Hapten, antigen and antibody of escitalopram, and preparation method and application of escitalopram

Abstract

The invention belongs to the technical field of in-vitro detection, and particularly relates to escitalopram hapten, antigen and antibody as well as a preparation method and application thereof. The hapten disclosed by the invention is shown as a formula I. The invention also provides an antigen prepared from the hapten and a corresponding antibody. The invention also provides preparation methods of the hapten, the antigen and the antibody. By utilizing the antigen and the antibody provided by the invention, a detection kit with good sensitivity and specificity on escitalopram in blood can be prepared, and immunological detection of escitalopram is realized. Therefore, the method has a good application prospect. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro detection, and specifically relates to an escitalopram hapten, antigen, antibody, and their preparation methods and uses. Background Art

[0002] Escitalopram oxalate is a selective serotonin reuptake inhibitor (SSRIs). Due to its significant effect and low side effects, it has become a first-line drug for current antidepressant treatment. Clinical applications have found that there are very large individual differences in the efficacy of escitalopram oxalate drugs. This difference mainly stems from the difference in the activity of drug-metabolizing enzymes. By monitoring the blood drug concentration, it helps to determine whether the drug concentration in the patient's body is within the therapeutic window, ensuring the efficacy while minimizing the occurrence of adverse reactions as much as possible. In addition, by monitoring the blood drug concentration, it can also determine whether the current blood drug concentration of the patient is consistent with the dosage, so as to understand whether the patient takes the medicine as prescribed.

[0003] According to the "Consensus Guidelines for Therapeutic Drug Monitoring in Neuropsychopharmacology: Update 2017" guideline consensus, the detection of escitalopram has a clear effective therapeutic concentration range of 15 - 80 ng / mL, and the warning concentration is 160 ng / mL.

[0004] For the detection of escitalopram blood drug concentration, the existing mainstream methods are liquid chromatography-tandem mass spectrometry (LC-MS / MS) method, high performance liquid chromatography (HPLC), homogeneous enzyme immunoassay (EMIT), etc. Among them, the LC-MS / MS method has complex sample pretreatment, high requirements for testers, and expensive instrument equipment, and this method is not conducive to clinical promotion.

[0005] Immunological detection methods mainly include the detection of antigen-antibody reactions, such as ELISA, immunofluorescence, immunohistochemistry, etc., and the detection of lymphocyte functions, such as lymphocyte proliferation test, cytokine determination, etc. These methods are widely used in fields such as medical pathological diagnosis, disease prognosis assessment, vaccine research and development, etc. Among them, ELISA is a highly sensitive in vitro antigen-antibody binding reaction detection method, which can be used for the detection of various pathogenic microorganisms and autoantigens. The ELISA detection has the advantages of simple operation, rapidity, simple experimental requirements, and convenient wide application compared with detection methods such as LC-MS / MS.

[0006] However, there is currently a lack of relevant research and reports on immunological detection of escitalopram. To achieve high-sensitivity and high-specificity detection, the construction of antigens and antibodies used in immunological detection is a particularly important key point. Therefore, developing antigens and antibodies for escitalopram to achieve high-sensitivity and high-specificity immunological detection of escitalopram is an urgent problem to be solved in this field. Summary of the Invention

[0007] In view of the problems of the prior art, the present invention provides an escitalopram hapten, antigen, antibody, and their preparation methods and uses.

[0008] A compound represented by Formula I, or a salt thereof, or a stereoisomer thereof, or a crystal form thereof, or a solvate thereof:

[0009]

[0010] Wherein, n is selected from 2 - 10;

[0011] R a is selected from amino or carboxyl.

[0012] Preferably, the structural formula of the compound represented by Formula I is:

[0013]

[0014] The present invention also provides a preparation method of the above compound, or a salt thereof, or a stereoisomer thereof, or a crystal form thereof, or a solvate thereof. The preparation method of the compound represented by Formula I includes the following steps:

[0015]

[0016] Step 1, reacting Compound 1 with water to obtain Compound 2;

[0017] Step 2, reacting Compound 2 with Compound 3 to obtain Compound 4;

[0018] Step 3, hydrolyzing Compound 4 to obtain the product.

