CN113004401B - Anti-carpain monoclonal antibody and kit for detecting carpain - Google Patents

Abstract

The invention relates to the field of biological detection, and discloses an anti-carpain monoclonal antibody and a kit for detecting carpain. The anti-katyalanine monoclonal antibody is obtained by secreting a hybridoma cell strain Kratom11B7, can simultaneously detect the cephalothin and the 7-hydroxyl cephalothin, and can better detect edible katyaline leaves, katyaline leaf extracts and derivatives thereof. And has the characteristics of high affinity, high specificity and high sensitivity. Compared with other existing products, the kit for detecting the katain in the saliva has the advantages of sensitivity, specificity, detection limit and the like.

Description

Anti-carpain monoclonal antibody and kit for detecting carpain

Technical Field

The invention relates to the field of biological detection, in particular to an anti-carpain monoclonal antibody and a kit for detecting carpain.

Background

Caragane, rubiaceae, is a psychoactive plant growing in southeast Asia, particularly in Thailand and Malaysia. Fresh or dried kathon leaves can be consumed by sucking, chewing or brewing tea. Low doses of carbine have a stimulating effect and have long been used by southeast asian physical workers as stimulants to counteract work fatigue; high doses of katain have sedative-anesthetic effects, so katain leaves are used as a replacement for traditional drugs and opiates.

The pharmacological functions of katain are mainly realized by alkaloid 7-hydroxy cephalothin (formula II) and cephalothin (formula I). Although the structures of these alkaloid molecules are similar to those of hallucinogens, these substances have no hallucinogen activity. Instead, these alkaloids interact primarily with the adrenergic and opioid receptors. Thus, it is often used to prevent or relieve withdrawal symptoms in opioid-dependent patients, or to reduce opioid dependence. It also has a certain risk of being abused as a drug due to its delayed withdrawal syndrome action. Studies have found that eating calorie pain is followed by dry mouth, sweating, dizziness, increased or decreased urination, decreased appetite, and nausea or vomiting. The side effects which may be caused by long-term use are anorexia and weight loss, insomnia, dysphoria, sleep disorder, hallucinations and dependence.

According to the New York Times of America, the source of the pain-relieving leaf is rich in Thailand, the advanced extraction method of alkaloid is simple, a drug cocktail made of the pain-relieving leaf is popular at present, and a plurality of teenagers have fallen into the addiction to the pain-relieving leaf. The sap extracted from the kathon leaves is usually mixed with cough syrup, coca-cola and ice cubes. Carbatine is relatively safe in its natural state, high concentrations of alkaloid extracts are more hazardous than raw leaves, and Carbatine may have additive effects if used in combination with sedatives such as alcohol. Contamination or contamination with any material that is co-swallowed is also one of the risks. In europe, certain brands of katain, illegally incorporating the unpublished synthetic opioid receptor agonist O-desmethyltramadol, as well as other substances such as caffeine, have been exposed. The death of 9 people in sweden in 2010-2011 was related to a captain preparation which incorporated several other substances rather than pure natural captain. Therefore, the establishment of the katain detection kit with strong specificity and high sensitivity has great significance.

Disclosure of Invention

In order to solve the technical problems, the invention provides an anti-carpain monoclonal antibody and a kit for detecting carpain. The anti-katyalanine monoclonal antibody is obtained by secreting a hybridoma cell strain Kratom11B7, can simultaneously detect the cephalothin and the 7-hydroxyl cephalothin, and can better detect edible katyaline leaves, katyaline leaf extracts and derivatives thereof. And has the characteristics of high affinity, high specificity and high sensitivity. Compared with other existing products, the kit for detecting the katain in the saliva has the advantages of sensitivity, specificity, detection limit and the like.

The specific technical scheme of the invention is as follows:

in a first aspect, the invention provides an anti-Katong monoclonal antibody, which is secreted by a hybridoma cell strain, wherein the hybridoma cell strain is named as Kratom11B7 and is preserved in China Center for Type Culture Collection (CCTCC) in 1 month and 31 months of 2021; the preservation number is CCTCC NO: C202146.

further, the anti-carpain monoclonal antibody belongs to an IgG2 subtype.

In a first aspect, the present invention provides a method for preparing an anti-carmine monoclonal antibody, comprising the following steps:

step 1: preparing a pillared suberin complete antigen: taking the quebracho-hattachinoline as a raw material, protecting a secondary amino group by trifluoroacetic anhydride, hydrolyzing methyl ester by lithium hydroxide to obtain a carboxyl group, introducing 5-aminopentanoic acid to a carboxyl site to increase the arm length to obtain a quebracho-hattachinoline hapten, coupling the quebracho-hattachinoline hapten with a carrier protein by a carbodiimide method, and removing a protecting group to obtain the quebracho-hattachinine complete antigen.

The activity of the existing katain holoantigen is low. For example, patent CN108732345A discloses a test paper for detecting quebracho, its derivatives and metabolites in human saliva and a preparation method thereof, the quebracho is prepared by introducing succinic anhydride on benzene ring to form quebracho hemisuccinate derivatives, which can be coupled with bovine serum albumin to prepare whole antigen. However, the research of the team of the invention finds that the secondary amine group in the caecum alkali is an important active group of the antigen, the secondary amine group is not protected in the method, two sites appear when succinic anhydride is introduced, the coupling site is unknown, and the activity is low. In addition, the introduced branched chain (the branched chain is used for coupling carrier protein) has too short arm length, so that the macromolecular carrier protein can cover the active site and cannot reserve the active group in the Carngan molecule, and the obtained whole antigen has the problems of low antigen activity, poor specificity and low sensitivity.

