Hybridoma cell line, anti-psilocybin monoclonal antibody secreted thereby, and applications thereof

Abstract

The present invention discloses a hybridoma cell line, the anti‑psilocybin monoclonal antibody secreted by the hybridoma cell line, and applications thereof. The hybridoma cell line is named PLCB‑5.2.1 and has a deposit number of CCTCC No. C2022364; the anti‑psilocybin monoclonal antibody is secreted by the above‑mentioned hybridoma cell line or its sub‑cultured cell line, and this antibody is of the IgG1 type and the light chain is of the kappa type. The above‑mentioned anti‑psilocybin monoclonal antibody can be used for preparing a psilocybin detection kit or for preparing a colloidal gold immunochromatographic test strip for detecting psilocybin. The anti‑psilocybin monoclonal antibody of the present invention has a high titer and strong specificity, and can be used for rapid and accurate immunoassay and immunoanalysis of psilocybin.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a hybridoma cell line, a monoclonal antibody against psilocybin secreted thereby, and applications thereof. Background Art

[0002] Psilocybin, also known as psilocin, baeocystin, or psilocine, has the chemical formula C 12 H 17 N 2 O 4 P, and is a neurotoxin with neuro-hallucinogenic effects.

[0003] Studies have found that this naturally occurring hallucinogenic agent, psilocybin, can induce therapeutic responses in patients with treatment-resistant depression by increasing the connectivity of the brain functional network. Depression patients often exhibit "negative cognitive bias", which is characterized by pessimism, poor cognitive flexibility, rigid thinking patterns, and negative fixation on "self" and the future. Treatment-resistant depression refers to major depressive disorder in which depressive symptoms cannot be improved after multiple antidepressant drug courses. Multiple clinical trials have reported test results showing improved depressive symptoms after treatment with psilocybin. However, the treatment mechanisms of psilocybin and related hallucinogenic agents have not been fully explained. Psilocybin therapy has rapid, significant, and lasting antidepressant effects, and the curative effect is significantly better than escitalopram. The remission of depressive symptoms is significantly correlated with the increased connectivity of the brain functional network. These significant changes in the brain module structure suggest that the rapid action of psilocybin has a "retention" effect on brain function. Such changes were not observed in patients using escitalopram. Thus, it is shown that psilocybin (and possibly other hallucinogenic agents) has a new action mechanism different from traditional antidepressant drugs. Specifically, psilocybin therapy may be able to relieve depressive symptoms by liberating the rigid brain network and stimulating coordinated and flexible brain functions beneficial to mental health.

[0004] The improper use of psilocybin is highly harmful. First, it enters the blood circulation, then reaches the nervous system, and then activates 5-hydroxytryptamine receptors, thereby causing nerve excitation. It mainly acts on the autonomic nerves, causing nerve excitation and hallucinations, creating illusions about time and space, until symptoms such as self-distortion, delusions, and thought dissociation occur. In severe cases, there may be tachycardia, dilated pupils, and difficulty urinating, but generally it does not endanger life.

[0005] Psilocybin also widely exists in wild mushrooms, and mushroom poisoning is likely to occur. Moreover, psilocybin is a first-class psychotropic substance, and its abuse constitutes drug-taking behavior.

[0006] The anti-psilocybin hybridoma cell line can be prepared by the hybridoma technology. The antibody prepared from this hybridoma cell line can be used to prepare a colloidal gold immunodetection test strip, meeting the requirement of rapid on-site detection of personnel. Summary of the Invention

[0007] The first object of the present invention is to provide a hybridoma cell line secreting anti-psilocybin monoclonal antibody, which can produce high-titer anti-psilocybin monoclonal antibody.

[0008] A hybridoma cell line secreting anti-psilocybin, named hybridoma cell line PLCB-5.2.1, has been deposited in the China Center for Type Culture Collection (CCTCC for short), and the deposit number is CCTCC No. C2022364; the address of the China Center for Type Culture Collection is: Preservation Center of Wuhan University, Wuchang District, Wuhan City, Hubei Province (opposite to the First Affiliated Primary School of Wuhan University).

