, and then mix the above two mixed solutions for activation, so that the molar ratio of the activator to the valproic acid derivative is 2:1, and stir and activate for 30 minutes at 37°C; ② after activation, add malate dehydrogenase, wherein the molar ratio of malate dehydrogenase to valproic acid is 1:10, and stir and couple at a temperature of 37°C for 2 hours to obtain a crude malate dehydrogenase-valproic acid conjugate; ③ the above crude malate dehydrogenase-valproic acid conjugate is dialyzed and purified with phosphate buffer for 12 hours to obtain a malate dehydrogenase-valproic acid conjugate;

[0019] The valproic acid derivative is a commercially available valproic acid derivative purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (named ABS205, valproic acid derivative, product number A275764-50mg).

[0020] The present invention has the following beneficial effects:

[0021] 1. The present invention replaces the glucose-6-phosphate dehydrogenase conjugate used in the valproic acid homogeneous enzyme immunoassay kit with a malate dehydrogenase-valproic acid conjugate. Compared with glucose-6-phosphate dehydrogenase, malate dehydrogenase has the advantages of heat resistance, wider pH adaptability, and less activity loss during the coupling process (i.e., high coupling efficiency), which can effectively solve the problem of poor stability of the valproic acid homogeneous enzyme immunoassay kit.

[0022] 2. The present invention replaces the glucose-6-phosphate in R1 with L-malic acid, so that the glucose-6-phosphate dehydrogenase in the serum cannot function due to the lack of substrate, thereby improving the anti-interference ability of the valproic acid homogeneous enzyme immunoassay kit and reducing the false positives in the clinical sample determination.

[0023] 3. The present invention improves the sensitivity of the overall reaction by adjusting the buffer system of R2 to promote the generation of NADH. The equation for malate dehydrogenase to metabolize L-malic acid to generate oxaloacetic acid is as follows:

[0024]

[0025] L-malic acid is the substrate of malate dehydrogenase, which is stable and inexpensive. The product produced after metabolism is oxaloacetic acid. However, oxaloacetic acid is unstable under weak acidic conditions and easily undergoes non-enzymatic decarboxylation reaction to degrade into carbon dioxide and water, which in turn causes the reaction to move further to the right, accelerates the reaction rate, produces NADH at a faster rate, and improves the reaction sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 This is a comparison chart of clinical sample serum of the valproic acid homogeneous enzyme immunoassay kit in Example 1 of the present invention;

[0028] Figure 2 This is a comparison chart of clinical sample serum of the valproic acid homogeneous enzyme immunoassay kit in Example 2 of the present invention;

[0029] Figure 3 This is a comparison chart of clinical sample serum of the valproic acid homogeneous enzyme immunoassay kit in Example 3 of the present invention;

[0030] Figure 4 This is a comparison chart of clinical sample serum of the valproic acid homogeneous enzyme immunoassay kit in Example 4 of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0032] Example 1

[0033] This embodiment provides a valproic acid homogeneous enzyme immunoassay kit and a preparation method thereof, wherein the components and contents are as follows:

[0034]

[0035]

[0036] The preparation method of the kit is:

[0037]

S1

[0038]

S2

[0039] [S3] Preparation of R2 reagent: Add 20 mM β-NAD+, 5.6 g / L malate dehydrogenase-valproate conjugate, 1 g / L sucrose and 1 g / L ProClin 300 to the buffer solution in sequence and adjust the volume to obtain R2 reagent.

[0040] Example 2

[0041] The difference between this embodiment and embodiment 1 is that the components and contents in the kit provided in this embodiment are as follows:

[0042]

[0043]

[0044] Example 3

[0045] The difference between this embodiment and embodiment 1 is that the components and contents in the kit provided in this embodiment are as follows:

[0046]

[0047] Example 4

[0048] The difference between this embodiment and embodiment 1 is that the components and contents in the kit provided in this embodiment are as follows:

[0049]

[0050] Validation results of analytical performance, clinical comparison and long-term stability of the valproic acid homogeneous enzyme immunoassay kit. (1) Preparation of calibrators: Using a storage buffer containing 50 mM phosphate buffer, 150 mM sodium chloride, 10% BSA, and 5% glycerol, valproic acid powder was prepared into a set of 6 calibrator solutions of 0.0, 10.0, 20.0, 40.0, 80.0 and 160.0 mg / L.