The method for preparing the pillarine complete antigen and the 7-hydroxyl pillarine complete antigen can be used for preventing the active sites from being covered after the carrier protein is coupled by increasing the length of the branched chain (the length of the branched chain needs to be optimal in a straight chain with 4-6 carbon atoms, the antigenic determinant can be increased when the branched chain is too long, and the antigenic determinant of a small molecule can be covered when the branched chain is too short), so that the obtained antigen has stronger specificity and higher sensitivity. In addition, the method has the advantages of simple process steps, high reaction yield, mild reaction conditions and no need of using reagents with strong toxicity and corrosivity.

And 2, step: preparation of 7-hydroxy-cephaeline complete antigen: taking 7-hydroxy-pillared alkali as a raw material, hydrolyzing methyl ester with lithium hydroxide to obtain carboxyl, introducing 5-aminopentanoic acid to increase the arm length at the carboxyl site to obtain 7-hydroxy-pillared alkali hapten, and coupling the 7-hydroxy-pillared alkali hapten with carrier protein by a carbodiimide method to obtain the 7-hydroxy-pillalkali complete antigen.

And 3, step 3: preparation of hybridoma cell strain: and (3) immunizing a mouse by using the pillarine complete antigen and the 7 hydroxyl pillarine complete antigen to obtain a hybridoma cell strain Kratom11B7.

The immunization method of the continuous enhanced stimulation method has the advantages of less antigen dosage, short immunization time, good stability, strong affinity and the like.

And 4, step 4: preparation of antibody: the hybridoma cell strain Kratom11B7 is used for secreting and producing the anti-katain monoclonal antibody, and the separation and purification treatment are carried out.

Preferably, step 3 comprises:

step 3.1: immunization;

step 3.2: fusion of splenocytes with myeloma cells;

step 3.3: and (4) screening hybridoma cells.

Further, step 3.1 specifically comprises:

mixing the pillared aristosine complete antigen and the 7-hydroxypillared aristosine complete antigen according to the mass ratio of (0.8-1.2) to 1, and then diluting the mixture to the concentration of 0.8-1.2mg/ml by using PBS buffer solution; and immunizing on the 1 st day, mixing the diluted antigen and the complete adjuvant according to the mass ratio of (0.8-1.2) to 1, injecting 3-7 ug/mouse subcutaneously into the mouse, injecting 8-12 ug/mouse near the lymph node on the 6 th day by using the incomplete adjuvant, injecting 13-17 ug/mouse near the lymph node on the 11 th day, injecting 18-22 ug/mouse near the lymph node on the 16 th day, injecting 23-27 ug/mouse near the lymph node on the 21 st day, and directly injecting 3-7 ug/mouse intravenously on the 24 th day.

Further, step 3.2 specifically includes:

step 3.2.1: preparation of a feeder cell suspension: killing a mouse by removing eyeballs and bloodletting, soaking the mouse in alcohol for disinfection, tearing the skin outside the abdomen of the mouse to expose the peritoneum of the mouse, injecting DMEM serum-free culture medium preheated at 35-40 ℃, slightly kneading the abdominal cavity of the mouse for 1-2 minutes, suspending abdominal cavity cells, and sucking out abdominal cavity liquid; cutting the chest cavity of the mouse, taking the thymus, grinding, collecting suspension, combining with the peritoneal fluid, centrifuging, and re-suspending the precipitate with HAT complete culture solution to obtain feeder cell suspension;

step 3.2.2: culture of mouse myeloma cells SP 2/0: subculturing the mouse myeloma cell SP2/0 in DMEM medium containing 8-12% by volume of FBS, subculturing the mouse myeloma cell SP2/0 one day before cell fusion to ensure that the mouse myeloma cell SP2/0 is suitable for the logarithmic phase of growth and the growth state is good for cell fusion;

step 3.2.3: preparation of splenocytes: killing the immunized mouse, disinfecting the mouse by soaking in alcohol, cutting the abdomen, taking out the spleen aseptically, placing the spleen on a filter screen connected with a centrifuge tube, immediately adding serum-free DMEM culture solution, shearing the spleen by ophthalmic scissors, lightly grinding the spleen by using a sterile syringe handle to filter spleen cells into the centrifuge tube through meshes, adding the serum-free culture solution, and centrifuging; discarding the supernatant, and repeatedly centrifuging with serum-free culture solution for later use;

step 3.2.4: fusion of splenocytes with myeloma cells: mixing splenocytes and mouse myeloma cell SP2/0, centrifuging to wash the cells, removing supernatant, placing in 35-40 deg.C water bath, adding 0.5-1.0ml of 35-40 deg.C pre-warmed PEG4000, adding the mixture within one minute, and standing for 80-100 s; adding 20-30ml of 35-40 ℃ pre-warmed DMEM serum-free culture solution to stop the PEG effect; centrifuging and taking the precipitate; then adding a suspension of feeder cells to the pelletInoculating into 96-well cell culture plate, standing at 35-40 deg.C, 3-7% ₂ Culturing in an incubator; after 20-30 hours, HAT medium is adopted, and after HAT selection culture solution is maintained and cultured for two weeks, HT culture solution is used, and then the maintenance and culture are carried out for two weeks, and common culture solution is used to obtain the fused cells.

Further, step 3.3 specifically comprises:

step 3.3.1: primary screening: changing the liquid once every 2-4 days after the fused cells, observing the growth condition of the fused cells in a 96-hole cell culture plate, absorbing the culture supernatant of the fused cells when the cells grow to cell clusters, and screening positive clones by adopting an indirect ELISA method; respectively taking the cephaeline complete antigen and the 7 hydroxyl cephaeline complete antigen as coating antigens, and screening cells with positive ELISA for rescreening;

step 3.3.2: re-screening: further using the pillared arisine and 7-hydroxy pillared arisine standard to perform re-screening on the screened positive clones by a competitive inhibition ELISA method, and screening hybridoma cell strains with the highest competitive inhibition rate for clone culture;

step 3.3.3: cloning of hybridoma cell line kratom: performing cloning culture of the hybridoma cell strain according to a limiting dilution method, accurately counting cells, diluting the cells into 3-5/ml cell suspension by using a DMEM medium containing 8-12% FBS, then inoculating 200 mu l of diluted cell suspension into a 96-well cell culture plate, observing the cell growth condition after 7 days, detecting the antibody level of cell culture supernatant, selecting a plurality of monoclones with the highest titer, and performing cloning culture until the monoclonal well antibody detection positive rate reaches 100 many times; to obtain an anti-Katong monoclonal cell line Kratom11B7 capable of stably secreting specific antibodies.