[0009] The present invention adopts a method combining small-dose intramuscular injection immunization and tail vein immunization, injects synthetic psilocybin antigen into mice to obtain immune mouse spleen cells, and then fuses the immune mouse spleen cells with mouse myeloma cells, and screens with psilocybin standard product to obtain the hybridoma cell line.

[0010] The molecular weight of psilocybin itself is too small and it has no immunogenicity, that is, it lacks T cell epitopes and cannot directly induce the animal body to produce specific antibodies. Therefore, the present invention chemically modifies psilocybin and conjugates it with BGG (bovine serum gamma globulin) to obtain an artificial antigen (PLCB-BGG) that can induce the proliferation and differentiation of B cells and then produce specific antibodies. The molecular structural formula of the psilocybin is shown in formula (I):

[0011]

[0012] The dosage of the psilocybin artificial antigen is 25 μg each time of injection.

[0013] The method of combining small-dose intramuscular injection immunization and intraperitoneal immunization is used to immunize mice, and the immunization titer is high.

[0014] The present invention also provides the application of the hybridoma cell line in preparing a test strip for detecting psilocybin.

[0015] The second object of the present invention is to provide an anti-psilocybin monoclonal antibody, which is secreted by the above hybridoma cell line PLCB-5.2.1 or its subculture cell line. This antibody is of IgG1 type and the light chain is of kappa type.

[0016] The third object of the present invention is to provide the use of the above-mentioned anti-psilocybin monoclonal antibody in the preparation of a psilocybin detection kit.

[0017] The detection kit described in the present invention is a competitive inhibition ELISA detection kit; it also contains a psilocybin standard and an enzyme-labeled secondary antibody. Due to the limitations of the molecular structure of psilocybin itself, the detection kit of the present invention is prepared based on the principle of competitive inhibition ELISA. When in use, first coat the psilocybin standard, then add the sample to be tested, and then sequentially add the anti-psilocybin monoclonal antibody and the enzyme-labeled secondary antibody for reaction, and detect the OD 450 value and calculate the competitive inhibition rate. The coating amount of the psilocybin standard is preferably the detection threshold of the anti-psilocybin monoclonal antibody for psilocybin. The enzyme-labeled secondary antibody can be selected as goat anti-mouse IgG-HRP.

[0018] The detection kit described in the present invention is a competitive enzyme-linked immunosorbent assay kit; it contains an enzyme-labeled plate coated with anti-psilocybin monoclonal antibody, a psilocybin standard, an enzyme-labeled antigen, a substrate and a termination solution. When in use, the sample to be tested and the enzyme-labeled psilocybin antigen are added to the enzyme-labeled plate for reaction. If there is residual psilocybin in the sample, it will compete with the enzyme-labeled psilocybin antigen for binding to the antibody on the plate, and then the substrate is added for reaction to terminate the color development. The color development intensity is inversely proportional to the amount of residual psilocybin in the sample. According to the standard curve made from the psilocybin standard, the content of psilocybin in the sample is calculated.

[0019] The fourth object of the present invention is to provide the use of the above-mentioned anti-psilocybin monoclonal antibody in the preparation of a colloidal gold immunochromatographic test strip for detecting psilocybin.

[0020] The colloidal gold immunochromatographic test strip for detecting psilocybin described in the present invention includes a sample pad, a marker pad, a reaction membrane and a sample absorption pad; the marker pad is coated with the anti-psilocybin monoclonal antibody labeled with colloidal gold, the test line (T line) of the reaction membrane is coated with a psilocybin-bovine serum albumin complex, and the quality control line (C line) of the reaction membrane is coated with a secondary antibody.

[0021] The test strip of the present invention is also prepared based on the principle of competitive inhibition ELISA, and the coating amount of the psilocybin-bovine serum albumin complex is also preferably the detection threshold of the anti-psilocybin monoclonal antibody for psilocybin. During detection, if only the C line shows color, it means that the sample to be tested contains psilocybin and the detected content of psilocybin is higher than the detection limit; if both the C line and the T line show color, it means that the detected content of psilocybin in the sample to be tested is lower than the detection threshold, or there is no psilocybin in the sample to be tested; if neither the C line nor the T line shows color, it means that the test strip has failed.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] The present invention immunizes mice with PLCB - BGG, prepares a hybridoma cell line using the spleen cells of the immunized mice. The anti - psilocybin monoclonal antibody secreted by this hybridoma cell line has a high titer and strong specificity, and can be used for rapid and accurate immunoassay and immunoanalysis of psilocybin.

