A cetirizine monoclonal antibody, nucleic acid molecule, vector, detection kit and application thereof

Abstract

The application belongs to the technical field of colloidal gold immunochromatography, and relates to cetirizine monoclonal antibody, nucleic acid molecules, vectors, detection kits and application thereof. The cetirizine monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises VH-CDR1, VH-CDR2 and VH-CDR3, the light chain variable region comprises VL-CDR1, VL-CDR2 and VL-CDR3, the sequence of VH-CDR1 is shown as SEQ ID NO. 1, the sequence of VH-CDR2 is shown as SEQ ID NO. 2, and the sequence of VH-CDR3 is shown as SEQ ID NO. 3. The monoclonal antibody of the application has high sensitivity to cetirizine, the minimum detection limit is 0.007 ng / mL, the quantitative range is 0.017-0.282 ng / mL, and the specificity is strong.

Description

Technical Field

[0001] This invention belongs to the field of colloidal gold immunochromatography technology, and relates to a cetirizine monoclonal antibody, nucleic acid molecule, vector, detection kit and its application. Background Technology

[0002] Cetirizine, chemical formula C 21 H 25 ClN2O3 is a second-generation H1 antihistamine, a long-acting, selective, oral, potent anti-allergic drug. It is used for seasonal or perennial allergic rhinitis, urticaria caused by allergens, and pruritus. Although cetirizine is an over-the-counter drug with high safety, blood drug concentration monitoring is still necessary in specific clinical scenarios: (1) Patients with renal insufficiency: Cetirizine is mainly excreted unchanged through the kidneys. Patients with impaired renal function have significantly reduced drug clearance, which can easily lead to drug accumulation and increased risk of adverse reactions; (2) Individualized dosing: Different individuals have different efficacy and tolerance to cetirizine. Some patients have poor efficacy or adverse reactions such as drowsiness and dry mouth. The dosage needs to be adjusted to balance efficacy and safety; (3) Drug interaction monitoring: When cetirizine is used in combination with central nervous system depressants (such as alcohol and benzodiazepines), the central inhibitory effect may be enhanced. Blood drug concentration monitoring helps to assess the risk of combined drug use; (4) Medication compliance assessment: In clinical trials or long-term treatment, blood drug concentration testing can objectively assess patient medication compliance and distinguish between poor compliance and poor efficacy caused by pharmacokinetic abnormalities. In recent years, some traditional Chinese medicines, health foods, and cosmetics claiming to have anti-allergic and antipruritic effects have been found to contain illegally added chemical drugs such as cetirizine. This illegal addition not only violates drug regulatory regulations but may also lead to consumers unknowingly using the same medication repeatedly or in excessive doses, causing serious adverse reactions. Therefore, establishing a rapid, sensitive, and specific detection method for cetirizine is of great significance for ensuring public safety.

[0003] Currently, the main methods for detecting cetirizine include chromatography and immunoassay. High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS) are classic methods for cetirizine detection. These methods have advantages such as high accuracy and the ability to distinguish between cetirizine and its enantiomer. However, chromatography has the following limitations: (1) complex sample pretreatment, requiring extraction, concentration, and other steps, which is cumbersome and time-consuming; (2) expensive equipment and high maintenance costs, making it unsuitable for grassroots laboratories and rapid on-site detection; (3) limited detection throughput, making it difficult to meet the needs of large-scale screening. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) have advantages such as simple operation, rapid detection, high throughput, low cost, and suitability for on-site screening, and are an important development direction for cetirizine detection. The core of immunoassay lies in obtaining highly specific and high-affinity anti-cetirizine antibodies. However, there is currently no research on monoclonal antibodies against cetirizine. Summary of the Invention

[0004] The purpose of this invention is to provide a highly specific and high-affinity cetirizine monoclonal antibody, nucleic acid molecule, vector, detection kit, and its applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A cetirizine monoclonal antibody includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region includes VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region includes VL-CDR1, VL-CDR2, and VL-CDR3. The sequence of VH-CDR1 is shown in SEQ ID NO.1, the sequence of VH-CDR2 is shown in SEQ ID NO.2, the sequence of VH-CDR3 is shown in SEQ ID NO.3, the sequence of VL-CDR1 is shown in SEQ ID NO.4, the sequence of VL-CDR2 is KVS, and the sequence of VL-CDR3 is shown in SEQ ID NO.5.

[0007] The sequence of SEQ ID NO.1 is: GFTFTDNY.

[0008] The sequence of SEQ ID NO.2 is: ISNKADGYTT.

[0009] The sequence of SEQ ID NO.3 is: ARDYGTMAWFAY.

[0010] The sequence of SEQ ID NO.4 is: QNTVHSDGNTY.

[0011] The sequence of SEQ ID NO.5 is: FQGSHVPLT.

[0012] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0013] 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.

[0014] The sequence of SEQ ID NO.6 is:

[0015] EVQLEESGGGLVQPGGSLRLSCATSGFTFTDNYMSWVRQPPGKALEWLGFISNKADGYTTEYSASVKGRFTISRDNSQSILYLQMNTLRAEDSGTYYCARDYGTMAWFAYWGQGTLVTVSA.