Preferably, in step 1, the preparation of the quebracho complete antigen comprises:

a) Protection of amino group: dissolving the pillared arietin in an organic solvent, stirring and dissolving at room temperature, slowly dropwise adding trifluoroacetic anhydride with the molar weight 1-3 times that of the pillared arietin in an ice-water bath under the protection of inert gas, removing the ice-water bath, and stirring and reacting at room temperature; after the reaction is finished, evaporating the solvent to dryness under reduced pressure in a water bath to obtain a light yellow oil liquid, adding n-hexane for dissolving, crystallizing into a white solid in an ice water bath, and washing the white solid with the n-hexane;

b) Hydrolysis of the ester group: adding methanol and water into the obtained white solid, adding lithium hydroxide in an amount which is 5 to 10 times of the molar weight of the white solid, stirring and dissolving, and continuing to react; after the reaction is finished, analyzing by using a thin plate chromatography, adjusting the pH of the raw material Rf =0.6-0.8 and the product Rf =0.2-0.4 by using dilute hydrochloric acid, and evaporating the solvent under reduced pressure in a water bath; dissolving in anhydrous alcohol, filtering, and evaporating the solvent under reduced pressure; separating by thin plate chromatography, collecting Rf =0.2-0.4 product spot under ultraviolet with petroleum ether/methanol mixed solution as developing agent, collecting obtained silica gel, repeatedly extracting with anhydrous ethanol, mixing, and evaporating under reduced pressure to remove anhydrous ethanol to obtain pale yellow oily substance, i.e. hatted pillar wood alkali acid;

c) Increasing arm length introduces carboxyl: dissolving the obtained quebracho in pyridine, adding 1-3 times of 5-aminovaleric acid, adding EDC with the same molar amount as 5-aminovaleric acid, and stirring at room temperature; after the reaction is finished, evaporating the solvent under reduced pressure, adding dichloromethane for dissolution, extracting for multiple times, taking a lower organic phase, drying the organic phase, filtering, and evaporating the solvent under reduced pressure; separating by thin plate chromatography, collecting product spots with Rf =0.4-0.5 under ultraviolet with 95wt% ethanol/ammonia water/dichloromethane/1, 4-dioxane mixed solution as developing agent, repeatedly extracting the collected silica gel with anhydrous ethanol, mixing, and evaporating under reduced pressure to remove anhydrous ethanol to obtain oily substance, namely calyx-shaped column lignan hapten;

d) Activating the coupled carrier protein: dissolving the obtained calophylline hapten in dimethylformamide, adding N-hydroxysuccinimide 0.4-0.5 times of the calophylline hapten and dicyclohexylcarbodiimide 0.7-0.8 times of the calophylline hapten, stirring at room temperature overnight, centrifuging, and taking the supernatant; adding the supernatant into 8-12mg/ml carrier protein/PBS solution according to the volume ratio of 1:3-5, uniformly mixing, stirring at room temperature for reaction, and standing overnight; putting the reaction solution into a sodium carbonate solution with the pH value of 11-13 for dialysis, and then dialyzing in a PBS buffer solution, wherein the buffer solution is changed every day; and after the dialysis is finished, centrifuging the reaction solution, and taking supernatant, namely the cap column lignan complete antigen.

In the process of preparing the pillared arietin complete antigen, too little trifluoroacetic anhydride can influence the reaction rate, the yield and the purity, and too much causes material waste and the next step of recrystallization; too little lithium hydroxide to hydrolyze the ester group affects the reaction rate.

Preferably, in step 2, the preparation of the 7 hydroxycoumarin complete antigen comprises:

a) Hydrolysis of the ester group: weighing 7-hydroxy pillared alkali powder in a container, adding methanol and water, adding 5-10 times of lithium hydroxide in molar weight, stirring for dissolving, and continuing to react; after the reaction is finished, analyzing by thin plate chromatography, wherein the raw material Rf =0.5-0.7, the product Rf =0.2-0.3, adjusting the pH =4.5-5.5 by using dilute hydrochloric acid, and evaporating the solvent under reduced pressure in a water bath; dissolving in anhydrous alcohol, filtering, and evaporating the solvent under reduced pressure; separating by thin plate chromatography, collecting product spot with Rf =0.2-0.3 under ultraviolet with petroleum ether/methanol mixed solution as developing agent, collecting obtained silica gel, repeatedly extracting with anhydrous ethanol, mixing, and evaporating anhydrous ethanol under reduced pressure to obtain light yellow oily 7 hydroxy hated pillar wood acid;

b) Increasing arm length introduces carboxyl: dissolving the obtained 7-hydroxy pillared wood alkali in pyridine, adding 1-3 times of 5-aminopentanoic acid in molar amount, adding EDC in equimolar amount with 5-aminopentanoic acid, and stirring at room temperature; after the reaction is finished, evaporating the solvent under reduced pressure, adding dichloromethane for dissolution, extracting for multiple times, taking a lower organic phase, drying the organic phase, filtering, and evaporating the solvent under reduced pressure; separating by thin plate chromatography, collecting product spots with Rf =0.3-0.4 under ultraviolet with 95wt% ethanol/ammonia water/dichloromethane/1, 4-dioxane mixed solution as developing agent, repeatedly extracting the collected silica gel with anhydrous ethanol, mixing, and evaporating under reduced pressure to remove anhydrous ethanol to obtain oily substance, namely 7 hydroxy pillared lignan hapten;

