A monoclonal antibody of citalopram, nucleic acid molecules, vectors, detection kits, detection reagents and application thereof

Abstract

The application belongs to the field of rapid detection of SSRIs drugs, and particularly relates to a monoclonal antibody of citalopram, a nucleic acid molecule, a carrier, a detection kit, a detection reagent and application thereof. The monoclonal antibody of citalopram comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, the light chain variable region comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3, the sequence of the heavy chain CDR1 is shown as SEQ ID NO. 1, the sequence of the heavy chain CDR2 is shown as SEQ ID NO. 2, and the sequence of the heavy chain CDR3 is shown as SEQ ID NO. 3. The monoclonal antibody of the application has high sensitivity for citalopram detection, the minimum detection limit is 0.06807 ng / mL, the quantitative detection range is 0.17567-4.49194 ng / mL, and the specificity is strong.

Description

Technical Field

[0001] This invention belongs to the field of rapid detection of SSRIs, specifically involving a monoclonal antibody, nucleic acid molecule, vector, detection kit, detection reagent and its application for citalopram. Background Technology

[0002] SSRIs (Selective Serotonin Reuptake Inhibitors) are a new class of antidepressants. Their scientific name is serotonin reuptake inhibitor, meaning selective serotonin reuptake inhibitor. SSRIs were developed and clinically tested in the 1980s. Currently, there are six commonly used SSRIs: fluoxetine, paroxetine, sertraline, fluvoxamine, citalopram, and escitalopram. These drugs selectively inhibit the reuptake of 5-HT at the presynaptic membrane, have little effect on NE, and almost no effect on dopamine (DA) reuptake.

[0003] Citalopram is one of the most selective SSRIs, with minimal or no effect on acetylcholine, histamine, GABA, oxaliplatin, opioid, and benzodiazepine receptors. It does not affect the cardiac conduction system or blood pressure, nor does it impair cognitive function or psychomotor function. It also has no effect on blood pressure, liver, or kidneys, making it suitable for long-term treatment and widely used. However, citalopram can cause numerous side effects, including nausea, dry mouth, dizziness, headache, drowsiness, shortened sleep duration, excessive sweating, decreased salivation, tremors, and diarrhea. Furthermore, it can cause hormonal imbalances, mania, tachycardia, and orthostatic hypotension, and there are even reports of it causing seizures. Therefore, monitoring citalopram levels in patients is necessary.

[0004] Currently, most techniques for detecting citalopram focus on high-performance liquid chromatography (HPLC). For example, prior art CN110646558A discloses a method for detecting escitalopram in blood, using HPLC-MS / MS to calculate the concentration of escitalopram by comparing the peak areas of the standard solution and the test solution. Prior art CN110455945A discloses a method and kit for detecting five psychotropic drugs and their main metabolites in blood, with the detection method being the same as the aforementioned prior art. All of these prior art techniques utilize chromatography to detect citalopram in blood, which is time-consuming and involves complex procedures. Furthermore, the components in the test sample are difficult to separate chromatographically, resulting in mixed absorption peaks and low detection sensitivity.

[0005] Meanwhile, immunological detection of citalopram is also employed. For example, prior art CN113105538B discloses a citalopram antigen and a citalopram colloidal gold test strip. The citalopram colloidal gold test strip uses citalopram antigen as a marker for the detection line and sheep / rat anti-IgG as a marker for the control line. The gold-labeled pad is prepared by spraying a citalopram monoclonal antibody-colloidal gold complex onto the conjugate pad. The detection limit of this invention's colloidal gold test strip is 500 ng / mL, and the color intensity of the detection line is negatively correlated with the concentration of citalopram. However, this technique directly conjugates the drug, which is not conducive to better generation of specific antibodies, resulting in lower detection sensitivity. Summary of the Invention

[0006] The purpose of this invention is to provide a highly sensitive monoclonal antibody, nucleic acid molecule, vector, detection kit, detection reagent, and its application for citalopram.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A monoclonal antibody of citalopram includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region includes light chain CDR1, light chain CDR2, and light chain CDR3. The sequence of heavy chain CDR1 is shown in SEQ ID NO.1, the sequence of heavy chain CDR2 is shown in SEQ ID NO.2, the sequence of heavy chain CDR3 is shown in SEQ ID NO.3, the sequence of light chain CDR1 is shown in SEQ ID NO.4, the sequence of light chain CDR2 is LMS, and the sequence of light chain CDR3 is shown in SEQ ID NO.5.

[0009] The sequence of SEQ ID NO.1 is: GFTFSSYG.

[0010] The sequence of SEQ ID NO.2 is: ISGGSFYI.

[0011] The sequence of SEQ ID NO.3 is: VRHGDYGTYVDSVLDYWGQGTSVTVSS.

[0012] The sequence of SEQ ID NO.4 is: KSLLYKDGKTY.

[0013] The sequence of the light chain CDR2 is: LMS.

[0014] The sequence of SEQ ID NO.5 is: QQLVESPFTFGSGTKLEIK.

[0015] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0016] In one preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 6; and / or the light chain variable region has the amino acid sequence shown in SEQ ID NO. 7.

[0017] The full-length VH region of the heavy chain variable region of the citalopram monoclonal antibody is:

[0018] EVKLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWVRQTPEKRLEWVATISGGSFYIYYLNSVKGRFTISSRDNAKNNLYLQMSSLRSEDTALYYCVRHGDYGTYVDSVLDYWGQGTSVTVSS (SEQ ID NO. 6).

[0019] The full-length VL region of the light chain of the citalopram monoclonal antibody is:

[0020] DIVITQDELSNPVTSGESVSMSCRSTKSLLYKDGKTYLNWFLQRPGQSPQLLIYLMSTRASGVSDRFSGSGSGTDFTLEISRVKAEDVGVYYCQQLVESPFTFGSGTKLEIK (SEQ ID NO. 7).

[0021] Based on the same inventive concept, the present invention also claims protection for a nucleic acid molecule that encodes a monoclonal antibody to the citalopram.

[0022] Based on the same inventive concept, the present invention also claims protection for the use of the monoclonal antibody of citalopram or the nucleic acid molecule in the preparation of a kit for assessing the level of citalopram.

[0023] Based on the same inventive concept, the present invention also claims protection for a carrier containing the aforementioned nucleic acid molecule.

[0024] Based on the same inventive concept, the present invention also claims a citalopram detection kit containing a monoclonal antibody against the citalopram or the nucleic acid molecule described herein.

[0025] Based on the same inventive concept, the present invention also claims protection for the application of the citalopram detection kit for assessing the level of citalopram in an individual's serum sample.

[0026] Based on the same inventive concept, the present invention also claims a citalopram detection reagent comprising a monoclonal antibody against citalopram, the nucleic acid molecule, the vector, or the citalopram detection kit.