[0024] The hybridoma cell line PLCB - 5.2.1 was deposited at the China Center for Type Culture Collection (CCTCC for short) on December 8, 2022, with the deposit number CCTCC No. C2022364; the address of the China Center for Type Culture Collection is: The Preservation Center of Wuhan University, Wuchang District, Wuhan City, Hubei Province (opposite the First Affiliated Primary School of Wuhan University). Brief Description of the Drawings

[0025] Figure 1 It is a schematic structural diagram of a psilocybin colloidal gold test strip of the present invention. Detailed Embodiments

[0026] The following further elaborates on the present invention in combination with specific embodiments.

[0027] Example 1 Preparation of Psilocybin Artificial Antigen

[0028] (1) Dissolve 10 mg of psilocybin artificial hapten, 20 mg of N - hydroxysuccinimide, and 15 mg of dicyclohexylcarbodiimide in 2 ml of N,N - dimethylformamide, and stir in a water bath at 25°C for 24 h, denoted as solution A.

[0029] (2) Dissolve 10 mg of bovine serum gamma - globulin (BGG) in 0.01 mol / L PBS solution, denoted as solution B.

[0030] (3) Slowly drip solution A into solution B under stirring, and then stir and react at room temperature for 2 h. Dialyze the mixed solution three times in PBS solution, with each dialysis duration exceeding 6 h, to obtain the artificial antigen PLCB - BGG.

[0031] Example 2 Preparation of Hybridoma Cell Line

[0032] (1) Mouse Immunization

[0033] Select 6-week-old female BALB / c mice. Mix 25 μg of PLCB-BGG antigen with an equal volume of quick antibody-5w adjuvant to make 100 μl, and inject it into the calf muscle of the mice; the calf muscle area is the part of the mouse leg where there is a large piece of muscle exposed to the outside, and most of the thigh of the mouse is wrapped by the abdominal skin, and the knee joint can also be found to distinguish the calf and thigh; inject the second dose in the same way two weeks later; two weeks later, perform intraperitoneal injection with 25 μg of antigen.

[0034] (2) Preparation of spleen cells

[0035] Take the spleen of the BALB / c female mice treated in step (1), grind the spleen cells into a suspension; centrifuge the spleen cell suspension and take the precipitate; add IMDM medium containing 15% fetal bovine serum to the precipitate and adjust the spleen cells to 10 ml.

[0036] (3) Fusion of spleen cells and myeloma cells:

[0037] (3-1) Preparation of feeder cells: Sacrifice the mice by removing the eyeballs, soak them in 75% alcohol for disinfection for 10 min, tear open the abdominal skin of the mice to expose the peritoneum, inject 8 mL of pre-warmed IMDM serum-free medium at 37 °C with a sterile syringe, gently knead the abdominal cavity of the mice to suspend the abdominal cavity cells, and aspirate the abdominal cavity fluid; centrifuge at 1500 rpm for 3 min, and resuspend the precipitate with 1×HAT IMDM culture medium to obtain a feeder cell suspension.

[0038] (3-2) Culture of mouse myeloma cell SP2 / 0: Passage the mouse myeloma cell SP2 / 0 with IMDM medium containing 10% (v / v) FBS, and passage it one day before cell fusion to ensure that the mouse myeloma cell SP2 / 0 is in a suitable growth state for cell fusion.

[0039] (3-3) Fusion of spleen cells and myeloma cells: Mix the spleen cells with adjusted concentration and mouse myeloma cell SP2 / 0 evenly at a cell number ratio of 4:1, centrifuge and wash the cells once at 1500 rpm, remove the supernatant, gently tap the bottom of the centrifuge tube to loosen the cell precipitate, place it in a 37 °C water bath, and slowly add 1 mL of pre-warmed 1 mL PEG1500 at 37 °C within 90 s while gently shaking; add 25 ml of pre-warmed IMDM serum-free medium at 37 °C to terminate the action of PEG; centrifuge at 1000 rpm for 5 min and take the precipitate; then add the feeder cell suspension prepared in step (3-1) to the precipitate, inoculate it into a 96-well cell culture plate, and place it in an incubator at 37 °C and 5% CO 2 for culture.