c) Activating the coupled carrier protein: dissolving the obtained 7 hydroxyl pillared suberine hapten in dimethylformamide, adding N-hydroxysuccinimide 0.4-0.5 times of the pillared suberine hapten and dicyclohexylcarbodiimide 0.7-0.8 times of the pillared suberine hapten, stirring at room temperature overnight, centrifuging, and taking the supernatant; adding the supernatant into 8-12mg/ml carrier protein/PBS solution according to the volume ratio of 1: 6-10, uniformly mixing, stirring at room temperature for reaction, and standing overnight; putting the reaction solution into a sodium carbonate solution with the pH value of 11-13 for dialysis, and then dialyzing in a PBS buffer solution, wherein the buffer solution is changed every day; and after the dialysis is finished, centrifuging the reaction solution, and taking supernatant, namely the 7-hydroxyl hattacharpin complete antigen.

In a third aspect, the present invention provides a kit for detecting katain in saliva, comprising:

a colloidal gold-labeled complex carrying the anti-katain monoclonal antibody,

nitrocellulose membrane coated with a cardamine-protein complex.

The kit for detecting the katain in the saliva is simpler, more convenient and quicker than the detection method used in the prior art. The reason is that the monoclonal antibody produced by the hybridoma cell strain prepared by cell fusion of mouse lymphocytes and mouse myeloma cells has high specific reactivity with the cephalomannine and the 7-hydroxyl cephalomannine.

In a fourth aspect, the invention provides a preparation method of the above kit, comprising the following steps:

preparing colloidal gold;

preparing a gold label;

preparing gold label strips;

coating a nitrocellulose membrane;

processing a glass cellulose membrane;

and assembling the reagent strip.

Preferably, the preparation method of the kit specifically comprises the following steps:

preparing colloidal gold: adding chloroauric acid solution into water, heating to boil, adding trisodium citrate solution, mixing, boiling until the color changes from blue to purple, and changing to mauve, and cooling to obtain colloidal gold solution containing 20-40nm colloidal gold particles;

preparation of gold labels: taking colloidal gold liquid, adjusting the pH value to 8-9, adding an anti-seizure monoclonal antibody, stirring, adding protein, and stirring for later use; centrifuging the colloidal gold solution coupled with the antibody, discarding the precipitate, and reserving the solution; re-dissolving the precipitate with sucrose-containing gold-labeled antibody diluent, filtering, mixing the solutions to obtain a gold-labeled working solution, and storing for later use;

preparing gold label strips: uniformly spraying the gold mark working solution on a gold mark pad, drying, cutting into strips to obtain gold mark strips, and sealing for later use; coating of nitrocellulose membrane: respectively loading a Caragana antigen conjugate (a hattacharzimine synthetic antigen) and a goat anti-mouse polyclonal antibody into a T column and a C column of a film spotting machine, and coating a solution of the T line and a solution of the C line on a nitrocellulose membrane;

and (3) glass fiber treatment: cutting the glass cellulose membrane into strips, soaking the strips in glass fiber treatment liquid, taking out the glass fiber treatment liquid, drying and sealing for later use; assembling the reagent strip: and respectively sticking the processed nitrocellulose membrane, gold label strip, glass cellulose membrane and absorbent paper onto a PVC plate, and cutting into strips to obtain the kit.

In a fifth aspect, the present invention provides a jam detection method, including the following steps: the kit of claim 8 is adopted, so that the sample to be detected and the capping column lignan synthetic antigen coated on the nitrocellulose membrane have competitive reaction with the colloidal gold labeled compound, and whether the content of the capping column lignan or the 7-hydroxy capping column lignan in the sample to be detected exceeds the standard is judged according to the color development intensity of the T line.

Compared with the prior art, the invention has the following technical effects:

(1) The anti-katain monoclonal antibody is secreted by a hybridoma cell strain Kratom11B7, can simultaneously detect the hattacrine and the 7-hydroxyl hattacrine, and can better detect edible katain leaves, katain leaf extracts and derivatives thereof. And has the characteristics of high affinity, high specificity and high sensitivity.

(2) The kit for detecting the Carbayonia in saliva is simpler, more convenient and quicker than the detection method used in the prior art, and is suitable for saliva detection with a15 ng/ml threshold value. Compared with other existing products, the detection product provided by the invention has more advantages in the aspects of sensitivity, specificity, detection limit and the like.

(3) In the process of preparing the katain antigen, active groups are firstly subjected to clustering protection, then, a methyl ester group is hydrolyzed by lithium hydroxide to obtain a carboxyl group, and 5-aminovaleric acid with a longer chain length is introduced into a carboxyl site. And then activating carboxyl by using carbodiimide as a crosslinking agent to obtain hapten, wherein the structure of the selected site and the crosslinking method are not obviously changed, and antigenic determinants are reserved. A bridge structure is introduced between the katain hapten and the carrier protein to expose the antigenic determinant, and the obtained katain artificial antigen keeps the structural specificity of the katain and is beneficial to the generation of a corresponding katain antibody.

Drawings

FIG. 1 is a color chart for measuring the color development intensity of the kit in example 6.

Detailed Description

The present invention will be further described with reference to the following examples.