[0016] The sequence of SEQ ID NO.7 is:

[0017] DIVMTQTTLSLPVSLGDHASISCRSRQNTVHSDGNTYFEWYLLKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK.

[0018] Based on the same inventive concept, the present invention also claims protection for a nucleic acid molecule encoding the cetirizine monoclonal antibody.

[0019] Based on the same inventive concept, the present invention also claims the use of the cetirizine monoclonal antibody or the nucleic acid molecule in the preparation of reagents for assessing cetirizine levels.

[0020] In one preferred embodiment, the reagent comprises a kit.

[0021] In a preferred embodiment of the present invention, the evaluation is based on the detection of cetirizine content in samples of traditional Chinese medicine, food, or cosmetics.

[0022] Based on the same inventive concept, the present invention also claims a carrier containing the aforementioned nucleic acid molecule.

[0023] Based on the same inventive concept, the present invention also claims a test kit containing the cetirizine monoclonal antibody or the nucleic acid molecule.

[0024] Based on the same inventive concept, the present invention also claims protection for the application of the test kit in detecting the content of cetirizine in samples of traditional Chinese medicine, food or cosmetics.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] This invention screened a novel monoclonal antibody against cetirizine, exhibiting high sensitivity for cetirizine detection, with a limit of detection (LOD) of 0.007 ng / mL, an IC50 of 0.061 ng / mL, and a quantitative detection range of 0.017-0.282 ng / mL. The antibody's detection sensitivity reaches the picogram per milliliter (pg / mL) level, meeting the requirements for accurate detection of low-concentration samples. Compared to existing anti-cetirizine monoclonal antibodies (with existing literature reporting IC50 values ​​of 0.007 ng / mL), this invention provides a significantly higher detection sensitivity. 50 Typically in the range of 1-5 ng / mL), the IC50 of the antibody of this invention is... 50 The value was reduced by more than an order of magnitude, significantly improving detection sensitivity. Furthermore, the anti-cetirizine monoclonal antibody provided by this invention shows no significant cross-reactivity with cetirizine structural analogs and other antihistamines, demonstrating high specificity. Attached Figure Description

[0027] Figure 1 These are ultraviolet (UV) scan images of the artificial antigens XTLQ-BSA and XTLQ-OVA; among them, Figure 1 In the image, 'a' is the ultraviolet scan identification image of the artificial antigen XTLQ-BSA; Figure 1 In the image, b is the ultraviolet scan identification image of the artificial antigen XTLQ-OVA.

[0028] Figure 2 This is a gel electrophoresis image of cetirizine monoclonal antibody.

[0029] Figure 3 This is a diagram illustrating the subtype identification of cetirizine monoclonal antibody.

[0030] Figure 4 This is the standard curve for the detection of cetirizine monoclonal antibody.

[0031] Figure 5 This is a scatter plot comparing the methodologies of the present invention and conventional methods. Detailed Implementation

[0032] 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.

[0033] Glossary of relevant technical terms

[0034] The term "antibody" broadly refers to what is medically known as immunoglobulin (Ig). It is a Y-shaped protein produced by our body's immune system (primarily B lymphocytes). An immunoglobulin (Ig) molecule consists of four polypeptide chains: two heavy chains (H chains) and two light chains (L chains). In a complete antibody, each heavy chain contains a variable region (VH) and a constant region (CH), with the constant region consisting of three domains: CH1, CH2, and CH3. Each light chain contains a variable region (VL) and a constant region (CL), with the light chain constant region consisting of only one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, namely complementarity-determining regions (CDRs), and framework regions (FRs). Each VH and VL is arranged in the order from the amino terminus to the carboxyl terminus as FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. For ease of distinction, the three CDRs in the VH domain are typically named CDR-H1, CDR-H2, and CDR-H3, while the three CDRs in the VL domain are named CDR-L1, CDR-L2, and CDR-L3. Immunoglobulins encompass a wide range of types (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), categories (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), and subclasses. Currently, the amino acid sequences of the constant regions of the heavy and light chains of murine and human immunoglobulins are known in the art.

[0035] The term "monoclonal antibody" refers to a population of antibodies that are essentially homogeneous, in which each antibody is identical except for the possible trace amounts of natural mutations. Such antibodies exhibit extremely high specificity, recognizing a single antigenic determinant (epitope), while traditional polyclonal antibodies are mixtures of multiple antibodies that target different epitopes.

[0036] The term “CDR” refers to the complementarity-determining region within the variable domain sequence of an antibody. Three CDRs are present on both the heavy and light chains, designated CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. As used herein, the term “CDR group” 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))) not only provides a definitive residue numbering system applicable to any variable domain of an antibody but also provides precise residue boundaries defining the three CDRs.

[0037] The term "plasmid" is a circular double-stranded DNA molecule that can be used as a vector to link exogenous DNA segments and introduce the target gene sequence into the host cell to achieve autonomous replication and expression.