[0019] Preferably, in Step 1, the reaction medium is a mixed solution of ethanol and water;

[0020] and / or, in Step 1, the pH condition of the reaction is 8 - 11;

[0021] and / or, in Step 1, the reaction temperature is 80 - 100 °C and the time is 12 - 18 h;

[0022] and / or, in Step 2, the reaction medium is DMF;

[0023] And / or, in step 2, the reaction is carried out under the action of a coupling agent, and the coupling agent is selected from T3P;

[0024] And / or, in step 2, the reaction is carried out under the action of a catalyst, and the catalyst is selected from DIEA;

[0025] And / or, in step 2, the temperature of the reaction is 10 - 30 °C and the time is 1 - 3 h;

[0026] And / or, in step 3, the hydrolysis medium is at least one of dichloromethane, DMSO or chloroform;

[0027] And / or, in step 3, the hydrolysis is carried out under the action of a catalyst, and the catalyst is selected from trifluoroacetic acid;

[0028] And / or, in step 3, the temperature of the hydrolysis is 10 - 30 °C and the time is 1 - 3 h.

[0029] The present invention also provides an escitalopram antigen, and its structure is as follows:

[0030]

[0031] Wherein,

[0032] n is selected from 2 - 10;

[0033] L is none or a linker;

[0034] R1 is a carrier protein.

[0035] Preferably, the value of n is 2, and the linker is

[0036] Preferably, the carrier protein is selected from keyhole limpet hemocyanin, ovalbumin or bovine serum albumin.

[0037] The present invention also provides a preparation method of an escitalopram antigen, including the following steps: coupling an escitalopram hapten with a carrier protein through a coupling agent to obtain an escitalopram antigen; the hapten is the above compound, or its salt, or its stereoisomer, or its crystal form, or its solvate.

[0038] Preferably, it includes the following steps:

[0039] Step A, take the escitalopram hapten and mix it with a coupling agent, react to obtain reaction solution A;

[0040] Step B, react the reaction solution A with a carrier protein to obtain reaction solution B;

[0041] Step C, separate to obtain the escitalopram antigen.

[0042] Preferably, in step A, the coupling agent is selected from EDC;

[0043] And / or, in step A, a stabilizer is further added, and the stabilizer is selected from NHS;

[0044] And / or, in step A, the reaction medium is DMF;

[0045] And / or, in step A, the reaction conditions are light avoidance, the temperature is 10 - 30 °C, and the time is 1 - 6 h;

[0046] And / or, in step B, the reaction medium is PBS solution;

[0047] And / or, in step B, the reaction conditions are light avoidance, the temperature is 10 - 30 °C, and the time is 6 - 12 h.

[0048] The present invention also provides an escitalopram antibody, which is an antibody produced by the above escitalopram antigen.

[0049] Preferably, the escitalopram antibody is a monoclonal antibody or a polyclonal antibody.

[0050] The present invention also provides a preparation method of the above escitalopram antibody, including the following steps:

[0051] Step a, after immunizing animals by the conventional Freund's adjuvant method with the above escitalopram antigen for multiple times, obtaining serum containing specific antibodies, and taking spleen cells for fusion after the titer detection is qualified;

[0052] Step b, screening positive fused cells and performing subcloning to obtain a monoclonal cell line;

[0053] Step c, producing ascites or culturing cells to produce, separating and purifying to obtain an escitalopram antibody.

[0054] The present invention also provides the application of the above escitalopram antigen and / or escitalopram antibody in the immunological detection of escitalopram for non-diagnostic purposes.

[0055] In the prior art, the immunological detection reagent for escitalopram can only use rabbit polyclonal antibody, and the specificity of the polyclonal antibody may be difficult to meet the performance requirements of the kit. The present invention first constructs a new escitalopram hapten and antigen, and prepares an escitalopram antibody by using this antigen. The antibody of the present invention is a monoclonal antibody against the escitalopram antigen, and has the advantages of high sensitivity and good specificity. Therefore, by using the escitalopram antigen and antibody of the present invention, the immunological detection of escitalopram can be carried out, and an immunological detection kit with excellent detection performance can be made. In addition, the present invention also has the advantages of good repeatability, simple operation, and better meeting the requirements of clinical promotion, and has a good application prospect.

[0056] Obviously, based on the above content of the present invention, and in accordance with the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can also be made.