General examples

The preparation of the katain antigen comprises the following steps:

preparing a quebracho complete antigen:

a) Protection of amino group: dissolving the pillared arietin in an organic solvent, stirring and dissolving at room temperature, slowly dropwise adding trifluoroacetic anhydride with the molar weight 1-3 times that of the pillared arietin in an ice-water bath under the protection of inert gas, removing the ice-water bath, and stirring and reacting at room temperature for 2-4h; after the reaction is finished, evaporating the solvent to dryness under reduced pressure in a water bath at 40-50 ℃ to obtain a light yellow oil liquid, adding 5-15ml of n-hexane for dissolving, crystallizing into a white solid in an ice water bath, and washing the white solid with the n-hexane;

b) And (3) hydrolysis of an ester group: adding 5-15mL of methanol and 1-3mL of water into the obtained white solid, adding 5-10 times of lithium hydroxide in molar weight, stirring to dissolve, and continuously reacting for more than 20 h; after the reaction is finished, analyzing by thin plate chromatography, wherein the raw material Rf =0.6-0.8, the product Rf =0.2-0.4, adjusting the pH =4.5-5.5 by using dilute hydrochloric acid, and evaporating the solvent under reduced pressure in a water bath at the temperature of 40-50 ℃; adding 5-15ml of absolute ethyl alcohol for dissolving, filtering, and evaporating the solvent to dryness under reduced pressure; separating by thin plate chromatography, collecting Rf =0.2-0.4 product spot under ultraviolet with petroleum ether/methanol mixed solution at volume ratio of 4-6: 1 as developing agent, collecting the obtained silica gel, repeatedly extracting with anhydrous ethanol, mixing, and evaporating under reduced pressure to remove anhydrous ethanol to obtain pale yellow oily substance cap column wood alkali acid;

c) Increasing arm length introduces carboxyl: dissolving the obtained pillared aristolic acid in 5-15ml of pyridine, adding 1-3 times of 5-aminopentanoic acid, adding EDC with the same molar amount as 5-aminopentanoic acid, and stirring at room temperature for more than 6 h; after the reaction is finished, evaporating the solvent under reduced pressure, adding 15-25ml of dichloromethane for dissolution, extracting for multiple times, taking a lower layer of organic phase, drying the organic phase, filtering, and evaporating the solvent under reduced pressure; separating by thin plate chromatography, collecting product points with Rf =0.4-0.5 under ultraviolet with 95wt% ethanol/ammonia water/dichloromethane/1, 4-dioxane mixed solution with volume ratio of (6-10): (0.5-1.5): (6-10): 1 as developing agent, collecting the obtained silica gel, repeatedly extracting with anhydrous ethanol, mixing, and evaporating under reduced pressure to remove anhydrous ethanol to obtain oily matter, namely cap column woodine hapten;

d) Activation of coupled carrier protein: dissolving the obtained quebracho hapten in dimethylformamide according to the solid-to-liquid ratio of 90-110mg/5ml, adding N-hydroxysuccinimide with the mass of 0.4-0.5 time of that of the quebracho hapten and dicyclohexylcarbodiimide with the mass of 0.7-0.8 time of that of the quebracho hapten, stirring overnight at room temperature, centrifuging at 8000-12000rpm at 1-5 ℃ for 1-10min, and taking a supernatant; adding the supernatant into 8-12mg/ml carrier protein/PBS solution according to the volume ratio of 1:3-5, uniformly mixing, stirring at room temperature for reaction for 1-3h, and standing at 2-8 ℃ overnight; dialyzing the reaction solution in sodium carbonate solution with pH of 11-13 for 2-4 days, and dialyzing in PBS buffer solution for 1-3 days, wherein the buffer solution is changed for 2-4 times every day; and after the dialysis is finished, centrifuging the reaction solution, and taking supernatant, namely the cap column lignan complete antigen.

Preparation of 7-hydroxy-cephaeline complete antigen:

a) Hydrolysis of the ester group: weighing 7 hydroxyl pillared alkali powder in a container, adding 5-15mL of methanol and 1-3mL of water, adding 5-10 times of lithium hydroxide in molar weight, stirring for dissolving, and continuing to react for more than 20 h; after the reaction is finished, analyzing by thin plate chromatography, wherein the raw material Rf =0.5-0.7, the product Rf =0.2-0.3, adjusting the pH =4.5-5.5 by using dilute hydrochloric acid, and evaporating the solvent under reduced pressure in a water bath at the temperature of 40-50 ℃; adding 5-15ml of absolute ethyl alcohol for dissolving, filtering, and evaporating the solvent to dryness under reduced pressure; separating by thin plate chromatography, collecting Rf =0.2-0.3 product spot under ultraviolet with petroleum ether/methanol mixed solution at volume ratio of 4-6: 1 as developing agent, collecting the obtained silica gel, repeatedly extracting with anhydrous ethanol, mixing, and evaporating anhydrous ethanol under reduced pressure to obtain light yellow oily 7 hydroxy pillared suberic acid;

b) Increasing arm length introduces carboxyl: dissolving the obtained 7-hydroxy pillared wood alkali in 5-15ml of pyridine, adding 1-3 times of 5-aminopentanoic acid, adding EDC with the same molar amount as 5-aminopentanoic acid, and stirring at room temperature for more than 6 h; after the reaction is finished, evaporating the solvent under reduced pressure, adding 15-25ml of dichloromethane for dissolution, extracting for many times, taking a lower organic phase, drying the organic phase, filtering, and evaporating the solvent under reduced pressure; separating by thin plate chromatography, taking a 95wt% ethanol/ammonia water/dichloromethane/1, 4-dioxane mixed solution with the volume ratio of (6-10) to (0.5-1.5) to (6-10) to 1 as a developing agent, collecting product points with Rf =0.3-0.4 under ultraviolet, collecting the obtained silica gel, repeatedly extracting with absolute ethanol, mixing, and evaporating the absolute ethanol under reduced pressure to obtain an oily substance, namely 7-hydroxy pillared alkali hapten;