[0051] (2) The parameters of the biochemical analyzer are as follows:

[0052]

[0053] (3) Analyze performance data table

[0054] Table 1 Analysis performance table of valproic acid homogeneous enzyme immunoassay kit of embodiment 1 of the present invention

[0055]

[0056] Table 2 Analysis performance table of valproic acid homogeneous enzyme immunoassay kit of embodiment 2 of the present invention

[0057]

[0058] Table 3 Analysis performance table of valproic acid homogeneous enzyme immunoassay kit of embodiment 3 of the present invention

[0059]

[0060] Table 4 Analysis performance table of valproic acid homogeneous enzyme immunoassay kit in Example 4 of the present invention

[0061]

[0062] As can be seen from Tables 1 to 4, the maximum blank absorbance change rate of the valproic acid homogeneous enzyme assay kit reagent of the present invention is 0.0057, which meets the requirements of blank absorbance change rate ≤ 0.0800 and blank absorbance ≤ 1.2000; the maximum values ​​of the repeatability CV at 20 mg / L and 40 mg / L are 4.37% and 2.50%, respectively; the minimum analytical sensitivity of the test sample of 10 mg / L is 0.0136, which meets the requirement of not less than 0.0050; the two quality control samples are tested 3 times each day, and the total test days are 5 days , the maximum precisions of the two levels were 4.76% and 2.54% respectively, meeting the requirement of ≤10%; in the linear data, in the range of [5-50) mg / L, the maximum absolute deviation was 1.81 mg / L, meeting the requirement of ≤5 mg / L, and the maximum relative deviation of [50-160] mg / L was -3.4%, meeting the requirement of ≤10%; the accuracy sample used was the working calibrator traceable by our company, with a target value of 58.2 mg / L, and the average value was taken after 3 measurements, and the maximum deviation relative to the target value was -6.53%. It can be seen from the data that the valproic acid homogeneous enzyme assay kit of the present invention has excellent performance and can accurately detect valproic acid with a concentration of 10 mg / L.

[0063] (4) Clinical serum comparison test

[0064] 100 clinical serum samples tested by Xiaolan People's Hospital were selected for correlation analysis with the values ​​measured by the homogeneous enzyme immunoassay kit of the present invention. The results are shown in Tables 1 to 4, and the correlation graphs are shown in Figure 1-4 As shown, the correlation equations are: Example 1, y = 1.0081x-0.2944, R2 = 0.9762; Example 2, y = 0.9983x + 0.9071R 2 =0.9738; Example 3, y = 0.9927x + 0.3126, R 2 =0.9759; Example 4, y = 1.0195x-0.5865, R 2 =0.9771. From the correlation R of the four examples 2 The values ​​all meet the requirement of >0.95, which can indicate that the two methods have a good correlation, further proving that the valproic acid homogeneous enzyme immunoassay kit of the present invention has accuracy.

[0065] The clinical data are shown in Table 5:

[0066] Table 5 Clinical Experimental Data of Examples 1-4

[0067]

[0068]

[0069]

[0070] (5) Interference test The valproic acid homogeneous enzyme immunoassay of the present invention uses malate dehydrogenase, which has a narrow distribution area and low quantity in the human body, as the main metabolic enzyme to reduce false positive interference of the kit. At the same time, the mouse anti-valproic acid monoclonal antibody used in the present invention has good specificity and can recognize valproic acid alone, but does not react to other anti-epileptic drugs that produce false positive interference.

[0071] The experimental method of the drug interference experiment is: 10 commonly used compounds and drugs are selected for drug interference detection, and their concentrations are adjusted to 100 mg / L. The valproic acid homogeneous enzyme immunoassay kit of the present invention is used for determination. The results show that the concentrations are all <1 mg / L, indicating that the detected drugs have no cross reaction with valproic acid, and the valproic acid homogeneous enzyme immunoassay kit of the present invention has specificity. Table 6 and Table 7 are the interference test data of the interfering compounds and each embodiment, respectively.

[0072] Table 6 Interference experiment of valproic acid homogeneous enzyme immunoassay kit

[0073]

[0074] Table 7 Anti-drug interference experimental data of Examples 1-4

[0075]

[0076] (6) Long-term stability test statistics

[0077] Long-term stability experimental method: The prepared valproate homogeneous enzyme assay kit was stored at 2-8°C, taken out at (1, 7, 9, 12) months, and the experiment was carried out according to the analytical performance requirements, and the results were statistically analyzed to determine the shelf life of the kit.