[0057] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 1H NMR characterization results of the escitalopram hapten for Example 1;

[0059] Figure 2 Mass spectrometry characterization results of the escitalopram hapten for Example 1;

[0060] Figure 3 Escitalopram concentration detection results for 30 samples in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0061] In the following examples and experimental examples, the reagents and raw materials not specifically described are all commercially available products.

[0062] Example 1 Escitalopram Hapten, Antigen and Antibody

[0063] I. Escitalopram Hapten

[0064] The structural formula of the escitalopram hapten in this example is as follows:

[0065]

[0066] Its preparation method is as follows:

[0067]

[0068] S1. Compound 1 reacts with water to obtain Compound 2;

[0069] S2. Compound 2 and Compound 3 undergo a nucleophilic substitution reaction to obtain Compound 4;

[0070] S3. Compound 4 undergoes a hydrolysis reaction to obtain Compound 5.

[0071] Step S1 specifically includes: Mix ethanol and water in a volume ratio of 1:1. Take 20 ml of the mixed ethanol and water, and add compound 1 (2.0 g, 6.17 mmol) to dissolve. Add NaOH (0.49 g, 12.34 mmol), and heat the reaction mixture at 80 °C for 16 h. The crude product of compound 2 is passed through anhydrous Na2SO4 (vacuum can reduce the boiling point of the liquid and has the function of rapid drying). Dry it with a rotary evaporator to obtain a solid powder. Purify the dried compound 2 by column chromatography to obtain the white solid, which is product compound 2 (1.27 g, yield 59.99%), and the specific mobile phase of the chromatography column is dichloromethane-methanol 15:1.

[0072] Step S2 specifically includes: Dissolve compound 2 (500 mg, 1.46 mmol) in anhydrous DMF (10 mL), add compound 3 (0.23 g, 1.21 mmol), add a mixed solution of T3P and ethyl acetate (T3P is mixed with 50% aqueous ethyl acetate solution in a volume ratio of 1:1, containing 1.39 g, 2.19 mmol of T3P) and DIEA (0.51 mL, 2.92 mmol), and stir at 25 °C for 1 h to obtain the crude product of compound 4; Primary purification: Dilute the crude product of compound 4 with water, and then subject the mixed solution to column chromatography for chromatographic separation to obtain the organic layer for standby (specifically, the chromatography column is a silica gel column, and the mobile phase in the chromatography column is dichloromethane); Secondary purification: Wash the organic layer 3 times with saturated NaHCO3 aqueous solution; Purify it by column chromatography (dichloromethane-methanol, 10:1) to obtain white oil, which is compound 4 (50.01 mg, yield 16.1%).

[0073] Step S3 specifically includes: Dissolve compound 4 (0.3 g, 0.64 mmol) in 6 ml of dichloromethane, add trifluoroacetic acid (3 mL, 38.515 mmol), and stir at room temperature for 1 h; Dry it with a rotary evaporator to obtain a solid powder; Dissolve the solid with ethyl acetate, and wash it with saturated NaHCO3 and saturated brine respectively; Dry it with anhydrous Na2SO4. After evaporation of the solvent, the residue is purified by column chromatography (dichloromethane:methanol = 10:1) to obtain compound 7, a white solid, with a yield of 42.3%.

[0074] Product characterization (1H NMR data is as Figure 1 shown, and the mass spectrometry data is as Figure 2As shown: 1H NMR (400 MHz, CD3OD) δ 7.77 (d, J = 8.0 Hz, 1H), 7.70 (s, 1H), 7.60 - 7.50 (m, 3H), 7.06 (t, J = 8.8 Hz, 2H), 5.21 (q, J = 12.8 Hz, 2H), 3.61 (t, J = 6.8 Hz, 2H), 3.13 (t, J = 8.2 Hz, 2H), 2.79 (s, 6H), 2.61 (t, J = 6.8 Hz, 2H), 2.33 (ddd, J = 14.8, 9.6, 5.4 Hz, 1H), 2.26 - 2.16 (m, 1H), 1.74 - 1.58 (m, 2H). LC-MS (ESI+) m / z 415.2 (M + H)+。

[0075] II. Escitalopram antigen

[0076] The structure is as follows:

[0077]