[0040] (4) Screening of hybridoma cell fusion

[0041] (4-1) Primary screening:

[0042] In step (3-3), after the fused cells are cultured in a 5% CO 2 incubator for 3 days, the IMDM culture medium with 1×HT is used for replacement, and after continuous culture for 4 days, the growth of the fused cells in the 96-well cell culture plate is observed. When the cells grow into cell clusters (observed under a 16× objective lens and a 10× eyepiece, and the cell size is preferably such that it fills 1 / 3 of the field of view), the supernatant of the fused cell culture is aspirated, and positive clones are screened by the indirect ELISA method.

[0043] The operating steps of the indirect ELISA method are as follows:

[0044] ① Dilute the PLCB-BGG antigen to 1 μg / mL with a carbonate buffer solution at pH 9.6 and a concentration of 0.05 M. Add 100 μl of the diluted antigen to each well of the 96-well enzyme-linked immunosorbent assay (ELISA) plate, and coat it overnight at 4°C. Wash the plate 3 times with a PBS buffer solution containing 0.05% tween20.

[0045] ② Add the PBS buffer solution to the supernatant of the fused cell culture and dilute it to 25 times the volume. Then add 100 μl of the diluted supernatant of the fused cell culture to each well of the 96-well cell culture plate. At the same time, set up a negative control. After reacting at 37°C for 60 minutes, wash the plate 1 time with a PBS buffer solution containing 0.05% tween20.

[0046] ③ Add the PBS buffer solution to the goat anti-mouse IgG-HRP and dilute it to 5000 times the volume. Then add 100 μl of the diluted goat anti-mouse IgG-HRP to each well of the 96-well cell culture plate. After reacting at 37°C for 30 minutes, wash the plate 3 times with a PBS buffer solution containing 0.05% tween20.

[0047] ④ Add 50 μl of the substrate TMB to each well and react at 37°C for 5 minutes.

[0048] ⑤ Add 50 μl of 1 M HCL to terminate the reaction, and measure its OD value at a UV wavelength of 450 nm. Take the OD 450 value of the supernatant of the fused cell culture / OD 450 value of the negative control > 2.5 as a positive clone, and further rescreen the screened positive clones by the competitive inhibition ELISA method.

[0049] (4-2) Rescreening:

[0050] The operating steps of the competitive inhibition ELISA method are as follows:

[0051] ① First, add 50 μl of 100 ng / ml psilocybin standard to the ELISA wells, then add 50 ul of positive clone culture supernatant, and design a blank control, that is, 50 μl PBS buffer and 50 μl positive clone culture supernatant are mixed and added to the ELISA wells. After incubation at 37°C for 60 minutes, wash the plate once with PBS buffer containing 0.05% tween20;

[0052] ② Add PBS buffer to goat anti-mouse IgG-HRP to dilute it to 5000 times volume, then add 100 μl of diluted goat anti-mouse IgG-HRP to each well of a 96-well cell culture plate, incubate at 37°C for 30 minutes, and wash the plate three times with PBS buffer containing 0.05% tween20;

[0053] ③ Add 50 μl of substrate TMB to each well and react at 37°C for 5 minutes;

[0054] ④ Add 50 μl of 1M HCL to terminate the reaction, measure the OD value at an ultraviolet wavelength of 450 nm, and calculate the competitive inhibition rate. The results are shown in Table 1.

[0055] The calculation formula for competitive inhibition rate is:

[0056] Table 1 Competitive inhibition ELISA results of each hybridoma cell line

[0057]

[0058]

[0059] As can be seen from Table 1, the competitive inhibition rate of hybridoma cell line PLCB-5 is the highest, so the hybridoma cell line PLCB-5 is selected for cloning culture.