c) Activating the coupled carrier protein: dissolving the obtained 7 hydroxyl capping column wood alkali hapten in dimethyl formamide according to the solid-to-liquid ratio of 90-110mg/5ml, adding N-hydroxysuccinimide with the mass of 0.4-0.5 time of that of the capping column wood alkali hapten and dicyclohexylcarbodiimide with the mass of 0.7-0.8 time of that of the capping column wood alkali hapten, stirring overnight at room temperature, centrifuging at 8000-12000rpm and 1-5 ℃ for 1-10min, and taking supernatant; adding the supernatant into 8-12mg/ml carrier protein/PBS solution according to the volume ratio of 1: 6-10, uniformly mixing, stirring at room temperature for reaction for 1-3h, and standing at 2-8 ℃ overnight; dialyzing the reaction solution in sodium carbonate solution with pH of 11-13 for 2-4 days, and dialyzing in PBS buffer solution for 1-3 days, wherein the buffer solution is changed for 2-4 times every day; and after the dialysis is finished, centrifuging the reaction solution, and taking supernatant, namely the 7-hydroxyl hattacharpin complete antigen.

A hybridoma cell strain capable of producing an anti-Katong monoclonal antibody, which is named as hybridoma cell strain Kratom11B7 and has been deposited in the China center for type culture Collection at 31.1.1.2021; the preservation number is CCTCC NO: C202146.

a preparation method of an anti-carpain monoclonal antibody comprises the following steps:

step 1: preparing a quebracho complete antigen;

step 2: preparing 7 hydroxyl hattacharpin complete antigen;

and step 3: preparation of hybridoma cell strain: the pillarine complete antigen and the 7 hydroxyl pillarine complete antigen are used for immunizing a mouse together to obtain a hybridoma cell strain Kratom11B7.

Preferably, step 3 comprises:

step 3.1: immunization: mixing the pillared aristosine complete antigen and the 7-hydroxypillared aristosine complete antigen according to the mass ratio of (0.8-1.2) to 1, and then diluting the mixture to the concentration of 0.8-1.2mg/ml by using PBS buffer solution; the diluted antigen and the complete adjuvant are mixed according to the mass ratio (0.8-1.2) to 1 after immunization on the 1 st day, 3-7 ug/mouse is injected subcutaneously on the mouse, the incomplete adjuvant is mixed with the antigen on the 6 th day, 8-12 ug/mouse is injected near the lymph node of the mouse, 13-17 ug/mouse is injected near the lymph node of the mouse on the 11 th day, 18-22 ug/mouse is injected near the lymph node of the mouse on the 16 th day, 23-27 ug/mouse is injected near the lymph node of the mouse on the 21 st day, and 3-7 ug/mouse is directly injected intravenously on the 24 th day.

Step 3.2: fusion of splenocytes with myeloma cells:

step 3.2.1: preparation of feeder cell suspension: killing a mouse by removing eyeballs and bloodletting, soaking the mouse in alcohol for disinfection, tearing the skin outside the abdomen of the mouse to expose the peritoneum of the mouse, injecting DMEM serum-free culture medium preheated at 35-40 ℃, slightly kneading the abdominal cavity of the mouse for 1-2 minutes, suspending abdominal cavity cells, and sucking out abdominal cavity liquid; cutting the chest cavity of the mouse, taking the thymus, grinding, collecting suspension, combining with the peritoneal fluid, centrifuging, and re-suspending the precipitate with HAT complete culture solution to obtain feeder cell suspension;

step 3.2.2: culture of mouse myeloma cells SP 2/0: subculturing the mouse myeloma cell SP2/0 by using a DMEM medium containing 8-12% FBS by volume fraction, and subculturing the mouse myeloma cell SP2/0 one day before cell fusion so as to ensure that the mouse myeloma cell SP2/0 is suitable for a logarithmic phase of growth and a good growth state is used for cell fusion;

step 3.2.3: preparation of splenocytes: killing the immunized mouse, disinfecting the mouse by soaking in alcohol, cutting the abdomen, taking out the spleen aseptically, putting the spleen on a filter screen connected with a centrifuge tube, immediately adding serum-free DMEM culture solution, shearing the spleen by using an ophthalmic scissors, then lightly grinding the spleen by using a sterile syringe handle, filtering the spleen cells into the centrifuge tube through meshes, adding the serum-free culture solution, and centrifuging; discarding the supernatant, and repeatedly centrifuging with serum-free culture solution for later use;

step 3.2.4: spleen cells fused with myeloma cells: uniformly mixing splenocytes with mouse myeloma cells SP2/0, centrifuging and washing the cells, removing supernatant, placing in a water bath at 35-40 ℃, adding 0.5-1.0ml of PEG4000 pre-warmed at 35-40 ℃, completing the addition within one minute, and then standing for 80-100 seconds; adding 20-30ml of 35-40 deg.C pre-warmed DMEM serum-free culture solution to stop PEG effect; centrifuging and taking the precipitate; then adding the suspension of feeder cells to the pellet, inoculating the pellet into a 96-well cell culture plate, subjecting the plate to 35-40 ℃ and 3-7% CO ₂ Culturing in an incubator; after 20-30 hours, HAT culture medium is adopted, after HAT selection culture solution is maintained and cultured for two weeks, HT culture solution is used, and then maintained and cultured for two weeks,the general culture medium was used instead to obtain fused cells.