[0027] In a preferred embodiment of the invention, the assessment is based on the detection of citalopram levels in the serum sample of the individual being tested.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] This invention provides a monoclonal antibody against citalopram, which has high sensitivity for the detection of citalopram, with a limit of detection (LOD) of 0.06807 ng / mL, an IC50 of 0.888 ng / mL, and a quantitative detection range of 0.17567~4.49194 ng / mL. It also has high specificity and no significant cross-reactivity with other similar drugs to citalopram. Attached Figure Description

[0030] Figure 1 This is a synthetic route diagram for the citalopram hapten CT-1; among which, Figure 1 In this context, 'a' represents the first step of the reaction. Figure 1 In this context, 'b' represents the second step reaction.

[0031] Figure 2 This is a synthetic route diagram for the citalopram hapten CT-2; among which, Figure 2 In this context, 'a' represents the first step of the reaction. Figure 2 In this context, 'b' represents the second step reaction.

[0032] Figure 3 The images show the UV scanning identification results of citalopram artificial antigen; in the images, a is the UV scanning identification result of CT-1-HAS, b is the UV scanning identification result of CT-2-OVA, and c is the UV scanning identification result of CT-1-LF.

[0033] Figure 4 This is a bar chart showing the subtype identification results of monoclonal antibodies.

[0034] Figure 5 This is an SDS-PAGE gel electrophoresis image of a monoclonal antibody.

[0035] Figure 6 This is a bar chart showing the results of different combinations of immunogens and coating agents.

[0036] Figure 7 This is a standard curve of indirect competitive ELISA for citalopram monoclonal antibody.

[0037] Figure 8 This is a scatter plot comparing the methodologies of the present invention and conventional methods. Detailed Implementation

[0038] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0039] Glossary of relevant technical terms

[0040] The term "antibody" broadly refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains (two heavy (H) chains and two light (L) chains). In a full-length antibody, each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs) and framework regions (FRs). Each VH and VL consists of three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, arranged from the amino terminus to the carboxyl terminus. The first, second, and third CDRs of the VH domain are typically denoted as CDR-H1, CDR-H2, and CDR-H3; similarly, the first, second, and third CDRs of the VL domain are typically denoted as CDR-L1, CDR-L2, and CDR-L3. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The amino acid sequences of the constant domains of the mouse and human Ig heavy and light chains are known in the art.

[0041] The term "monoclonal antibody" refers to antibodies derived from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical, except for a small number of potentially naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic determinant (epitope). Furthermore, unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen.

[0042] The term “CDR” refers to the complementarity-determining region within the variable domain sequence of an antibody. Three CDRs exist in each variable domain of the heavy and light chains, designated CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The term “CDR group” as used herein refers to a group of three CDRs present in a single variable domain capable of binding the antigen. The exact boundaries of these CDRs have been defined differently depending on the system. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (1987) and (1991)) provides not only a definitive residue numbering system applicable to any variable domain of an antibody but also precise residue boundaries defining the three CDRs.

[0043] The term "plasmid" refers to a circular double-stranded DNA molecule that can be linked to another DNA segment. Vectors can carry the target DNA sequence into host cells, replicate autonomously, and be expressed in the host cells.

[0044] The term "transformation" refers to any process by which exogenous DNA enters a host cell. Transformation can be performed under natural or artificial conditions using a variety of methods known in the art. Transformation can rely on any known method to introduce a exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell to be transformed and may include, but is not limited to, viral infection, electroporation, lipid transfection, and particle bombardment.

[0045] The reagents used in this invention are all commonly used reagents, purchased from Aladdin or Maclean's. For example, citalopram hydrobromide was purchased from Aladdin.

[0046] Example 1

[0047] Synthesis and Identification of Citalopram Hapten

[0048] 1. Synthesis of citalopram hapten CT-1

[0049] The synthetic route of the citalopram hapten CT-1 involves two steps, such as... Figure 1 As shown, where, Figure 1 In this context, 'a' represents the first step of the reaction. Figure 1 In this context, 'b' represents the second step of the reaction, and the specific steps are as follows:

[0050] One equivalent of citalopram hydrobromide was dissolved in an aqueous solution, extracted, and the organic phase was collected. The organic phase was then dissolved in 1,2-dichloroethane and 1.2 equivalents of 1-chloroethyl chloroformate and refluxed at high temperature. The mixture was evaporated to dryness, and methanol was added and refluxed under the same conditions. After the reaction was completed, 1.2 equivalents of triethylamine were added, followed by 1.2 equivalents of ethyl 4-bromobutyrate. Finally, the product was hydrolyzed with an aqueous sodium hydroxide solution to obtain CT-1.

[0051] 2. Identification of citalopram hapten CT-1

[0052] The 1H NMR spectrum of the citalopram hapten was as follows: 1H NMR (600 MHz, DMSO-d6) δ 11.68 (s, 1H), 7.72 (dd, J = 2.3, 1.1 Hz, 1H), 7.58 (dd, J = 7.9, 2.3 Hz, 1H), 7.41–7.21 (m, 3H), 7.20–6.97 (m, 2H), 4.90 (d, J = 1.1 Hz, 2H), 2.57 (td, J = 5.9, 1.5 Hz, 2H), 2.30 (t, J = 6.1 Hz, 2H), 2.28–2.21 (m, 5H), 2.13 (dt, J = 14.4, 7.8 Hz, 1H), 2.06 (dt, J = 14.7, 8.0 Hz, 1H), 1.67 (tt, J = 7.7, 5.9Hz, 2H), 1.58 - 1.46 (m, 4H), 1.40 - 1.31 (m, 2H).

[0053] The mass spectrometry results for the citalopram hapten are as follows: + ESI-MS + [M+H] + 397.5.

[0054] The mass spectrometry and NMR results show that the mass spectrometry results correspond to the molecular weight of the citalopram hapten, and the proton NMR spectra correspond to the proton NMR spectra of the citalopram hapten backbone structure. This indicates that the citalopram hapten, denoted as CT-1, was successfully prepared, and its structural formula is shown in formula (I). .

[0055] 3. Synthesis of citalopram hapten CT-2

[0056] The synthetic route of the citalopram hapten CT-2 consists of two steps, such as... Figure 2 As shown, where, Figure 2 In this context, 'a' represents the first step of the reaction. Figure 2 In this context, 'b' represents the second step of the reaction, and the specific steps are as follows:

[0057] One equivalent of citalopram hydrobromide was dissolved in an aqueous solution, extracted, and the organic phase was collected. The organic phase was then dissolved in 1,2-dichloroethane and 1.2 equivalents of 1-chloroethyl chloroformate and refluxed at high temperature. The mixture was evaporated to dryness, and methanol was added and refluxed under the same conditions. After the reaction was completed, 1.2 equivalents of triethylamine were added, followed by 1.2 equivalents of ethyl 6-bromohexanoate. Finally, the product was hydrolyzed with an aqueous sodium hydroxide solution to obtain CT-2.