[0078] Wherein, L is a linker, and the linker is R1 is a carrier protein. The carrier protein is selected from keyhole limpet hemocyanin, ovalbumin or bovine serum albumin. In this example, bovine serum albumin and ovalbumin are taken as examples to illustrate its preparation method:

[0079] S1. Activation of the hapten:

[0080] Dissolve the escitalopram hapten in dimethylformamide solution to prepare an 800 mL escitalopram antigen solution with a concentration of 5×10 -5 mg / μL, then add 5 mg EDC and 5 mg NHS, and react at 25°C in the dark for 1 - 6 hours to obtain a reaction solution, and the solution is colorless, clear and transparent;

[0081] S2. Small molecule labeling;

[0082] Weigh 10 mg of bovine serum albumin (or 10 mg of ovalbumin), dissolve it in 5 ml of 0.01 M PBS (pH 7.4) solution, and the solution is colorless, clear and transparent; mix the reaction solution in step S1 with the bovine serum albumin (or ovalbumin) solution to obtain a slightly milky white turbid solution, and react at 25°C in the dark for 8 h, and the solution is still a slightly milky white turbid solution;

[0083] S3. Centrifuge the turbid solution (6000 r / min, 5 min), and extract the supernatant;

[0084] S4. Dialyze the supernatant with PBS buffer solution for 2 d, the pH of the PBS buffer solution is 7.4, and the dialysis temperature is 2 - 8°C;

[0085] S5. Centrifuge the retained solution after dialysis (6000 r / min, 5 min), and extract the supernatant to obtain the escitalopram-carrier protein complex, namely the escitalopram antigen.

[0086] III. Escitalopram Antibody

[0087] Use the above-mentioned escitalopram antigen to immunize animals to prepare paired monoclonal antibodies, which specifically include the following steps:

[0088] Step a. After immunizing animals (mice are selected in this example) multiple times by the conventional Freund's adjuvant method with the escitalopram antigen, obtain the serum containing specific antibodies. After the titer test is qualified, take spleen cells for fusion;

[0089] Step b. Screen positive fused cells and perform subcloning to obtain monoclonal cell lines;

[0090] Step c. Produce ascites or produce by cell culture, separate and purify to obtain monoclonal escitalopram antibodies.

[0091] Use the ELISA method to test the titer of the prepared escitalopram antibody. The specific detection method is as follows:

[0092] 1.1 Reagents

[0093] 1.1.1 Escitalopram antibody (produced using the escitalopram antigen with bovine serum albumin as the carrier protein) and escitalopram antigen (ovalbumin as the carrier protein);

[0094] 1.1.2 HRP-labeled goat anti-mouse enzyme-labeled secondary antibody;

[0095] 1.1.3 TMB chromogenic solution (when in use, the TMB chromogenic solution needs to be restored to room temperature. Note: Place it in an environment of 18 - 28 °C for 30 - 45 min);

[0096] 1.1.4 0.1M PH = 9.6 sodium bicarbonate buffer solution;

[0097] 1.1.5 0.01M PBS;

[0098] 1.1.6 Termination solution (2M H2SO4 solution or 1M HCl solution);

[0099] 1.1.7 Standard solution;

[0100] 1.2 Method

[0101] 1.2.1 Indirect ELISA (detecting the titer of antigen and antibody)

[0102] a) Dilute the escitalopram antigen with 0.1M PH = 9.6 sodium bicarbonate buffer solution to

[0103] Dilute to 1 μg / mL, add to a 96-well ELISA plate, 100 μL per well, react at 37 °C for 3 h or let stand overnight at 4 °C.

[0104] b) Discard the liquid in the wells of the plate, add 250 μL of ELISA washing solution, let stand for 30 s, discard the liquid in the plate, and repeat 3 times. (Wash away the uncoated free antigen).

[0105] c) Dilute the escitalopram antibody to 1 mg / mL with 0.01 M PBS, and then dilute again with

[0106] 0.01 M PBS to 1k, 2k, 4k, 8k, 16k, 32k, 64k in labeled centrifuge tubes for standby.

[0107] d) Add the diluted escitalopram antibody from the above step (negative control is 0.01 M PBS), 100 μL per well, make three replicates for each concentration gradient, and react at 37 °C for 45 min.

[0108] e) Discard the liquid in the wells of the plate, add 250 μL of ELISA washing solution, let stand for 30 s, discard the liquid in the plate, and repeat 3 times.