[0038] The term “transformation” refers to all processes by which exogenous DNA enters host cells, typically using a variety of techniques known in the art under natural or artificial conditions. These techniques encompass a range of methods for introducing exogenous nucleic acid sequences into prokaryotic or eukaryotic host cells, such as viral infection, electroporation, lipid transfection, and particle bombardment, the choice of which depends on the type of host cell to be transformed.

[0039] The reagents used in this invention are all commonly used reagents, purchased from Bitmain or McLean.

[0040] Example 1

[0041] Complete antigen preparation and identification

[0042] (1) Add cetirizine (18.8 mg) to a brown reaction vial, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC (14 mg), N-hydroxysuccinimide NHS (8.4 mg), dissolve in 400 μL N,N-dimethylformamide DMF, add 300 μL of primary water, sonicate to dissolve, and stir at room temperature for 3-4 h.

[0043] (2) Add bovine serum albumin (BSA) and ovalbumin (OVA) (10 mg each, dissolved in 1 mL of 0.1 mol / L carbonate buffer at pH 9.6) and stir overnight at 4°C.

[0044] (3) Collect the reaction solution, transfer it to a dialysis bag that has been boiled, and then put it into 5L of 0.01M PBS buffer.

[0045] (4) Dialyze for 3 days at 4℃, and change the solution with pre-cooled 0.01M PBS buffer solution every 6-8 hours.

[0046] (5) The artificial antigens purified by dialysis were designated as XTLQ-BSA and XTLQ-OVA, respectively. XTLQ-BSA is cetirizine-conjugated BSA protein, which serves as an immunogen in the embodiments of this invention. XTLQ-OVA is cetirizine-conjugated OVA protein, which serves as a coating agent in the embodiments of this invention. The concentrations of XTLQ-BSA and XTLQ-OVA were determined and identified by ultraviolet spectroscopy. The results are as follows: Figure 1 As shown, where, Figure 1 In the image, 'a' is the ultraviolet scan identification image of the artificial antigen XTLQ-BSA; Figure 1 Figure 'b' shows the UV scan identification of the artificial antigen XTLQ-OVA. By comparing the highest absorbance values ​​of each substance before and after conjugation, it was found that the absorption curve of XTLQ-BSA was significantly different from that of the carrier protein BSA. The absorption peak of XTLQ-BSA was similar to that of BSA, but significantly higher at 280 nm, and showed a significant shift relative to the curve of the hapten XTLQ. Since all unreacted components were removed by dialysis after conjugation, the characteristic peak of the conjugated product was contributed by the protein-bound drug molecule, indicating that the reaction product is a complex of the carrier protein BSA and XTLQ. This demonstrates that the present invention successfully prepared the cetirizine artificial antigen XTLQ-BSA. The results for XTLQ-OVA were similar, also demonstrating the successful preparation of the cetirizine artificial antigen XTLQ-OVA. The protein was then recovered and stored at -20°C.

[0047] Example 2

[0048] Antibody preparation

[0049] 1. Animal immunization

[0050] (1) Preparation of immunogen: Take 100 μg XTLQ-BSA equivalent / animal, dilute with PBS to 200 μL, and mix with 200 μL Freund's adjuvant in a 2.5 mL syringe. Emulsify with an emulsifier for 5-10 min. Take a drop of the emulsion and drop it onto the water surface until the emulsion does not spread on the water surface.

[0051] (2) Immunization: Balb / C female mice (purchased from Zhuhai Baishengtong Experimental Animal Company) were used as immunization animals. Immunogen was injected at multiple sites on the abdomen and back of each mouse, 130 μL / mouse. The selection of immunization adjuvants and immunization cycle are shown in Table 1.

[0052] (3) Serum detection: One week after the three immunizations, blood was collected from the tail vein of the mice, and the supernatant was collected after centrifugation to obtain antiserum.

[0053] (4) The antibody titer and inhibition rate of the antiserum were examined by ic-ELISA.

[0054] The specific steps are as follows:

[0055] 1) Plate coating: Dilute the coating agent to 1000 ng / mL with coating buffer (0.1 M carbonate buffer), and add 100 μL to each well of a 96-well microplate. Then incubate at 4°C for 12 h, wash twice with a plate washer, and pat dry.

[0056] 2) Sealing: Dilute skim milk powder with PBST solution to 6%, add 120 μL to each well of the plate, and seal at 4°C for 3 hours. Finally, discard the liquid, pat dry, and place in a 37°C oven to dry. After drying, transfer to a 4°C storage area.

[0057] 3) Primary antibody competitive incubation: Add 50 μL of blank PBST to the first column of the microplate (negative wells); dilute cetirizine to 1 μg / mL and add 50 μL to each well in the second column (positive wells); finally, dilute the collected blood supernatant with PBST and add it to each row of wells in ascending order of dilution, with 50 μL of PBST added to the eighth row as a blank control. Incubate at 37°C for 40 min, then discard the liquid in the wells, wash 5 times with a plate washer, and pat dry.