[0058] 4. Identification of citalopram hapten CT-2

[0059] The 1H NMR spectrum of the citalopram hapten was as follows: 1H NMR (600 MHz, DMSO-d6) δ 11.68 (s, 1H), 7.72 (dd, J = 2.3, 1.1 Hz, 1H), 7.58 (dd, J = 7.9, 2.3 Hz, 1H), 7.41–7.21 (m, 3H), 7.20–6.97 (m, 2H), 4.90 (d, J = 1.1 Hz, 2H), 2.57 (td, J = 5.9, 1.5 Hz, 2H), 2.30 (t, J = 6.1 Hz, 2H), 2.28–2.21 (m, 5H), 2.13 (dt, J = 14.4, 7.8 Hz, 1H), 2.06 (dt, J = 14.7, 8.0 Hz, 1H), 1.67 (tt, J = 7.7, 5.9Hz, 2H), 1.58 - 1.46 (m, 4H), 1.40 - 1.31 (m, 2H).

[0060] The mass spectrometry results for the citalopram hapten are as follows: + ESI-MS + [M+H] + :425.5.

[0061] The mass spectrometry and NMR results show that the mass spectrometry results correspond to the molecular weight of the citalopram hapten, and the proton NMR spectra correspond to the proton NMR spectra of the citalopram hapten backbone structure. This indicates that the citalopram hapten, denoted as CT-2, was successfully prepared, and its structural formula is shown in formula (II). .

[0062] Example 2

[0063] Synthesis and Identification of Citalopram Artificial Antigen

[0064] I. Synthesis of Citalopram Artificial Antigen

[0065] 1. The CT-1 (3.156 mg, 0.012 mol) prepared in Example 1 was added to a brown reaction vial, along with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC (3.5 mg, 0.018 mol) and N-hydroxysuccinimide NHS (2.1 mg, 0.018 mmol), dissolved in 100 μL of N,N-dimethylformamide DMF, and stirred at room temperature for 3 h to obtain the hapten activation solution;

[0066] 2. Dissolve lactoferrin LF (10 mg, 0.00015 mol) in 1 mL of PBS buffer (0.01 mol / L, pH = 7.4) to obtain LF solution. Stir the LF solution and hapten activation solution overnight at 4°C.

[0067] 3. Collect the reaction solution, transfer it to the prepared dialysis bag, and then place it in 5L of 0.01M PBS buffer;

[0068] 4. Dialyze at 4℃ for 3 days, changing the solution with pre-cooled 0.01M PBS buffer every 8 hours;

[0069] 5. After dialysis purification, citalopram artificial antigen, denoted as CT-1-LF, was obtained. After concentration determination and ultraviolet spectroscopy identification, it was stored in a -20℃ freezer.

[0070] The only difference in the above method is that ovalbumin OVA is used instead of LF; and CT-2 is used instead of CT-1 to prepare citalopram artificial antigen, which is denoted as CT-2-OVA.

[0071] II. Identification of Citalopram Artificial Antigen

[0072] LF, OVA, CT-1, CT-2, CT-1-LF, and CT-2-OVA were scanned and identified using the ultraviolet full-wavelength method (200-350nm).

[0073] The identification results of CT-1-LF are as follows: Figure 3 As shown in c, by comparing the highest absorbance values ​​of each substance before and after coupling, it was found that the absorption curve of CT-1-LF is significantly different from that of the carrier protein LF. CT-1 has a strong absorption peak above 300 nm, while after coupling with LF, the absorption peak of CT-1-LF is similar to that of LF at 230 nm, but significantly higher than that of LF at 280 nm, and the curve of CT-1 relative to the hapten is significantly shifted.

[0074] Since all unreacted components have been removed by dialysis during the post-coupling dialysis process, the characteristic peaks of the coupling product are contributed by the protein-bound drug molecules, indicating that the reaction product is a complex of the carrier protein LF and CT-1.

[0075] The identification results of CT-2-OVA are as follows: Figure 3 As shown in b, similarly, the absorption curve of CT-2-OVA showed characteristic peaks that were different from those of OVA and CT-2, indicating that the reaction product was a complex of the carrier protein OVA and CT-2.

[0076] The above results demonstrate that the present invention successfully prepared citalopram artificial antigens CT-1-LF and CT-2-OVA, the structural formulas of which are shown in formulas (V) and (VI): ; .

[0077] Protein is either the carrier protein LF or OVA.

[0078] Using the same method, citalopram artificial antigens were prepared using carrier proteins OVA, LF, HAS, BSA, and CT-2 or CT-1, respectively. These antigens were then identified by ultraviolet full-wavelength scanning (200–350 nm), yielding CT-1-HAS, CT-1-BSA, CT-1-OVA, CT-2-HAS, CT-2-BSA, and CT-2-LF. The structural identification results of CT-1-HAS are as follows: Figure 3 As shown in 'a'.

[0079] Example 3

[0080] Preparation of citalopram monoclonal antibody

[0081] 1. Animal immunization

[0082] Using CT-1-LF prepared in Example 2 as the immunogen, CT-1-LF was diluted to 5 mg / mL with 0.01 mol / L PBS, then mixed with an equal volume of Freund's complete adjuvant and thoroughly emulsified. This mixture was then used to immunize 8-week-old female BALB / c mice (commercially available, such as those from Beijing Vital River Laboratory Animal Technology Co., Ltd.). For the first immunization, three female BALB / c mice were subcutaneously inoculated at multiple sites in the abdomen, with an antigen dose of 100 μg / mouse and 0.1 mL per mouse. A second immunization was performed 14 days later, emulsified with an equal volume of Freund's incomplete adjuvant and the immunogen, using the same dose as the first immunization. Three booster immunizations were administered. After immunization, tail serum was collected. Its titer and inhibition rate were determined. Mice with the best results were selected for a pulse immunization, with an antigen dose of 100 μg / mouse.

[0083] 2. Cell fusion

[0084] Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 1450) method, with the following specific steps:

[0085] a. After euthanizing mice by blood collection from the eyeballs, immediately disinfect them in 75% alcohol for about 5 minutes. Aseptically remove the spleen of the mice, grind it moderately with the rubber tip of a syringe and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect the suspension, centrifuge (1000 rpm, 7 minutes), wash the spleen cells three times with RPMI-1640 medium, and after the last centrifugation, dilute the spleen cells to a certain volume, count them, and set them aside for later use.

[0086] b. Collect SP2 / 0 cells;

[0087] c. Mix the two cell lines at a ratio of 1:5, bring the volume to 20 mL, and centrifuge (1000 rpm / min, 7 min). Discard the supernatant.

[0088] Remove the centrifuge tube and begin fusion by rotating it at a medium speed of 60°. After fusion, centrifuge the cell culture (1000 rpm / min, 7 min). Remove the 200 mL of HAT medium that has been incubated and place it in a clean bench for later use.