Step 3.3: screening of hybridoma cells:

step 3.3.1: primary screening: changing the liquid once every 2-4 days after the fused cells, observing the growth condition of the fused cells in a 96-hole cell culture plate, absorbing the culture supernatant of the fused cells when the cells grow to cell clusters, and screening positive clones by adopting an indirect ELISA method; respectively taking the cephaeline complete antigen and the 7 hydroxyl cephaeline complete antigen as coating antigens, and screening cells with positive ELISA for rescreening;

step 3.3.2: re-screening: the screened positive clones are further rescreened by competitive inhibition ELISA method with the hattacroline and 7-hydroxy hattacroline standard substance, and hybridoma cell strains with highest competitive inhibition rate are screened out for clone culture;

step 3.3.3: cloning of hybridoma cell line kratom: performing cloning culture of the hybridoma cell strain according to a limiting dilution method, accurately counting cells, diluting the cells into 3-5/ml cell suspension by using a DMEM medium containing 8-12% FBS, then inoculating 200 mu l of diluted cell suspension into a 96-well cell culture plate, observing the cell growth condition after 7 days, detecting the antibody level of cell culture supernatant, selecting a plurality of monoclones with the highest titer, and performing cloning culture until the monoclonal well antibody detection positive rate reaches 100 many times; to obtain an anti-Katong monoclonal cell line Kratom11B7 capable of stably secreting specific antibodies.

And 4, step 4: preparation of antibody: the hybridoma cell strain Kratom11B7 is used for secreting and producing the anti-katain monoclonal antibody, and the separation and purification treatment are carried out.

A kit for detecting katain in saliva, comprising:

a colloidal gold-labeled complex carrying the anti-katain monoclonal antibody,

nitrocellulose membrane coated with a cardamine-protein complex.

The preparation method of the kit comprises the following steps:

preparing a colloidal gold solution: adding 2ml of 1% chloroauric acid solution into 100ml of water, heating to boil, adding 1-3ml of 1% trisodium citrate solution, mixing and boiling for 5-10 minutes until the color of the colloidal gold solution changes from blue to purple and then to mauve, cooling to obtain a colloidal gold solution containing 20-40nm colloidal gold particles, and storing at 2-8 ℃ for later use;

preparing a gold-labeled solution: taking 1ml of colloidal gold solution, adjusting the pH value to 8-9 by using 1M NaOH solution, then adding 10-20ug of anti-katain monoclonal antibody, stirring, then adding 100-200ul of 10% BSA protein solution, and stirring for later use; centrifuging the colloidal gold solution coupled with the antibody, discarding the precipitate, and reserving the solution; re-dissolving the precipitate with 20% sucrose-containing gold-labeled antibody diluent, filtering, mixing the solutions to obtain gold-labeled working solution, and storing at 2-8 deg.C;

preparing the gold label strip: uniformly spraying the gold-labeled working solution on a gold-labeled pad, drying at 35-40 ℃, cutting into strips to obtain gold-labeled strips, sealing in an aluminum foil bag filled with a drying agent, and storing at room temperature for later use;

coating of nitrocellulose membrane: respectively loading a carpain-protein compound (a hattacharpin synthetic antigen) and a goat anti-mouse polyclonal antibody into a T column and a C column of a film spotting machine, and coating a solution of the T line and a solution of the C line on a nitrocellulose membrane;

and (3) glass fiber treatment: cutting the glass cellulose membrane into strips, soaking the strips in glass fiber treatment liquid, taking out the glass fiber treatment liquid, drying and sealing for later use; assembling the reagent strip: and respectively sticking the processed nitrocellulose membrane, gold label strip, glass cellulose membrane and absorbent paper to a PVC plate, and cutting into strips of 3-4mm to obtain the kit.

A method for detecting stuck pain, comprising the steps of: by adopting the kit, a sample to be detected and the capping column wood alkali synthetic antigen coated on the nitrocellulose membrane are subjected to competitive reaction with the colloidal gold labeled compound, and whether the content of the capping column wood alkali or the 7-hydroxyl capping column wood alkali in the sample to be detected exceeds the standard or not is judged according to the color development intensity of the T line.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present invention will be further analyzed with reference to the following examples (in the following examples, PBS buffer is an aqueous solution containing 0.008M disodium hydrogen phosphate dodecahydrate, 0.15M sodium chloride, and 0.002M sodium dihydrogen phosphate dihydrate, pH 7.4; subcutaneous part near lymph node is axillary, forearm or neck of four limbs; 1 XHAT DMEM medium contains 15% by volume of FBS; and 1 XHT DMEM medium contains 15% by volume of FBS).

Example 1: preparation of Caragan antigen

Preparation of complete Antigen of quebracho (Antigen 1):

a) Protection of amino groups

398.5mg (1 mmol) of pillared arisin powder is weighed into a150 ml three-neck flask, 60ml of dichloromethane is added, stirring and dissolving are carried out at room temperature, under the condition of ice water bath and under the protection of nitrogen, 420mg (2 mmol) of trifluoroacetic anhydride is slowly dropped, the ice water bath is removed, and stirring is carried out at room temperature for 3 hours. After the reaction, the solvent was evaporated to dryness under reduced pressure in a water bath at 45 ℃ to obtain a pale yellow oily liquid, which was dissolved in 10ml of n-hexane. It can be crystallized into white solid in ice-water bath. 420mg (0.86 mmol) of the white solid was washed with n-hexane.

b) Hydrolysis of ester group

To the white solid, 10mL of methanol and 2mL of water were added, and 158mg (6.88 mmol) of lithium hydroxide was added, and after dissolution by stirring, the reaction was continued for 20 hours or more.

At the end of the reaction, analysis by thin-plate chromatography (TLC) showed starting material Rf =0.7 and product Rf =0.3, pH =5 with dilute hydrochloric acid and solvent was evaporated under reduced pressure in a water bath at 45 ℃. Dissolving in 10ml of absolute ethanol, filtering, and evaporating the solvent to dryness under reduced pressure. Separation by TLC, developing solvent 1: collecting Rf =0.3 product spot under ultraviolet with petroleum ether/methanol = 5: 1 (v/v) as developing agent, repeatedly extracting the collected silica gel with anhydrous ethanol, mixing, and evaporating anhydrous ethanol under reduced pressure to obtain pale yellow oily substance of cap column wood acid 365mg (0.77 mmol) for use.

c) Introduction of carboxyl groups by increasing arm length

The above-obtained pillared aristolic acid was dissolved in 10ml of pyridine, 158mg (1.54 mmol) of 5-aminopentanoic acid was added thereto, and 295mg (1.54 mmol) of EDC was added thereto and the mixture was stirred at room temperature for 6 hours or more.