[0058] 4) Secondary antibody conjugation: HRP-labeled goat anti-mouse antibody (purchased from Beijing TransGen Biotech Co., Ltd.) was diluted 5000 times with PBST, 100 μL was added to each well, and incubated in a 37℃ water bath for 30 min. The antibody was washed 5 times and patted dry.

[0059] 5) Color development: Add 100 μL of color development solution (purchased from Shenzhen Zhenrui Biotechnology Co., Ltd.) to each well and incubate in a 37℃ water bath for 10 min;

[0060] 6) Termination: Add 50 μL of stop solution (10% H2SO4) to each well and immediately read the absorbance at 450 nm using a microplate reader. Wells with absorbance values ​​between 0.8 and 1.2 in the negative column are considered valid. Calculate the inhibition rate using the following formula.

[0061] .

[0062] 2. Cell fusion

[0063] (1) Preparation of myeloma cells: Mix myeloma cells (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) and complete culture medium (RPMI-1640, Gbico) and place them in a 10cm culture dish. Observe the cell state and select large, round and transparent cells for expansion culture to 3-4 dishes. When each dish is almost full, change the medium once to discard the cells that have not adhered. Cell fusion can be used after 12 hours.

[0064] (2) Sprint immunization: Before formal cell fusion, mice are immunized once to obtain a strong short-term stimulus.

[0065] The sprint immunization process is as follows:

[0066] Injection concentration: Immunogen diluted to 1 mg / mL;

[0067] Injection volume: 100µL;

[0068] Injection site: abdominal cavity;

[0069] Injection method: direct injection, no adjuvant required.

[0070] (3) Collection of myeloma cells: Remove the supernatant from 3-4 culture dishes after medium replacement. Add approximately 2 mL of basal culture medium using a 1 mL extended pipette tip, and repeatedly pipette from left to right and from top to bottom until most of the myeloma cells are removed. At this point, the bottom of the culture plate will no longer be blurred and will become transparent. Collect the myeloma cells into 50 mL centrifuge tubes, seal them, and centrifuge at 1000 rpm for 7 minutes. Discard the supernatant after centrifugation and set aside for later use.

[0071] (4) Preparation of immune spleen cells: Place the basal culture medium (RPMI-1640, Gbico) in a culture dish and place the disposable cell grinding mesh in the culture dish for later use. The mice with the best antiserum effect selected in Example 1 were sacrificed, soaked in 75% alcohol for 2 minutes, and then transferred to a clean bench to remove the spleen. The removed spleen was placed in the cell grinding mesh, and the spleen was first ground with the tip of a syringe until it was completely broken up. The basal culture medium (RPMI-1640, Gbico) was then used to rinse the mesh and syringe. The spleen cells were collected into a 50 mL centrifuge tube, sealed, and centrifuged at 1000 r / min for 7 minutes. The supernatant was discarded after centrifugation and the cells were ready for use.

[0072] (5) Cell mixing: Mix myeloma cells and immune spleen cells (after centrifugation and discarding the supernatant) in a centrifuge tube at a ratio of 1:5 to 1:10, add about 15 mL of basal culture medium, seal the tube, and centrifuge at 1000 r / min for 7 min. Discard the supernatant after centrifugation and set aside for later use.

[0073] (6) Cell fusion: After centrifugation, the supernatant of the mixed myeloma cells and immune spleen cells was discarded and the tubes were sealed. The precipitated cells were loosened by tapping with fingers to ensure thorough mixing. The centrifuge tubes were incubated in 37°C water for 5 minutes, then removed and placed in 45°C water. 1 mL of PEG 1500 preheated to 37°C was pipetted into the precipitated cells over 1 minute, gently stirring after each drop of PEG to ensure even mixing. Shaking continued for 1 minute. 1 mL of preheated basal culture medium was added over 1 minute, gently stirring along the wall. Then, 3 mL, 8 mL, and 8 mL of basal culture medium were added over 3 minutes, respectively, while gently stirring up and down to separate the PEG. After adding the contents, seal the centrifuge tube and centrifuge at 1000 rpm for 7 minutes. After centrifugation, discard the supernatant and add 200 mL of HAT medium (RPMI-1640, Gbico). Gently aspirate the liquid with a pipette tip and stir gently. Spread the complete culture medium containing the fused cells into 10 96-well cell culture plates, 200 μL / well.

[0074] 3. Cell selection and cell line establishment

[0075] The cells were cultured in an incubator for about 10 days. Starting from day 3, the cell growth and culture medium status should be observed daily. On day 5, or if the cells grew too fast, the medium was partially replaced with HAT medium. On day 8, the medium was completely replaced with HT medium. On day 10, the medium was partially replaced with HT medium. At this time, the cell supernatant was subjected to the first ic-ELISA test. Wells with high titers were selected for subcloning and preliminary cell purification.

[0076] The specific steps are as follows:

[0077] (1) Plate coating: Dilute the coating agent to a certain concentration using coating buffer (0.01 mol / L carbonate buffer, pH 9.6) (adjust the coating concentration according to actual experimental needs), and then add it to a 96-well microplate at 100 μL / well. Incubate the microplate in a 37°C water bath for 12 h. After incubation, wash the plate twice and then pat it dry.