[0060] (4-3) Cloning of hybridoma cell line PLCB-5

[0061] The cloning culture of hybridoma cell line PLCB-5 was carried out by limiting dilution method. The cells were accurately counted and diluted into 4 cells / mL cell suspension with IMDM medium containing 15% FBS by volume. Then 200 μl per well was inoculated into 96-well cell culture plates. After 7 days, the cell growth was observed and the antibody level of the cell culture supernatant in the cell culture plate was detected. Three monoclonal clones with the highest antibody titer were selected and cloned again until the positive rate of antibody detection in the monoclonal wells reached 100%. Then the monoclonal cells were picked and named PLCB-5.2.1. After expanded culture, the anti-psilocybin monoclonal antibody was purified.

[0062] (5) Purification of anti-psilocybin monoclonal antibodies

[0063] Take the hybridoma cell line PLCB-5.2.1 after expansion culture, inoculate 1×10 6 cells into the abdominal cavity of mice. After 10 days, collect the ascites. Take 10 ml of ascites, dilute it with 2 volumes of PBS, then add 3 volumes of saturated ammonium sulfate. Centrifuge the resulting mixture and take the precipitate. Dissolve the precipitate with PBS at pH 7.4, and then dialyze it 4 times overnight with PBS at pH 7.4. Collect the antibody solution and filter it. Use OD 280 to measure the protein content, and the protein concentration is 3.56 mg / ml.

[0064] Example 3 Reactivity Test of Monoclonal Antibody Against Psilocybin

[0065] The indirect ELISA method was used to detect the reactivity of the monoclonal antibody against psilocybin prepared in Example 1. The detection steps include:

[0066] ①Dilute the PLCB-BGG antigen with carbonate buffer at pH 9.6 and a concentration of 0.05 M to 50 ng / mL, and then add 100 μl / well of the diluted antigen to a 96-well enzyme-linked immunosorbent assay (ELISA) plate and coat it overnight at 4°C;

[0067] ②Wash the plate 3 times with PBS buffer containing 0.05% Tween 20; then add 100 μl of the monoclonal antibody solution against psilocybin at a concentration of 0.3 μg / ml and make a 1:3 dilution. At the same time, set up a negative control. After reacting at 37°C for 60 minutes, wash the plate once with PBS buffer containing 0.05% Tween 20;

[0068] ③Dilute the goat anti-mouse IgG-HRP with PBS buffer to 5000 times the volume, and then add 100 μl / well of the diluted goat anti-mouse IgG-HRP to a 96-well cell culture plate. React at 37°C for 30 minutes, and wash the plate 3 times with PBS buffer containing 0.05% Tween 20; then add 50 μl of the substrate TMB to each well and react at 37°C for 5 minutes, and terminate the reaction with 1 M HCl. Measure the OD value at 450 nm. Use the OD 450 value of the monoclonal antibody solution against psilocybin / OD 450 value of the negative control > 2.5 is a positive value; the detection results are shown in Table 2.

[0069] Table 2 OD 450 values of monoclonal antibody against psilocybin at different concentrations

[0070] Concentration of anti - psilocybin monoclonal antibody <![CDATA[OD 450 value]]> 0.3 μg / ml 2.382 0.1 μg / ml 2.312 0.03 μg / ml 2.086 0.01 μg / ml 1.634 0.003 μg / ml 1.036 0.001 μg / ml 0.434 0.0003 μg / ml 0.263 0.0001 μg / ml 0.082 PBS (blank control) 0.098

[0071] As can be seen from Table 2, the reactivity of the monoclonal antibody against psilocybin prepared in Example 1 can reach 0.001 μg / ml.

[0072] Example 4 Detection Limit Test of the Anti-psilocybin Monoclonal Antibody

[0073] The indirect ELISA method was used to detect the reactivity of the anti-psilocybin monoclonal antibody prepared in Example 1. The detection steps were as follows:

[0074] ① Dilute PD-BGG antigen to 1 μg / mL with carbonate buffer at pH 9.6 and a concentration of 0.05 M. Then add 100 μL / well of the diluted antigen to a 96-well ELISA plate and coat overnight at 4°C;

[0075] ② Wash the plate 3 times with PBS buffer containing 0.05% tween20; then add 50 μL of psilocybin standards at concentrations of 50 ng / ml, 40 ng / ml, 30 ng / ml, 20 ng / ml, and 10 ng / ml to the ELISA wells respectively. Then add 100 μL of the anti-psilocybin monoclonal antibody solution at a concentration of 0.3 μg / ml. At the same time, a blank control was designed, that is, first add 50 μL of PBS buffer and then add 50 μL of positive clone culture supernatant to the ELISA wells. After incubating at 37°C for 60 minutes, wash the plate once with PBS buffer containing 0.05% tween20;