[0109] f) Dilute the HRP-labeled goat anti-mouse enzyme-linked secondary antibody according to the instructions (dilute with 0.01 M PBS), add the diluted HRP-labeled goat anti-mouse enzyme-linked secondary antibody, 100 μL per well, and react at 37 °C for 45 min.

[0110] g) Discard the liquid in the wells of the plate, add 250 μL of ELISA washing solution, let stand for 30 s, discard the liquid in the plate, and repeat 3 times.

[0111] h) Add the chromogenic solution (store in the dark) (TMB chromogenic solution needs to be restored to room temperature), 100 μL per well, and react in the dark at room temperature (18 - 28 °C environment) for 15 min.

[0112] i) Add the stop solution (1 M HCl solution or 2 M H2SO4 solution), 100 μL per well, OD 450 Read the OD value.

[0113] Note: The 1 M HCl solution is prepared from the 6 M HCl solution, and it should be prepared and used immediately. Taking 12 mL of the preparation amount as an example, add 2 mL of 6 M HCl solution to 10 mL of purified water and vortex to mix evenly.

[0114] 1.2.2 Competitive ELISA (detecting antigen-antibody competition)

[0115] a) Dilute the escitalopram antigen with 0.1 M sodium bicarbonate buffer with pH = 9.6 to

[0116] 1 μg / mL, add it to a 96-well ELISA plate, 100 μL per well, react at 37 °C for 3 h or let it stand overnight at 4 °C.

[0117] b) Discard the liquid in the wells of the plate, add 250 μL of ELISA washing solution, let it stand for 30 s, discard the liquid in the plate, and repeat 3 times. (Wash away the uncoated free antigen)

[0118] c) Dilute the escitalopram antibody to 1 mg / mL with 0.01 M PBS, and then dilute it again to 1k, 2k, 4k, 8k, 16k, 32k, 64k with 0.01 M PBS in labeled centrifuge tubes for standby.

[0119] d) Add 50 μL of the diluted standard solution (negative control is 0.01 M PBS) to each well, and then add 50 μL of the diluted escitalopram antibody to each well (negative control is 0.01 M PBS). Make three replicates for each concentration gradient and react at 37 °C for 45 min.

[0120] e) Discard the liquid in the wells of the plate, add 250 μL of ELISA washing solution, let it stand for 30 s, discard the liquid in the plate, and repeat 3 times.

[0121] f) Dilute the HRP-labeled goat anti-mouse enzyme-linked secondary antibody according to the instructions (dilute with 0.01 M PBS), add the diluted HRP-labeled goat anti-mouse enzyme-linked secondary antibody, 100 μL per well, and react at 37 °C for 45 min.

[0122] g) Discard the liquid in the wells of the plate, add 250 μL of ELISA washing solution, let it stand for 30 s, discard the liquid in the plate, and repeat 3 times.

[0123] h) Add the chromogenic solution (store in the dark) (TMB chromogenic solution needs to be restored to room temperature), 100 μL per well, and react in the dark at room temperature (18 - 28 °C environment) for 15 min.

[0124] i) Add the stop solution (1 M HCl solution or 2 M H2SO4 solution), 100 μL per well, OD 450 Read the OD value.

[0125] Note: The 1 M HCl solution is prepared from 6 M HCl solution and should be used immediately. Taking 12 mL of the preparation amount as an example, add 2 mL of 6 M HCl solution to 10 mL of purified water and vortex to mix well.

[0126] The results are as follows:

[0127] Table 1 Results of the titer inhibition assay of escitalopram antibody

[0128]

[0129] The prepared escitalopram antibody was subjected to IC50 testing. The results showed that the IC50 was 8.10 ng / ml, indicating that the escitalopram antibody had high sensitivity and good specificity.