[0089] d. After centrifuging the hybridoma cells, discard the supernatant, aspirate 2 mL of HAT medium into a centrifuge tube, gently and slowly pipette to mix evenly, then transfer the cell solution in the centrifuge tube to 200 mL of HAT medium, gently shake the medium to disperse the cells evenly, and then plate them.

[0090] 3. Cell selection and cell line establishment:

[0091] On day 3 of cell fusion, the fused cells were partially replaced with RPMI-1640 screening medium (Gbico). On day 5, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 2% 50×HT. On day 7, the cell supernatant was collected for screening.

[0092] The screening process involves two steps: First, positive cell wells are selected using the ic-ELISA method. Positive cell wells are then selected based on the serum titer and inhibition rate obtained through the indirect competitive ELISA method.

[0093] The specific operating steps are as follows:

[0094] 1. Dilute the original CT-2-OVA coating solution to 1000 ng / mL with coating buffer (0.05 M carbonate buffer, pH 9.6), add 100 μL / well to coat a 96-well microplate, incubate overnight at 4°C, discard the coating buffer, and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)).

[0095] 2. Add 120 μL of blocking buffer (PBST containing 7% skim milk powder) to each well, block at 37°C for 1 hour, discard the blocking buffer, plate, and dry in a drying oven at 37°C for later use.

[0096] 3. On day 7, cell supernatants from different culture wells were collected and diluted with PBST at volume ratios of 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:25600, and 1:512000. Blank control wells were also set up (using PBST instead). 1 mg / mL citalopram standard was diluted 1000 times with PBST to obtain a 1 μg / mL citalopram standard dilution.

[0097] 4. Potency column setup: First, add 50 μL of PBST to each well, then add 50 μL of cell supernatant taken from different culture wells at different dilution ratios on day 7 to each well, and finally add 50 μL of PBST to the last well instead of antibody.

[0098] 5. Incubate at 37℃ for 40 minutes, wash 5 times, and then plate.

[0099] 6. Add goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST), incubate at 37℃ for 30 min, wash 5 times, and plate.

[0100] 7. Add colorimetric reagent (TMB single-component colorimetric reagent from Nanjing Detai Bioengineering Co., Ltd., product number DTT0298) and incubate at 37℃ for 10 min for color development;

[0101] 8. Add 10% v / v H2SO4 to terminate the reaction and read the OD value at 450 nm; calculate the potency, which is OD. 450 The antibody dilution factor corresponding to approximately 1.0 was used to select 96 cell wells with high titers as positive cell wells.

[0102] In the experiments of this invention, each mouse can obtain approximately 10 8 After fusion, the cell viability rate is approximately 10%. -5 Only about 1000 fused cells can survive. Furthermore, the antibodies secreted by the hybridoma are highly random. This makes the screening process highly random and uncertain. Through screening, 96 positive cell wells containing monoclonal cell clusters capable of producing antibodies with high titers were obtained.

[0103] The second step uses citalopram as a standard and employs ic-ELISA to determine the inhibitory effect on the positive cells screened in the first step. Cell wells showing good inhibition of the citalopram standard are selected, and subcloning is performed using the limiting dilution method. Seven days later, the cells are detected using the same method as in the first step. The specific steps are as follows:

[0104] 1. Dilute the original CT-2-OVA coating solution to 1000 ng / mL with coating buffer (0.05 M carbonate buffer, pH 9.6), add 100 μL / well to coat a 96-well microplate, incubate overnight at 4°C, discard the coating buffer, and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)).

[0105] 2. Add 120 μL of blocking buffer (PBST containing 7% skim milk powder) to each well, block at 37°C for 1 hour, discard the blocking buffer, plate, and dry in a drying oven at 37°C for later use.

[0106] 3. Dilute the cell supernatant from the positive cell wells selected in step 1 with PBST at volume ratios of 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:25600, and 1:512000. Simultaneously, set up blank control wells (using PBST instead). Dilute 1 mg / mL citalopram standard 1000 times with PBST to obtain a 1 μg / mL citalopram standard dilution.

[0107] 4. Potency column setup: First, add 50 μL of PBST to each well, then add 50 μL of cell supernatant from the positive cells selected in the first step at different dilution ratios to each well, and finally add 50 μL of PBST to the last well instead of the supernatant.

[0108] 5. Inhibition column setup: First, add 50 μL of citalopram standard dilution to each well, then add 50 μL of cell supernatant from the positive cells selected in the first step at different dilution ratios to each well, and finally add 50 μL of PBST to the last well instead of the supernatant.

[0109] 6. Incubate at 37℃ for 40 minutes, wash 5 times, and then plate.

[0110] 7. Add goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST), incubate at 37℃ for 30 min, wash 5 times, and plate.

[0111] 8. Add the color developing solution and incubate at 37°C for 10 minutes;

[0112] 9. Add 10% v / v H2SO4 to terminate the reaction and read the OD value at 450 nm; calculate the titer and inhibition rate. The titer is OD. 450 The antibody dilution factor corresponding to approximately 1.0 is: inhibition rate = (OD value of titer - OD value of inhibition) / OD value of inhibition × 100%.

[0113] After three rounds of subcloning and ic-ELISA testing, five monoclonal cell lines were finally obtained, and the data are shown below:

[0114] Through repeated screening, five monoclonal cell clusters that could stably produce antibodies were obtained.

[0115] The ability of the supernatants from these five positive clones to recognize drugs was analyzed after serial dilution. The results showed that, after a 512k-fold dilution, the antibody produced by clone 12H still exhibited the highest sensitivity for drug recognition. While other clones could also recognize drugs (i.e., produce monoclonal antibodies), the KL antibodies they produced had significantly lower sensitivity than those produced by clone 12H. Therefore, the antibody derived from clone 12H was selected for subsequent sequencing.

[0116] 4. Preparation and identification of citalopram monoclonal antibody

[0117] BALB / c mice aged 8-10 weeks were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6 Citalopram hybridoma cells were used, and ascites fluid was collected starting from day 7. The ascites fluid was then purified for antibody purification using immunoaffinity chromatography.

[0118] The purification steps for monoclonal antibodies are as follows:

[0119] All solutions used for purification were filtered through a 0.22 μm pore size membrane and sonicated for 30 min (including ultrapure water, equilibration buffer PBS, and elution buffer Gly-HCl) to remove insoluble solids and air bubbles. The collected ascites fluid was diluted 1-fold with PBS, passed through a 0.45 μm pore size nylon microporous membrane to remove impurities, and then purified using an AKA protein purifier. The purification steps are as follows:

[0120] (1) System and column equilibration: Replace the protective solution in the column with equilibration buffer PBS and wash the column;

[0121] (2) Sample loading: Flow the ascites fluid through the proteinG affinity chromatography column at a rate of 1 mL / min until the UV detection line and Cond detection line return to the baseline and remain stable;

[0122] (3) Elution: Wash the protein G affinity chromatography column with 0.1 mol / L Gly-HCl elution buffer at pH 2.7 at a rate of 2 mL / min to elute the antibody adsorbed on the column. Adjust the pH of the collected liquid to about 7 with 1 mol / L Tris-HCl (pH 9.0).