[0076] ③ Dilute goat anti-mouse IgG-HRP with PBS buffer to 5000-fold volume. Then add 100 μL / well of the diluted goat anti-mouse IgG-HRP to a 96-well cell culture plate and react at 37°C for 30 minutes. Wash the plate 3 times with PBS buffer containing 0.05% tween20; then add 50 μL of substrate TMB to each well and react at 37°C for 8 minutes. Then terminate the reaction with 1 M HCL and measure the OD value at 450 nm. Take the OD value of the anti-psilocybin monoclonal antibody solution / the OD value of the negative control 450 value > 2.5 as the positive value; the detection results are shown in Table 3. 450 value > 2.5 as the positive value; the detection results are shown in Table 3.

[0077] Table 3 Competitive Inhibition ELISA Results of the Anti-psilocybin Monoclonal Antibody against Different Concentrations of Standards

[0078]

[0079]

[0080] As can be seen from Table 3, the lowest concentration of psilocybin that can be detected by the anti-psilocybin monoclonal antibody prepared in Example 1 is 500 ng / ml.

[0081] Application Example 1 Psilocybin Colloidal Gold Detection Strip

[0082] As Figure 1As shown, a psilocybin colloidal gold test strip in this embodiment includes a sample pad, a colloidal gold pad, a nitrocellulose membrane, and an absorbent pad; the sample pad, the colloidal gold pad, the nitrocellulose membrane, and the absorbent pad are sequentially and end-to-end overlapped on the bottom plate. Among them, the colloidal gold pad is coated with the monoclonal antibody against psilocybin prepared in Example 1 labeled with colloidal gold, the test line (T) of the nitrocellulose membrane is coated with a psilocybin-bovine serum albumin complex, and the quality control line (C) is coated with goat anti-mouse IgG. The preparation method of the test strip is a conventional method in the art.

[0083] During detection, insert one end of the sample pad of the test strip into the sample to be tested at room temperature, and note that the liquid level of the sample to be tested should not exceed the MAX line on the test strip; observe with the naked eye and record the color development of the C line and the T line.

[0084] If only the C line shows color, it means that the sample to be tested contains psilocybin and the content of psilocybin is higher than 500 ng / ml; if both the C line and the T line show color, it means that the content of psilocybin detected in the sample to be tested is lower than 500 ng / ml; if neither the C line nor the T line shows color, it means that the test strip has failed.

[0085] The above embodiments are not limitations on the present invention. The present invention is not limited to the above embodiments. As long as it meets the requirements of the present invention, it belongs to the protection scope of the present invention.

Claims

1. A hybridoma cell line secreting anti - psilocybin antibody, characterized in that, named hybridoma cell line PLCB - 5.2.1, and the deposit number is CCTCC No.C2022364.

2. An anti - psilocybin monoclonal antibody, characterized in that, it is secreted by the hybridoma cell line or its sub - cultured cell line described in claim 1.

3. An anti - psilocybin monoclonal antibody according to claim 2, characterized in that, the antibody is of IgG1 type and the light chain is of kappa type.

4. Use of the anti - psilocybin monoclonal antibody described in claim 2 in the preparation of a psilocybin detection kit.

5. The use according to claim 4, characterized in that, the detection kit includes a competitive inhibition ELISA detection kit and a competitive enzyme - linked immunosorbent assay kit.

6. Use of the anti - psilocybin monoclonal antibody described in claim 2 in the preparation of a colloidal gold immunochromatographic test strip for detecting psilocybin.

7. The use according to claim 6, characterized in that, the colloidal gold immunochromatographic test strip for detecting psilocybin includes a sample pad, a conjugate pad, a reaction membrane and an absorbent pad; the conjugate pad is coated with the colloidal gold - labeled anti - psilocybin monoclonal antibody.

8. The use according to claim 7, characterized in that, the detection line of the reaction membrane is coated with a psilocybin - bovine serum albumin complex, and the quality control line of the reaction membrane is coated with a secondary antibody.

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