[0130] Example 2 Escitalopram Detection Kit and Its Detection Method

[0131] I. Escitalopram Detection Kit

[0132] The composition of the kit in this example is as follows (for 10 person - times):

[0133] Table 2

[0134] Composition Specification M1 Reagent 0.5ml R1 Reagent 1ml

[0135] Among them,

[0136] 1. Biotin - ester - labeled antibody

[0137] (1) Put 0.1 mg of the escitalopram antibody prepared in Example 1 (produced using escitalopram antigen with bovine serum albumin as the carrier protein) into PBS buffer and dialyze for 24 - 48 hours, changing the buffer twice in the middle;

[0138] (2) Dilute the antibody recovered after dialysis to 0.2 mg / mL, add a certain amount of biotin - ester, react at room temperature for 40 min, terminate the reaction with a termination solution (1 M lysine or 1 M Tris) for 30 min, and then transfer it into a dialysis bag and dialyze for 24 - 48 hours.

[0139] (3) Recover the dialyzed biotinylated antibody, add an equal volume of glycerol, label it and store it at - 20 ± 5 °C.

[0140] 2. Preparation of Reagent M1

[0141] (1) Take 100 mg of magnetic particles and wash them 2 - 5 times with Tris - HCL buffer;

[0142] (2) Add 0.1 mg of biotinylated antibody at a ratio of 1 μg / mg and mix well at room temperature for 40 min;

[0143] (3) Add 1 mL of magnetic particle blocking solution at a ratio of 10 μL / mg. The blocking solution is a 3 g / L vitamin H solution, and react at room temperature for 40 min;

[0144] (4) Wash 2 - 5 times with Tris - HCL buffer and suspend, with a final concentration of 0.4 mg / mL.

[0145] 3. Acridinium - ester - labeled escitalopram antigen

[0146] (1) Take 0.1 mg of the escitalopram antigen prepared in Example 1 (with ovalbumin as the carrier protein), dilute the concentration to 1 mg / mL, add 4 μL of 5 mmol / l acridinium ester, react at room temperature in the dark for 2 hours, and then add 1 M lysine or 1 M Tris to terminate the reaction for 30 min.

[0147] (2) Filter and purify the acridine-labeled substance with a filter column, add an equal volume of glycerol, affix a label, and store it at -20 ± 5 °C.

[0148] 4. Preparation of Reagent R1:

[0149] Dilute the acridine-labeled substance of the above escitalopram antigen with PBS buffer at a volume ratio of 1:2000.

[0150] II. Escitalopram Detection Method

[0151] The method for detecting the concentration of escitalopram in a blood sample using the above kit is as follows:

[0152] (1) Mix the blood sample to be tested, Reagent M1, and Reagent R1 in a volume ratio of 1∶5∶10;

[0153] (2) Perform luminescence detection on the mixed solution in step (1) to obtain a detection signal value;

[0154] (3) Calculate the content of escitalopram in the sample to be tested by converting the signal value according to the standard curve.

[0155] Among them, the standard curve is drawn as follows: Use the escitalopram calibrator, Reagent R1, and Reagent M1 for chemiluminescence detection to obtain the detection signal value and draw the standard curve. In this example, the data points for drawing the standard curve are as follows:

[0156] Table 3 Data Points of the Standard Curve

[0157] Calibrator Concentration (ng / mL) RLU 0 1180620 4 912444 8 712781 20 402498 50 188780 200 45760

[0158] Thirty samples were respectively detected by the method of this example and the existing LC / MS clinical method, and the results are compared as shown in Table 4 and Figure 3 shown.

[0159] Table 4 Detection Results of Escitalopram Concentrations in 30 Samples

[0160]

[0161]

[0162] As can be seen from the above data, the escitalopram concentration results obtained by the detection method of this embodiment are close to those obtained by the existing LC / MS clinical method, indicating that the detection method of this embodiment has good accuracy.

[0163] Repeatability experiments were carried out on standard samples with escitalopram concentrations of 10 ng / mL and 100 ng / mL, and the results are shown in the following table:

[0164] Table 5 Results of repeatability experiments

[0165]

[0166] It can be seen that for samples with different concentrations, the CV values of the concentration detection results of 10 repeated experiments are 1.23%-2.52%, indicating that the detection method of this embodiment has good repeatability and can meet the requirements of clinical detection.

[0167] As can be seen from the above embodiments, the present invention constructs a new escitalopram hapten and antigen, and further prepares the corresponding antibody. Using the antigen and antibody provided by the present invention, a detection kit with good sensitivity and specificity for blood escitalopram can be prepared to realize the immunological detection of escitalopram. The detection method of the present invention has good accuracy and repeatability, can meet the requirements of clinical detection, and has good application prospects.