[0123] (4) Dialysis: After pH adjustment, the solution was dialyzed in PBS solution at 4℃ for two days; the molecular weight cutoff for dialysis was 5000kda, and high-purity citalopram monoclonal antibody was finally obtained.

[0124] (5) Identification of monoclonal antibodies

[0125] (5.1) Determination of monoclonal antibody purity: Mouse antibody subtypes were classified as IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3. This experiment used a commercially available antibody subtype identification kit (Sigma) for antibody subtype identification. The results are as follows: Figure 4 As shown, the results indicate that the antibody subtype is IgG1.

[0126] (5.2) Determination of monoclonal antibody purity: The purity of the purified antibody was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using the PAGE gel rapid preparation kit from Yamei Biotechnology. The steps are as follows: Take 8 μL of antibody and ascites fluid, mix them with 2 μL of 5×SDS loading buffer, heat at 100℃ for 10 min, load 10 μL of sample and 5 μL of standard protein marker, perform SDS-PAGE gel electrophoresis, stain with Coomassie brilliant blue staining solution, destain overnight on a shaker, and then image using a gel imaging system. The SDS-PAGE gel electrophoresis results are shown below. Figure 5 The theoretical molecular weight of IgG via non-reducing electrophoresis is 150 kDa. Reducing electrophoresis separates it into two 50 kDa heavy chains and a 25 kDa light chain. The electrophoresis results match the theoretical value, indicating successful purification and successful preparation of the monoclonal antibody. The antibody derived from clone 12H was sent to Nanjing Zhongding Biotechnology Co., Ltd. for sequencing, and the results are shown in Table 2 below.

[0127] The full length of the heavy chain variable region VH of the monoclonal antibody is: EVKLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWVRQTPEKRLEWVATISGGSFYIYYLNSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCVRHGDYGTYVDSVLDYWGQGTSVTVSS (SEQ ID NO.6).

[0128] The full length of the variable region (VL) of the light chain of a monoclonal antibody is:

[0129] DIVITQDELSNPVTSGESVSMSCRSTKSLLYKDGKTYLNWFLQRPGQSPQLLIYLMSTRASGVSDRFSGSGSGTDFTLEISRVKAEDVGVYYCQQLVESPFTFGSGTKLEIK (SEQ ID NO. 7).

[0130] The purified monoclonal antibody was stored at -20°C.

[0131] 5. Sensitivity evaluation of citalopram monoclonal antibody (indirect competitive ELISA method)

[0132] Using CT-2-OVA prepared in Example 2 as the coating agent, it was diluted with coating buffer (0.05M carbonate buffer, pH 9.6) to a concentration of 1µg / mL, and 100μL / well was spread onto a 96-well microplate and incubated at 4°C overnight (12h).

[0133] Discard the coating solution, wash twice with PBST (0.01M PBS, 0.06% v / v Tween-20), and blot dry; add 120μL of blocking solution (PBST containing 6% skim milk powder) to each well, and block at 37℃ for 1h; discard the blocking solution, blot the plate, dry at 37℃, and then pack it in a resealable bag for later use.

[0134] The antibody derived from clone 12H was diluted with PBST at a volume ratio of 1:128000; the citalopram standard was diluted with PBS to different concentrations, resulting in citalopram standard dilutions with concentrations of 1000 ng / mL, 250 ng / mL, 62.5 ng / mL, 15.63 ng / mL, 3.9 ng / mL, 0.98 ng / mL, 0.244 ng / mL, 0.06 ng / mL, 0.015 ng / mL, and 0 ng / mL.

[0135] Add 50 μL / well of citalopram standard dilution buffer at different concentrations (three sets in parallel), then add 50 μL / well of diluted citalopram monoclonal antibody (antibody derived from clone 12H), incubate at 37°C for 40 min, wash five times, and pat dry;

[0136] Add 100 μL / well of goat anti-mouse secondary antibody-HRP (HS201, HRP-labeled goat anti-mouse IgG (H+L) antibody, Beijing TransGen Biotech Co., Ltd., diluted 5000 times with PBST), incubate at 37℃ for 30 min, wash five times, and pat dry;

[0137] Add 100 μL / well of colorimetric reagent and develop for 10 min;

[0138] The reaction was terminated by adding 50 μL of 10% H2SO4 solution, and the OD value was read at 450 nm.

[0139] ELISA standard curve plotting: Plot B / B0 as the ordinate (B represents the absorbance OD of different concentrations of citalopram standards). 450 B0 is the absorbance value (OD) of the blank control well. 450 A standard curve was obtained by fitting the logarithm of the concentration of citalopram standard to the logarithm on the x-axis using the Logistic function, as shown below. Figure 7 As shown, the formula for the standard curve is obtained. Regression equation: Y = 0.07522 + [0.86439 / (1 + (x / 0.08017)^0.88)]. The limit of detection (LOD) is 0.06807 ng / mL, and the half-inhibitory concentration (IC50) is... 50 The concentration was 0.888 ng / mL, and the quantitative detection range was 0.17567~4.49194 ng / mL.

[0140] On the other hand, ultraviolet spectroscopy and immunological structure data also proved that the present invention successfully prepared haptens and antigens.

[0141] 6. Specificity evaluation of citalopram monoclonal antibody

[0142] Paroxetine, sertraline, fluoxetine and other drugs are analogues. The specificity of the citalopram monoclonal antibody (antibody derived from clone 12H) prepared in this embodiment was evaluated by cross-reactivity experiments.

[0143] Following the sensitivity evaluation method described above, the only difference is that citalopram standard is replaced with other drug standards, and detection is performed at the same dilution factor to obtain the IC50 of each structural analog. 50 value.

[0144] The cross-reactivity rate (CR) of citalopram is calculated using the following formula: CR (%) = IC 50 (Citalopram) / IC 50 (Similar drugs) × 100%, the smaller the cross-reactivity rate, the stronger the specificity.

[0145] Note: NR indicates no reaction, meaning the antibody does not recognize the analogue.

[0146] As shown in Table 3, the cross-reactivity of the citalopram monoclonal antibody of the present invention with citalopram is 100%, IC50... 50The concentration was 0.888 ng / mL, with no cross-contamination with paroxetine, fluoxetine, and sertraline. This indicates that the antibody used to detect citalopram has high recognition ability and specificity for citalopram, effectively eliminating interference from citalopram analogs such as paroxetine, fluoxetine, and sertraline, and can be specifically used for the detection of citalopram.