[0078] (2) Blocking: Add blocking solution (6% skim milk powder), 120 μL / well, and then incubate in a 37°C water bath for 3 h. Discard the liquid in the plate and invert it in a 37°C oven for 30 min.

[0079] (3) Competitive reaction: The cell supernatant was serially diluted with 0.01 mol / L PBST solution at dilution factors of 1000, 2000, 4000, 8000, 16000, 32000 and 64000 (the specific dilution factor can be adjusted according to the actual experimental needs). After adding the solution, the plate was incubated in a 37℃ water bath for 40 min, then washed 5 times and patted dry.

[0080] (4) Add secondary antibody: Dilute HRP-labeled goat anti-rabbit or goat anti-mouse (purchased from Beijing TransGen Biotech Co., Ltd.) 5000 times with 0.01 mol / L PBST, 100 μL / well, and incubate in a 37℃ water bath for 30 min. After incubation, wash the plate 5 times and pat dry.

[0081] (5) Color development: Add color development solution (purchased from Shenzhen Zhenrui Biotechnology Co., Ltd.), 100 μL / well, and incubate in a 37℃ water bath for 10 min.

[0082] (6) Termination: Add stop solution (10% sulfuric acid solution), 50 μL / well, and read the absorbance value at 450 nm wavelength in the microplate reader.

[0083] (7) Result determination: OD of the negative well 450 The cell supernatant dilution factor corresponding to 1.0~1.5 is defined as the titer, and the titer is determined based on the OD of the well. 450 Values ​​and their corresponding OD values ​​of the suppression pores 450 Value, select 96 cell wells with high titer as positive cell wells.

[0084] In an embodiment of the invention, approximately 1 × 10⁻⁶ mice can be obtained from each immunized mouse. 9 One splenic lymphocyte. After fusing these splenic lymphocytes with myeloma cells using cell fusion technology, approximately 1 × 10⁶ cells were obtained. 4 Hybridoma cells survived and formed clones. Due to the random binding of different B lymphocytes to myeloma cells during the fusion process, the resulting hybridoma cells exhibited high heterogeneity in antibody secretion characteristics, with key performance indicators including antibody specificity, affinity, and subtype being unpredictable. Therefore, the subsequent screening and cloning processes were significantly random and uncertain. To obtain monoclonal antibodies with the target characteristics, multiple rounds of screening and cloning culture were required on the fused hybridoma cells. Following this screening process, 96 positive cell wells exhibiting high-titer antibody secretion activity and identified as monoclonal in origin were ultimately obtained for subsequent antibody function validation.

[0085] The second step uses cetirizine 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 against the cetirizine 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:

[0086] (1) Plate coating: Dilute the coating agent to a certain concentration using coating buffer (0.01 mol / L CB, pH 9.6) (adjust the coating concentration according to actual experimental needs), and then add it to a 96-well microplate at 100 μL / well. Incubate the microplate in a 37℃ water bath for 12 h. After incubation, wash the plate twice and then pat it dry.

[0087] (2) Blocking: Add blocking solution (6% skim milk powder), 120 μL / well, then incubate in a 37℃ water bath for 3 h, discard the liquid in the plate, and invert it in a 37℃ oven for 30 min;

[0088] (3) Competitive reaction: Cell supernatant was serially diluted with 0.01 mol / L PBST solution at dilution factors of 1000, 2000, 4000, 8000, 16000, 32000 and 64000 (the specific dilution factor can be adjusted according to the actual experimental needs). The standard of the detection drug was diluted with 0.01 mol / L PBST solution to the required concentration for later use (the concentration of the detection drug was adjusted according to the actual experimental needs). 50 μL of 0.01 mol / L PBST was added to each negative well, followed by 50 μL of diluted antibody. 100 μL of 0.01 mol / L PBST was added to each negative control well. 50 μL of diluted detection drug was added to each positive well, followed by 50 μL of diluted antibody. 50 μL of 0.01 mol / L PBST and 50 μL of diluted detection drug were added to each positive control well. After adding the liquid, place the plate in a 37°C water bath for 40 minutes, then wash the plate 5 times and pat it dry.

[0089] (4) Add secondary antibody: Dilute HRP-labeled goat anti-rabbit or goat anti-mouse antibody 5000 times with 0.01 mol / L PBST, 100 μL / well, and incubate in a 37℃ water bath for 30 min. After incubation, wash the plate 5 times and pat dry;

[0090] (5) Color development: Add color development solution, 100 μL / well, and incubate in a water bath at 37°C for 10 min;

[0091] (6) Termination: Add stop solution, 50 μL / well, and read the absorbance value at 450 nm wavelength in the microplate reader.

[0092] (7) Result determination: OD of the negative well 450The cell supernatant dilution factor corresponding to 1.0~1.5 is defined as the titer, and the titer is determined based on the OD of the well. 450 Values ​​and their corresponding OD values ​​of the suppression pores 450 Calculate the inhibition rate. Inhibition rate = [(OD value of potency - OD value of inhibition) / OD value of inhibition] × 100%.