[0078] Table 8 Statistical results of long-term stability experiment of valproic acid homogeneous enzyme immunoassay kit in Example 1

[0079]

[0080] Table 9 Statistical results of long-term stability experiment of valproic acid homogeneous enzyme immunoassay kit in Example 2

[0081]

[0082] Table 10 Statistical results of long-term stability experiment of valproic acid homogeneous enzyme immunoassay kit in Example 3

[0083]

[0084] Table 11 Statistical results of long-term stability test of valproic acid homogeneous enzyme immunoassay kit in Example 4

[0085]

[0086] It can be seen from the data in Tables 8 to 11 that the valproic acid homogeneous enzyme immunoassay kits of Examples 1-4 can meet the analytical performance requirements of the kit detection in each experiment during the storage period of 12 months at 2-8°C, indicating that the valproic acid homogeneous enzyme immunoassay kits of Examples 1-4 of the present invention can maintain stable performance within 12 months.

[0087] The embodiments described above are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention shall be subject to the claims.

Claims

1. A valproic acid homogeneous enzyme immunoassay kit, comprising R1 reagent and R2 reagent, characterized in that: The R1 reagent includes a buffer, a mouse anti-valproic acid monoclonal antibody, L-malic acid, a stabilizer, a preservative and a surfactant; The R2 reagent includes a buffer, β-NAD+, a malate dehydrogenase-valproic acid conjugate, a protective agent and a preservative.

2. The valproic acid homogeneous enzyme immunoassay kit according to claim 1, characterized in that: The R1 reagent specifically includes: 50-250 mM buffer, 1-5 g / L mouse anti-valproic acid monoclonal antibody, 1-5 g / L L-malic acid, 0.1-5 g / L stabilizer, 0.5-5 mL / L preservative, and 0.1-10 g / L surfactant; The R2 reagent specifically includes: 150-250 mM buffer, 20 mM β-NAD+, 0.1-10 g / L malate dehydrogenase-valproic acid conjugate, 1-10 g / L stabilizer, and 0.1-5 g / L preservative.

3. The valproic acid homogeneous enzyme immunoassay kit according to claim 1 or 2, characterized in that: The buffer is one or more of HEPES buffer, PBS buffer, acetic acid-sodium acetate buffer, Tris buffer, MOPSO buffer, TAPSO buffer, MES buffer, and PIPES buffer.

4. The valproic acid homogeneous enzyme immunoassay kit according to claim 1 or 2, characterized in that: The stabilizer is one or more of casein, sorbitol, trehalose, mannitol and sucrose.

5. The valproic acid homogeneous enzyme immunoassay kit according to claim 1 or 2, characterized in that: The preservative is one or more of sodium azide and ProClin 300.

6. The valproic acid homogeneous enzyme immunoassay kit according to claim 1 or 2, characterized in that: The surfactant is one or more of Tween-20, Tween-80, Triton X-100, Brij-35, and NP-9.

7. The valproic acid homogeneous enzyme immunoassay kit according to claim 1 or 2, characterized in that: The malate dehydrogenase-valproic acid conjugate was prepared by the following method: ① First, the valproic acid derivative is fully dissolved in a dimethyl sulfoxide organic solvent, and then 4-morpholineethanesulfonic acid is added to the activator 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to fully dissolve, and the above two mixed solutions are mixed again for activation, so that the molar ratio of the activator to the valproic acid derivative is 2:1, and the activation is stirred at 37°C for 30 minutes; ② After activation, malate dehydrogenase was added, wherein the molar ratio of malate dehydrogenase to valproic acid was 1:10, and the mixture was stirred and coupled at 37°C for 2 hours to obtain a crude malate dehydrogenase-valproic acid conjugate; ③ The crude malate dehydrogenase-valproic acid conjugate was dialyzed and purified with phosphate buffer for 12 hours to obtain the malate dehydrogenase-valproic acid conjugate.

8. The method for preparing the valproic acid homogeneous enzyme immunoassay kit according to claims 1-7, characterized in that: The steps include: Preparation of S1 and R1 reagents L-malic acid, mouse anti-valproic acid monoclonal antibody, stabilizer, preservative and surfactant were sequentially added to the phosphate buffer to obtain R1 reagent; Preparation of S2 and R2 reagents β-NAD+, malate dehydrogenase-valproic acid conjugate, stabilizer and preservative were added to the buffer in sequence and the volume was fixed to obtain R2 reagent.

9. Use of the valproic acid homogeneous enzyme immunoassay kit according to claims 1-7 in preparing a reagent for detecting valproic acid.

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