[0147] The above results indicate that the citalopram monoclonal antibody prepared in this invention exhibits excellent detection performance for citalopram, with high sensitivity and strong specificity.

[0148] Example 4

[0149] Combination of immunogen and coating antigen

[0150] The specific steps of the synthetic route for citalopram artificial antigen are as follows:

[0151] 1. Dissolve 1 equivalent of citalopram hydrobromide in an aqueous solution, extract, collect the organic phase, dissolve the organic phase in 1,2-dichloroethane and 1.2 equivalents of 1-chloroethyl chloroformate, reflux at high temperature, evaporate to dryness, add methanol and reflux under the same conditions, after which 1.2 equivalents of triethylamine are added, followed by 1.2 equivalents of ethyl 2-bromoacetate or ethyl 4-bromophenylacetate. Finally, hydrolyze the product with an aqueous sodium hydroxide solution to obtain CT-2C or CT-benzene ring.

[0152] The structure of CT-2C is as follows: ;

[0153] The structure of the CT-benzene ring is as follows: .

[0154] 2. Citalopram and 2,2,2-trichloroethyl chloroformate were dissolved in an organic solvent at a molar ratio of 1:1.8, with a solute mass to organic solvent volume ratio of 0.6 g:5 ml. After reflux for 8 hours, the mixture was separated by chromatography. The solvent was then evaporated to obtain the citalopram hapten, whose structural formula is as follows: It was named CT-C.

[0155] The CT-C, CT-2C, or CT-benzene ring (0.012 mol) prepared in step 1 above was added to a brown reaction vial, along with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC (3.5 mg, 0.018 mol) and N-hydroxysuccinimide NHS (2.1 mg, 0.018 mmol), dissolved in 100 μL of N,N-dimethylformamide DMF, and stirred at room temperature for 3 h to obtain the hapten activation solution.

[0156] Lactoferrin LF (10 mg, 0.00015 mol) was dissolved in 1 mL of PBS buffer (0.01 mol / L, pH = 7.4) to obtain LF solution. The LF solution and hapten activation solution were stirred overnight at 4 °C.

[0157] Collect the reaction solution, transfer it to a prepared dialysis bag, and then place it in 5L of 0.01M PBS buffer;

[0158] Dialysis was performed at 4°C for 3 days, with the solution changed every 8 hours using pre-cooled 0.01M PBS buffer.

[0159] After dialysis purification, citalopram artificial antigens CT-C-LF, CT-2C-LF, and CT-benzene ring-LF were obtained. After concentration determination and ultraviolet spectroscopy identification, they were stored in a -20°C freezer.

[0160] The only difference from the above method is that ovalbumin OVA, bovine serum albumin BSA, or human serum albumin HSA are used instead of LF to obtain CT-C-HAS, CT-2C-HAS, CT-benzene ring-HAS, CT-C-BSA, CT-2C-BSA, CT-2C-OVA, CT-C-OVA, CT-benzene ring-BSA, and CT-benzene ring-OVA.

[0161] Using the prepared citalopram artificial antigens CT-2-OVA, CT-2C-HAS, CT-2C-LF, CT-1-HAS, CT-1-LF, CT-2-HAS, CT-2-LF, CT-benzene ring-HAS, and CT-benzene ring-LF as coating agents, and CT-1-LF, CT-2C-HAS, CT-2C-BSA, CT-2C-LF, CT-2C-OVA, CT-1-HAS, CT-1-BSA, and CT-1-L as coating agents... F, CT-1-OVA, CT-2-HAS, CT-2-BSA, CT-2-LF, CT-2-OVA, CT-benzene ring-HAS, CT-benzene ring-BSA, CT-benzene ring-LF, CT-benzene ring-OVA, and CT-C-LF were used as immunogens to prepare the most effective citalopram monoclonal antibody. The antibody preparation method was the same as in Example 3. The combined effect of different immunogens and coating agents was evaluated by the serum titer and inhibition rate obtained by the indirect competitive ELISA method.

[0162] The specific operating steps are as follows:

[0163] 1. Dilute the coating agent to a concentration of 1000 ng / mL with coating buffer (0.05 M carbonate buffer, pH 9.6), add 100 μL / well to coat a 96-well microplate, incubate overnight at 4°C, discard the coating buffer, and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)).

[0164] 2. Add 120 μL of blocking buffer (PBST containing 7% skim milk powder) to each well, block at 37°C for 1 hour, discard the blocking buffer, plate, and dry in a drying oven at 37°C for later use.

[0165] 3. The citalopram monoclonal antibodies with the best efficacy prepared from different immunogens were diluted with PBST at volume ratios of 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:25600, and 1:512000. Blank control wells (replaced with PBST) were also set up. The 1 mg / mL citalopram standard was diluted 1000 times with PBST to obtain a 1 μg / mL citalopram standard dilution.

[0166] 4. Titer column setup: First, add 50 μL of PBST to each well, then add 50 μL of citalopram monoclonal antibody at different dilutions to each well, and finally add 50 μL of PBST to the last well instead of the antibody.

[0167] 5. Inhibition column setup: First, add 50 μL of citalopram standard dilution buffer to each well, then add 50 μL of citalopram monoclonal antibody at different dilution ratios to each well, and finally add 50 μL of PBST to the last well instead of antibody.

[0168] 6. Incubate at 37℃ for 40 minutes, wash 5 times, and then plate.

[0169] 7. Add goat anti-mouse secondary antibody-HRP (HS201, HRP-labeled goat anti-mouse IgG (H+L) antibody, Beijing TransGen Biotech Co., Ltd., diluted 5000 times with PBST), incubate at 37℃ for 30 min, wash 5 times, and plate.

[0170] 8. Add the color developing solution and incubate at 37°C for 10 minutes;

[0171] 9. Add 10% v / v H2SO4 to terminate the reaction and read the OD value at 450 nm; calculate the titer and inhibition rate. The titer is OD. 450 The antibody dilution factor corresponding to approximately 1.0 is: inhibition rate = (OD value of titer - OD value of inhibition) / OD value of inhibition × 100%.

[0172] The results of different combinations of immunogens and coating antigens are shown in Tables 4 and 5. Figure 6 As shown.

[0173] As shown in Tables 4 and 5, the monoclonal antibody prepared using the artificial antigen CT-1-LF of citalopram as an immunogen and T-2-OVA is the optimal combination of immunogen and coating agent. Under this combination, the monoclonal antibody can not only specifically recognize the target analyte citalopram, but also has good antibody sensitivity and can be used to specifically recognize the target analyte citalopram.