[0093] After three rounds of subcloning and ic-ELISA testing, five monoclonal cell lines were obtained and numbered according to their original well sequence. The data are shown below:

[0094] Through repeated screening, five monoclonal cell clusters that could stably produce antibodies were obtained.

[0095] Comparing the drug recognition capabilities of the supernatants from these five positive clones after serial dilution, the results showed that after a 32K-fold dilution, the antibody produced by clone 8D2 still exhibited the highest drug recognition sensitivity. While other clones could also recognize the drug (i.e., produce monoclonal antibodies), the sensitivity of their antibodies was significantly lower than that of the antibody produced by clone 8D2. Therefore, the antibody derived from clone 8D2 was selected for subsequent sequencing.

[0096] The antibody derived from clone 8D2 was purified and identified using the following specific steps:

[0097] Nine-week-old female Balb / c mice were selected. On the first day of the experimental cycle, each mouse was injected with 500 μL of liquid paraffin. Seven days after paraffin injection, cultured cells were injected into the peritoneal cavity of the mice, causing the cells to proliferate in the peritoneal cavity in the form of ascites tumors, thus obtaining a large amount of ascites. After cell injection, the mice's condition needed to be observed frequently. When the mice's abdomens were significantly swollen and their viability decreased, the ascites was dissected and the fluid was centrifuged (4℃, 10000 rpm, 10 min) to remove the upper fat and lower protein layers, collecting the middle ascites layer. The collected ascites was stored at -20℃ for later use. The ascites was purified according to the following steps:

[0098] (1) Cleaning the system and column: Clean the system and column with ultrapure water;

[0099] (2) Equilibrate the system and column: Replace the liquid phase in the system and column with equilibration buffer PBS;

[0100] (3) Sample loading: Flow the ascites fluid through the protein G 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;

[0101] (4) Elution: The protein G affinity chromatography column was washed with 0.1 mol / L Gly-HCl buffer (pH 2.7) at a flow rate of 2 mL / min to elute the antibody adsorbed on the column. The collected antibody was adjusted to pH 7 with 1 mol / L Tris-HCl (pH 9.0).

[0102] (5) Dialysis: After pH adjustment, the antibody was dialyzed with PBS solution at 4°C for three days;

[0103] (6) Determination of monoclonal antibody purity: The purity of the purified antibody was determined using SDS-PAGE. For example... Figure 2 As shown, the theoretical molecular weight of IgG in non-reducing electrophoresis is 150 kDa. Reducing electrophoresis will separate it into two 50 kDa heavy chains and a 25 kDa light chain. The electrophoresis results are consistent with the theoretical value, indicating that the purification was successful and the monoclonal antibody was successfully prepared.

[0104] (7) Monoclonal antibody subtype identification: Mouse antibodies are classified into IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA. This experiment used a commercially available antibody subtype identification kit to identify the antibody subtypes in ascites fluid. The results are as follows: Figure 3 As shown, the results indicate that the antibody subtype is IgG1.

[0105] (8) Antibody sequencing: The antibody derived from clone 8D2 was sent to Nanjing Zhongding Biotechnology Co., Ltd. for sequencing. The results are shown below:

[0106] The full length of the heavy chain variable region VH of the monoclonal antibody is: EVQLEESGGGLVQPGGSLRLSCATSGFTFTDNYMSWVRQPPGKALEWLGFISNKADGYTTEYSASVKGRFTISRDNSQSILYLQMNTLRAEDSGTYYCARDYGTMAWFAYWGQGTLVTVSA (SEQ ID NO.6).

[0107] The full length of the variable region (VL) of the light chain of a monoclonal antibody is:

[0108] DIVMTQTTLSLPVSLGDHASISCRSRQNTVHSDGNTYFEWYLLKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK (SEQ ID NO. 7).

[0109] Example 3

[0110] Antibody performance testing

[0111] 1. Cetirizine antibody sensitivity evaluation (indirect competitive ELISA method)

[0112] An indirect competitive ELISA method for detecting cetirizine includes the following steps:

[0113] (1) The artificial antigen XTLQ-OVA was used as the coating agent, diluted to 62.5 ng / mL with coating buffer (0.1 M carbonate buffer), and coated with 100 μL of the coating agent in each well of a 96-well microplate. The plate was incubated at 37°C overnight (12 h).

[0114] (2) Discard the coating solution, wash twice, and pat dry;

[0115] (3) Add 120 μL of blocking solution (i.e., 6% skim milk powder) to each well and seal at 37°C for 3 hours;

[0116] (4) Discard the sealing liquid, pat the plate, dry at 37°C for 30 minutes, and then pack it in a self-sealing bag for later use;

[0117] (5) Dilute cetirizine antibody with PBST 1:4000, and dilute cetirizine to 10000ng / mL, 5000ng / mL, 2000ng / mL, 285.71429ng / mL, 40.81633ng / mL, 5.8309ng / mL, 0.83299ng / mL, 0.119ng / mL, 0.017ng / mL, 0.00243ng / mL, and 0.00034714ng / mL;

[0118] (6) Add 50 μL of cetirizine dilution to each row (three parallel groups), then add 50 μL of antibody per well, incubate at 37°C for 40 min, wash five times, and pat dry;

[0119] (7) Add 100 μL / well of goat anti-rabbit secondary antibody-HRP (5000-fold dilution, purchased from Beijing TransGen Biotech Co., Ltd.), incubate at 37℃ for 30 min, wash five times, and pat dry;

[0120] (8) Add 100 μL of colorimetric solution to each well and develop the color for 10 min;

[0121] (9) Add 50 μL of 10% H2SO4 solution to terminate the reaction and read the OD value at 450 nm.