Claims

1. A compound of formula I, or a salt, stereoisomer, crystal form, or solvate thereof: Among them, n is selected from 2 - 10; R a Selected from amino or carboxyl groups.

2. The compound according to claim 1, or a salt thereof, or a stereoisomer thereof, or a crystal form thereof, or a solvate thereof, characterized in that, The structural formula of the compound of formula I is:

3. A process for preparing the compound according to claim 2, or a salt thereof, or a stereoisomer thereof, or a crystal form thereof, or a solvate thereof, characterized in that, The preparation method of the compound of formula I comprises the following steps: Step 1, reacting compound 1 with water to obtain compound 2; Step 2, reacting compound 2 with compound 3 to obtain compound 4; Step 3, hydrolyzing compound 4 to obtain the product.

4. According to the preparation method described in claim 3, characterized in that: In step 1, the reaction medium is a mixed solution of ethanol and water; and / or, in step 1, the pH condition of the reaction is 8 - 11; and / or, in step 1, the reaction temperature is 80 - 100 °C and the time is 12 - 18 h; and / or, in step 2, the reaction medium is DMF; and / or, in step 2, the reaction is carried out under the action of a coupling agent, and the coupling agent is selected from T3P; and / or, in step 2, the reaction is carried out under the action of a catalyst, and the catalyst is selected from DIEA; and / or, in step 2, the reaction temperature is 10 - 30 °C and the time is 1 - 3 h; and / or, in step 3, the hydrolysis medium is at least one of dichloromethane, DMSO, or chloroform; and / or, in step 3, the hydrolysis is carried out under the action of a catalyst, and the catalyst is selected from trifluoroacetic acid; and / or, in step 3, the hydrolysis temperature is 10 - 30 °C and the time is 1 - 3 h.

5. An escitalopram antigen, characterized in that, Its structure is as follows: Wherein, n is selected from 2 - 10; L is none or a linker; R1 is a carrier protein.

6. The escitalopram antigen according to claim 5, characterized in that: The value of n is 2, and the linker is 7. The escitalopram antigen according to claim 5, wherein: The carrier protein is selected from keyhole limpet hemocyanin, ovalbumin, or bovine serum albumin.

8. A method for preparing an escitalopram antigen, characterized in that, Comprises the following steps: coupling the escitalopram hapten with a carrier protein through a coupling agent to obtain an escitalopram antigen; the hapten is the compound described in claim 1, or a salt, stereoisomer, crystal form, or solvate thereof.

9. The preparation method of the escitalopram antigen according to claim 8, characterized in that, Comprises the following steps: Step A, mixing the escitalopram hapten with a coupling agent and reacting to obtain reaction solution A; Step B, reacting reaction solution A with a carrier protein to obtain reaction solution B; Step C, separating to obtain the escitalopram antigen.

10. According to the preparation method described in claim 9, characterized in that: In step A, the coupling agent is selected from EDC; and / or, in step A, a stabilizer is further added, and the stabilizer is selected from NHS; and / or, in step A, the reaction medium is DMF; and / or, in step A, the reaction conditions are light avoidance, the temperature is 10 - 30 °C, and the time is 1 - 6 h; and / or, in step B, the reaction medium is a PBS solution; and / or, in step B, the reaction conditions are light avoidance, the temperature is 10 - 30 °C, and the time is 6 - 12 h.

11. An escitalopram antibody, characterized in that, It is an antibody produced by the escitalopram antigen described in any one of claims 5 - 7.

12. The escitalopram antibody according to claim 11, wherein The escitalopram antibody is a monoclonal antibody or a polyclonal antibody.

13. The method for preparing an escitalopram antibody according to claim 11 or 12, characterized in that, Comprises the following steps: Step a, after immunizing an animal by multiple injections using the conventional Freund's adjuvant method with the escitalopram antigen, obtaining a serum containing specific antibodies, and fusing spleen cells after passing the titer test; Step b, screening positive fused cells and performing subcloning to obtain a monoclonal cell line; Step c: Produce ascites or perform cell culture production, followed by separation and purification to obtain the escitalopram antibody.

14. Use of the escitalopram antigen according to any one of claims 5 to 7 and / or the escitalopram antibody according to claim 11 or 12 in the immunoassay of escitalopram for non-diagnostic purposes.

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