[0174] Example 5

[0175] An ELISA immunoassay for detecting citalopram

[0176] 1. Composition

[0177] (1) The enzyme-labeled plate coated with the coating agent is prepared by the following method:

[0178] The CT-2-OVA prepared in Example 2 was used as the coating agent. It was diluted to 500 ng / mL with the coating stock solution (0.05 M carbonate buffer, pH 9.6). 100 μL of the solution was added to coat 96-well microplates and incubated overnight at 4°C in the dark. The liquid in the wells was poured off, and the plates were washed twice with the washing solution in this kit for 30 seconds each time. The plates were then patted dry. Then, the blocking solution in this kit was added at 120 μL / well and incubated at 37°C in the dark for 1 hour. The liquid in the wells was poured off, and the plates were patted dry. After drying, the plates were vacuum sealed with aluminum foil for storage.

[0179] (2) Standards: Citalopram standard dilutions of 12 different concentrations: 100 ng / mL, 250 ng / mL, 62.5 ng / mL, 15.63 ng / mL, 3.9 ng / mL, 0.98 ng / mL, 0.244 ng / mL, 0.06 ng / mL, 0.015 ng / mL, 0.00375 ng / mL, 0.0009 ng / mL and 0 ng / mL.

[0180] (3) Antibody: Monoclonal antibody prepared in Example 3 (antibody derived from clone 12H);

[0181] (4) Enzyme-labeled secondary antibody: Horseradish peroxidase-labeled goat anti-mouse secondary antibody (HS201, HRP-labeled goat anti-mouse IgG (H+L) antibody, Beijing TransGen Biotech Co., Ltd.);

[0182] (5) Substrate colorimetric solution: composed of solution A and solution B, where solution A is urea peroxide and solution B is tetramethylbenzidine;

[0183] (6) Termination solution: 2 mol / L H2SO4;

[0184] (7) Washing solution: pH value is 7.4, containing 0.8% Tween-20 by volume, 0.02% sodium azide preservative by mass, and 0.2 mol / L phosphate buffer; dilute the washing solution 20 times with water before use (i.e., add 1 part washing solution to 19 parts water, prepare fresh before use) to obtain the washing solution working solution;

[0185] (8) Diluent: 0.2 mol / L phosphate buffer; dilute the diluent 20 times with water before use (i.e., add 1 part of diluent to 19 parts of water, prepare fresh before use) to obtain the working solution of the diluent;

[0186] (9) Blocking solution: 0.2 mol / L phosphate buffer with pH 7.3 and containing 2% casein.

[0187] 2. Instructions for use

[0188] (1) Sample testing

[0189] Twenty serum samples from clinical cases received from Xiangya Third Hospital of Hunan Province and the corresponding wells of the standard in this kit were numbered sequentially. Three parallel wells were prepared for each sample and standard, and the positions of the standard and sample wells were recorded. The antibody was diluted with the working solution at a volume ratio of 1:40 (i.e., 1 part antibody to 40 parts working solution, prepared fresh for each use) to obtain the antibody working solution. The enzyme-labeled secondary antibody was diluted with the working solution at a volume ratio of 1:10 (i.e., 1 part enzyme-labeled secondary antibody to 10 parts working solution, prepared fresh for each use) to obtain the enzyme-labeled secondary antibody working solution.

[0190] Add 50 μL of standard or sample to the corresponding well, then add 50 μL of antibody working solution to the corresponding well, gently shake to mix, cover with a cover plate and incubate at 25°C in the dark for 40 min.

[0191] Shake off the liquid in the well and add 250 μL of washing working solution per well. Wash thoroughly 4-5 times, with 10-second intervals between each wash. Discard the washing working solution in the well and pat dry with absorbent paper (any air bubbles not removed after patting can be punctured with an unused pipette tip).

[0192] Add 100 μL of enzyme-labeled secondary antibody working solution to the corresponding microwell, gently shake to mix, cover with a cover plate and incubate at 25°C in the dark for 30 min.

[0193] Shake off the liquid in the well and add 250 μL of washing working solution per well. Wash thoroughly 4-5 times, with 10-second intervals between each wash. Discard the washing working solution in the well and pat dry with absorbent paper (any air bubbles not removed after patting can be punctured with an unused pipette tip).

[0194] Add 50 μL of substrate development solution A per well, then add 50 μL of substrate development solution B per well, gently shake to mix, cover with a cover plate and incubate at 25°C in the dark for 10 min.

[0195] Add 50 μL of stop solution per well, gently shake to mix, set the microplate reader to 450 nm, and measure the OD value of each well.

[0196] (2) Drawing the standard curve

[0197] Plot B / B0 as the ordinate (B represents the absorbance OD of standards at different concentrations). 450 B0 is the absorbance value (OD) of the blank control well. 450 The logarithm of the standard concentration was plotted on the x-axis, and a logistic function was used for curve fitting. The regression equation was: Y = 0.07522 + [0.86439 / (1 + (x / 0.08017)^0.88)]. The limit of detection (LOD) was 0.06807 ng / mL, and the half-maximal inhibitory concentration (IC50) was [missing value]. 50 The concentration was 0.888 ng / mL, and the quantitative detection range was 0.17567~4.49194 ng / mL.

[0198] (3) Calculation of sample concentration

[0199] OD of the sample 450 Substituting the average value into the formula of the standard curve above, we obtain the concentration of the sample. Multiplying this by the corresponding dilution factor gives the actual concentration of citalopram in the sample.

[0200] Example 6

[0201] A chemiluminescent immunoassay method for detecting citalopram

[0202] 1. Composition

[0203] (1) The solid support coated with the coated element is prepared by the following method:

[0204] The CT-2-OVA prepared in Example 2 was used as the coating agent. It was diluted to 500 ng / mL with the coating stock solution (0.05 M carbonate buffer, pH 9.6). The CT-2-OVA was added at a rate of 100 μL / well to coat a 96-well high-binding white chemiluminescent microplate. The plate was incubated overnight at 4°C in the dark. The liquid in the wells was discarded, and the plate was washed twice with the washing solution provided in this kit for 30 seconds each time. The plate was then patted dry. The blocking solution provided in this kit was then added at a rate of 120 μL / well, and the plate was incubated at 37°C in the dark for 1 hour. The liquid in the wells was discarded, and the plate was patted dry. After drying, the plate was vacuum sealed with an aluminum foil for storage.

[0205] (2) Standard products:

[0206] Twelve different concentrations of citalopram standards were prepared, with concentrations of 100 ng / mL, 250 ng / mL, 62.5 ng / mL, 15.63 ng / mL, 3.9 ng / mL, 0.98 ng / mL, 0.244 ng / mL, 0.06 ng / mL, 0.015 ng / mL, 0.00375 ng / mL, 0.0009 ng / mL, and 0 ng / mL.

[0207] (3) Antibody

[0208] Antibody 1 is the monoclonal antibody (antibody derived from clone 12H) prepared in Example 3 of this invention.