[0122] (10) The standard curve of the indirect competitive ELISA for detecting cetirizine is shown in the figure. Figure 4 As shown, the half-inhibitory concentration (IC50) of the antibody used to detect cetirizine is known. 50The detection limit was 0.061 ng / mL, the quantitative detection range was 0.017-0.282 ng / mL, and the detection limit was 0.007 ng / mL; this indicates that the antibody prepared by this invention for detecting cetirizine can meet the detection requirements and has high detection sensitivity for cetirizine.

[0123] 2. Evaluation of the specificity of cetirizine antibodies

[0124] The specificity of cetirizine for detection was determined by cross-reactivity experiments with cetirizine and its analogues. The specificity of the antibody was expressed as the cross-reactivity rate (CR); the lower the CR, the stronger the specificity. Cetirizine and its analogues (amlodipine, ibastine, etc.) were serially diluted and measured using an indirect competitive ELISA method to obtain the IC50 values ​​for each analogue. 50 The cross-reactivity rate (CR) of cetirizine is calculated using the following formula: CR(%) = IC 50 (Cetirizine) / IC 50 (Similar substances) × 100%. The results are shown in Table 3.

[0125] Note: ND indicates no reaction, meaning the antibody does not recognize the analogue.

[0126] Cross-reactivity results of cetirizine and its analogues showed that the antibody used to detect cetirizine had a 100% cross-reactivity with cetirizine, IC50. 50 The concentration was 0.061 ng / mL, and it showed no cross-reactivity with other drugs, indicating good specificity.

[0127] Example 4

[0128] Establishment of an ELISA method for detecting cetirizine

[0129] 1. Solution preparation:

[0130] (1) Coating solution (0.05mol / L carbonate buffer solution, pH 9.6): Accurately weigh 3.4g of Na2CO3 and 5.8g of NaHCO3, and dilute to 2000mL with distilled water.

[0131] (2) 0.01mol / L PBS (pH 7.4): Accurately weigh 42.5g NaCl, 1g KCl, 14.5g Na2HPO4•12H2O, and 1g KH2PO4, and dilute to 5000mL with distilled water.

[0132] (3) 0.01 mol / L PBST (pH 7.4): Add 1% Tween-20 to PBS.

[0133] (4) Blocking solution: Accurately weigh 0.6g of skim milk powder and dissolve it in 100mL of PBST.

[0134] (5) Termination solution: Take 100 mL of concentrated sulfuric acid and slowly add it to 900 mL of distilled water.

[0135] (6) Washing buffer (pH 7.4): Accurately weigh 32.0g NaCl, 12.0g Na2HPO4•12H2O and 2.4mL Tween-20, and dilute to 5000mL with distilled water.

[0136] 2. Composition

[0137] (1) The enzyme-labeled plate coated with the coating agent is prepared by the following method:

[0138] The XTLQ-OVA prepared in Example 2 was used as the coating agent. It was diluted to 100 ng / mL with the coating stock solution and added at a rate of 100 μL / well to coat a 96-well microplate. The plate was incubated at 37°C in the dark for 4 hours. The liquid in the wells was discarded, and the plate was washed twice with the washing buffer provided in this kit for 30 seconds each time, then patted dry. Then, the blocking buffer provided in this kit was 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 membrane for storage. Washing buffer: pH 7.4, containing 0.8% Tween-20 (v / v), 0.02% sodium azide preservative (w / w), and 0.2 mol / L phosphate buffer. Before use, the washing buffer was diluted 20 times with water to obtain the working solution. Diluent: 0.2 mol / L phosphate buffer. Before use, the diluent was diluted 20 times with water to obtain the working solution. Blocking solution: pH 7.3, 0.2 mol / L phosphate buffer containing 6% skim milk powder.

[0139] (2) Standards: Cetirizine standards of 10 different concentrations: 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.

[0140] (3) Antibody: XTLQ-BSA monoclonal antibody prepared in Example 2.

[0141] (4) Enzyme-labeled secondary antibody: horseradish peroxidase-labeled goat anti-mouse secondary antibody (purchased from Beijing TransGen Biotech Co., Ltd.).

[0142] (5) Substrate color development solution: It consists of solution A and solution B. Solution A is urea peroxide and solution B is tetramethylbenzidine. The recommended volume ratio of solution A to solution B is 1:1.

[0143] (6) Termination solution: 10% H2SO4.