[0209] (4) Chemiluminescent labeled secondary antibody:

[0210] Acridinium Ester (AE)-labeled goat anti-mouse secondary antibody (Tianjin Sanjian Biotechnology Co., Ltd.);

[0211] (5) Chemiluminescent substrate solution:

[0212] It includes trigger solution I and trigger solution II, wherein trigger solution I is a hydrogen peroxide solution and trigger solution II is an alkaline trigger solution;

[0213] (6) Washing solution:

[0214] The pH value is 7.4, and it contains 0.8% Tween-20 by volume, 0.02% sodium azide preservative by mass, and 0.2 mol / L phosphate buffer. Before use, dilute the washing solution 20 times with water (i.e., add 1 part washing solution to 19 parts water, prepare fresh before use) to obtain the washing solution working solution.

[0215] (7) Diluent:

[0216] 0.2 mol / L phosphate buffer; dilute the diluent 20 times with water before use (i.e., add 1 part diluent to 19 parts water, prepare fresh before use) to obtain the working solution of the diluent;

[0217] (8) Sealing liquid:

[0218] A 0.2 mol / L phosphate buffer solution with a pH of 7.3 containing 2% casein.

[0219] 2. Instructions for use

[0220] (1) Sample testing

[0221] The sample inclusion criteria are:

[0222] 1. Age ≥ 18 years old, gender not limited;

[0223] 2. Participate in this study voluntarily and sign a written informed consent form;

[0224] 3. Able to provide a sufficient amount of venous blood sample (≥3mL);

[0225] 4. Meet any of the following conditions:

[0226] It is confirmed that citalopram was taken for ≥5 half-lives and the last dose was traceable;

[0227] The healthy control group had not used any SSRIs in the past month.

[0228] 5. Sample collection, processing, and preservation comply with laboratory standard operating procedures.

[0229] The sample exclusion criteria are:

[0230] 1. Those who have not signed or have withdrawn their informed consent form;

[0231] 2. The sample contains severe hemolysis, lipemia, or jaundice, which may affect the test results;

[0232] 3. Patients with severe liver or kidney failure (ALT / AST > 3 times the upper limit of normal or eGFR < 30 mL / min); pregnant or lactating women;

[0233] 4. Combining structurally similar psychotropic drugs that may seriously interfere with test results (unless required by the study design);

[0234] 5. Samples are not stored properly or the preset storage time has been exceeded.

[0235] The steps for sample collection and processing are as follows:

[0236] 1. Collect 3-5 mL of venous blood;

[0237] 2. Centrifuge at 3000 rpm for 10 min to separate serum;

[0238] 3. After aliquoting, serum should be stored at -20℃ or -80℃;

[0239] 4. All samples were anonymized and coded for use only in this study.

[0240] Twenty clinical samples received from Xiangya Third Hospital of Hunan Province and the corresponding wells of the standard of this kit were numbered sequentially. Two parallel wells were prepared for each sample and the standard, and the positions of the standard wells and sample wells were recorded.

[0241] Dilute the antibody (antibody 1) with the diluent at a volume ratio of 1:40 (i.e., add 1 part antibody to 40 parts diluent, prepare fresh before use) to obtain the antibody working solution; dilute the chemiluminescent labeled secondary antibody with the diluent at a volume ratio of 1:10 (i.e., add 1 part chemiluminescent labeled secondary antibody to 10 parts diluent, prepare fresh before use) to obtain the chemiluminescent labeled secondary antibody working solution.

[0242] Add 50 μL of standard or sample to the corresponding microwell, then add 50 μL of antibody working solution, gently shake to mix, seal with a cover plate membrane, and incubate at 25°C in the dark for 40 min.

[0243] Shake off the liquid in the well, add 250 μL of washing solution / well, and wash thoroughly 4 to 5 times with 10 seconds between each wash. Discard the washing solution and working solution in the well and pat dry with absorbent paper.

[0244] Add 100 μL of chemiluminescent labeled secondary antibody working solution to the corresponding microwell, gently shake to mix, seal the plate with a cover film, and place it in a 25°C dark environment for 30 min to react.

[0245] Shake off the liquid in the well, add 250 μL of washing solution / well, and wash thoroughly 4 to 5 times with 10 seconds between each wash. Discard the washing solution and working solution in the well and pat dry with absorbent paper.

[0246] Add chemiluminescent substrate trigger solution I and trigger solution II (100 μL / well) to each well in sequence, and immediately place the well in a chemiluminescence detector to measure the relative luminescence intensity (RLU) of each well.

[0247] (2) Drawing the standard curve

[0248] Plotting B / B0 as the ordinate (B being the luminescence intensity value RLU of different concentration standards, and B0 being the luminescence intensity value RLU of the blank control well), and the logarithm of the standard concentration as the abscissa, a standard curve was plotted using the Logistic function: y = 4210.620 + (601889.190 - (4210.620)) / 1 + (X / 6.394)^1.264, R 2 =0.999. The data is as follows:

[0249] (3) Calculation of the lowest detection limit

[0250] Following the operating instructions CLSI EP05-A3, the limit of detection was determined by measuring the luminescence values ​​of 20 negative serum samples. The mean value was calculated, and then +2SD was added to the standard curve to obtain the limit of detection. The data are as follows.

[0251] (4) Twenty samples were tested using both the method described in this embodiment and the existing LC / MS clinical method (Li Jiong, Jia Yanbo, Qiu Hongyu, et al. Determination of 8 illegally added antidepressant chemical components in sedative traditional Chinese medicine and health food by UPLC-MS / MS [J]. Chinese Journal of Modern Applied Pharmacy, 2019, 36(08):956-960.). The results are shown in the table.

[0252] A correlation scatter plot was created using the test results of 20 samples obtained through this embodiment and existing LC / MS clinical methods. The results are as follows: Figure 8 As shown in the figure. The results indicate that the method in the example is basically consistent with the instrumental method for measuring concentration, and the correlation is good.

[0253] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A monoclonal antibody against citalopram, comprising a heavy chain variable region and a light chain variable region, characterized in that, The heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 6; and the light chain variable region has the amino acid sequence shown in SEQ ID NO.

7.

2. A nucleic acid molecule, characterized in that, It encodes a monoclonal antibody of citalopram as described in claim 1.

3. The use of the monoclonal antibody of citalopram according to claim 1 or the nucleic acid molecule according to claim 2 in the preparation of a kit for assessing citalopram levels.

4. The application according to claim 3, characterized in that, The assessment is based on the detection of citalopram levels in the serum samples of the tested individuals.

5. A carrier, characterized in that, It contains the nucleic acid molecule as described in claim 2.

6. A citalopram detection kit, characterized in that, It contains a monoclonal antibody of citalopram as described in claim 1 or a nucleic acid molecule as described in claim 2.

7. A citalopram detection reagent, characterized in that, It includes the monoclonal antibody of citalopram as described in claim 1, the nucleic acid molecule as described in claim 2, the vector as described in claim 5, or the citalopram detection kit as described in claim 6.

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