[0144] 3. Instructions for use

[0145] (1) Sample testing

[0146] Number the corresponding wells on the coated ELISA plate according to the sample and the standard of this kit, and perform triple replicates for each sample and standard. Dilute the antibody with the diluent at a volume ratio of 1:50 to obtain the antibody working solution. Dilute the enzyme-labeled secondary antibody with the diluent at a volume ratio of 1:100 to obtain the enzyme-labeled secondary antibody working solution.

[0147] 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.

[0148] Shake off the liquid in the wells. Add 250 μL of washing working solution to the corresponding microwell. Wash thoroughly 4-5 times, with 10-second intervals between each wash. Discard the washing working solution in the wells and pat dry with absorbent paper.

[0149] Add 100 μL of enzyme-labeled secondary antibody working solution to the corresponding microwell, gently shake to mix, cover with a cover plate membrane, and incubate at 25°C in the dark for 30 min.

[0150] Shake off the liquid in the wells. Add 250 μL of washing working solution to the corresponding microwell. Wash thoroughly 4-5 times, with 10-second intervals between each wash. Discard the washing working solution in the wells and pat dry with absorbent paper.

[0151] Add 50 μL of substrate chromogenic solution A to each well, then add 50 μL of substrate chromogenic solution B to each well. Gently shake to mix, cover with a cover plate, and incubate at 25°C in the dark for 10 min.

[0152] Add 50 μL of the stop solution to the corresponding well, gently shake to mix, set the microplate reader to 450 nm, and measure the OD value of each well.

[0153] (2) Drawing the standard curve

[0154] 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. 450Using the logarithm of the concentration of the standard as the abscissa, a standard curve was obtained by fitting the curve using the Logistic function: y = 0.09708 + (0.9366 - 0.09708) / 1 + (X / 0.06108)^1.01255, R0 2 =0.99.

[0155] (3) Calculation of sample concentration

[0156] OD of the sample 450 Substituting the average value into the formula for the standard curve above (y = 0.09708 + (0.9366 - 0.09708) / 1 + (X / 0.06108)^1.01255, R...), 2 =0.99) to obtain the concentration of the sample, and then multiply it by the corresponding dilution factor to obtain the actual concentration of cetirizine in the test sample.

[0157] Example 5

[0158] Testing of actual samples

[0159] (1) Verification of method accuracy

[0160] Ten commercially available food and cosmetic samples were mixed with a known amount of cetirizine standard solution. The resulting test solutions were then analyzed using the ic-ELISA method established in Example 4, with three parallel determinations performed at each concentration level. The measured values ​​were substituted into the standard curve (y = 0.09708 + (0.9366 - 0.09708) / 1 + (X / 0.06108)^1.01255, R0). 2 The actual concentration was calculated using the formula (=0.99), and the results are shown in Table 4.

[0161] The results showed that the actual content of the sample measured by this method was consistent with the added content, with an absolute deviation of <5%, proving that this method has sufficient accuracy.

[0162] (2) Verification of method reliability

[0163] Ten samples were tested using both the method described in this embodiment and the existing LC / MS method (Peng Yan, Yang Zhi, Jin Mengna, et al. Simultaneous determination of 17 anti-allergic chemical components added to traditional Chinese medicine and health food by solid phase extraction purification-UPLC-MS / MS method [J]. Journal of Pharmaceutical Analysis, 2019, 39(6):1115-1126.). The results are shown in Table 5.

[0164] A correlation scatter plot was created using the test results of 10 samples obtained through this embodiment and existing LC / MS methods. The results are as follows: Figure 5 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.

[0165] 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 cetirizine monoclonal antibody, characterized in that, It includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region includes VL-CDR1, VL-CDR2, and VL-CDR3. The sequence of VH-CDR1 is shown in SEQ ID NO.1, the sequence of VH-CDR2 is shown in SEQ ID NO.2, the sequence of VH-CDR3 is shown in SEQ ID NO.3, the sequence of VL-CDR1 is shown in SEQ ID NO.4, the sequence of VL-CDR2 is KVS, and the sequence of VL-CDR3 is shown in SEQ ID NO.

5.

2. The cetirizine monoclonal antibody according to claim 1, characterized in that, 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.

3. Use of the cetirizine monoclonal antibody according to claim 1 or 2 in the preparation of reagents or kits for assessing cetirizine levels.

4. A nucleic acid molecule, characterized in that, It encodes the cetirizine monoclonal antibody as described in claim 1 or 2.

5. The use of the nucleic acid molecule of claim 4 in the preparation of reagents or kits for assessing cetirizine levels.

6. The application according to claim 3 or 5, characterized in that, The assessment is based on the detection of cetirizine content in samples of traditional Chinese medicine, food, or cosmetics.

7. A carrier, characterized in that, It contains the nucleic acid molecule as described in claim 4.

8. A test kit, characterized in that, It contains the cetirizine monoclonal antibody as described in claim 1 or 2 or the nucleic acid molecule as described in claim 4.

9. The application of the detection kit according to claim 8, characterized in that, The application is to detect the content of cetirizine in samples of traditional Chinese medicine, food, or cosmetics.

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