After the reaction was completed, the solvent was distilled off under reduced pressure, 20ml of dichloromethane was added to dissolve the solvent, the mixture was extracted with 20ml _ 3, the lower organic phase was taken out, the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure. Separation by TLC, developing agent 2: a developing solvent was prepared by collecting a spot of the product having Rf =0.4-0.5 under ultraviolet irradiation at a ratio of 95% ethanol, ammonia water, dichloromethane, 1, 4-dioxane = 8: 1: 8: 1 (v/v), repeatedly extracting the collected silica gel with absolute ethanol, mixing, and evaporating the absolute ethanol under reduced pressure to obtain an oily substance, namely, 396mg (0.71 mmol) of the pillarine hapten.

d) Activated conjugated carrier proteins

100mg of the pillarine hapten is dissolved in 5ml of Dimethylformamide (DMF), and 45mg of N-hydroxysuccinimide (NHS) and 75mg of Dicyclohexylcarbodiimide (DCC) are added and stirred at room temperature overnight. Centrifugation was carried out at 4 ℃ and set at 10000rpm for 5min. And taking the supernatant for later use.

50ml of 10mg/ml BSA/PBS solution were prepared.

The supernatant was added to 10mg/ml BSA/PBS solution at a ratio of 1: 5 (V/V), mixed well, stirred at room temperature for 2 hours, and then left to stand at 2-8 ℃ overnight. The reaction solution was dialyzed against 0.5% sodium carbonate solution at pH 12 for three days, and then against PBS buffer solution at pH 7.4 and 0.01M for two days, during which the buffer was changed three times a day. And after dialysis, centrifuging the reaction solution, and taking supernatant, namely the capping column lignan synthetic antigen with the concentration of 6.9mg/ml.

Preparation of 7-hydroxy-pillarine complete Antigen (Antigen 2):

a) Hydrolysis of ester group

414.5mg (1 mmol) of 7 hydroxycoumarin powder was weighed into a 100mL single-neck flask, 10mL of methanol and 2mL of water were added, 184mg (8 mmol) of lithium hydroxide was added, and the reaction was continued for 20 hours or more after dissolution with stirring.

At the end of the reaction, analysis by thin-plate chromatography (TLC) showed starting material Rf =0.6 and product Rf =0.25, pH =5 with dilute hydrochloric acid and solvent was evaporated under reduced pressure in a water bath at 45 ℃. Dissolving in 10ml of absolute ethanol, filtering, and evaporating the solvent to dryness under reduced pressure. Separation by TLC, developing solvent 1: collecting Rf =0.25 product spot under ultraviolet with petroleum ether/methanol = 5: 1 (v/v) as developing agent, repeatedly extracting the collected silica gel with anhydrous ethanol, mixing, and evaporating the anhydrous ethanol under reduced pressure to obtain a light yellow oily substance, namely 349mg (0.87 mmol) of 7-hydroxy-capped-column-type lignanic acid for standby.

b) Introduction of carboxyl group by increasing arm length

The above-obtained pillared Corynoic acid was dissolved in 10ml of pyridine, 179mg (1.74 mmol) of 5-aminopentanoic acid was added thereto, and 334mg (1.74 mmol) of EDC was added thereto and stirred at room temperature for 6 hours or more.

After the reaction, the solvent was evaporated under reduced pressure, dissolved in 20ml of dichloromethane, extracted with 20ml × 3, and the lower organic phase was taken out, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Separation by TLC, developing agent 2: the ratio of 95% ethanol, ammonia water, dichloromethane, 1, 4-dioxane = 8: 1: 8: 1 (v/v) was used as a developing solvent, the product spot with Rf =0.3-0.4 was collected under UV, the collected silica gel was repeatedly extracted with absolute ethanol, and after mixing, the absolute ethanol was evaporated under reduced pressure to give an oily substance, i.e., 380mg (0.78 mmol) of 7 hydroxypillarine hapten.

c) Activated conjugated carrier proteins

100mg of 7 hydroxycoumarin hapten was dissolved in 5ml of Dimethylformamide (DMF), and 45mg of N-hydroxysuccinimide (NHS) and 75mg of Dicyclohexylcarbodiimide (DCC) were added thereto, followed by stirring at room temperature overnight. Centrifugation was carried out at 4 ℃ and set at 10000rpm for 5min. And taking the supernatant for later use.

50ml of 10mg/ml BSA/PBS solution were prepared.

The supernatant was added to 10mg/ml BSA/PBS solution at a ratio of 1: 5 (V/V), mixed well, stirred at room temperature for 2 hours, and then left to stand at 2-8 ℃ overnight. The reaction solution was dialyzed against 0.5% sodium carbonate solution at pH 12 for three days, and then against PBS buffer solution at pH 7.4 and 0.01M for two days, during which the buffer was changed three times a day. And after the dialysis is finished, centrifuging the reaction solution, and taking supernatant, namely 7 hydroxyl capping column wood alkali synthetic antigen with the concentration of 7.2mg/ml.

Example 2: preparation of Caragan antigen

Preparation of complete Antigen of quebracho (Antigen 1):

a) Protection of amino groups

398.5mg (1 mmol) of pillared arisin powder is weighed into a150 ml three-neck flask, 60ml of dichloromethane is added, stirring and dissolving are carried out at room temperature, 210mg (1 mmol) of trifluoroacetic anhydride is slowly dropped under the condition of ice water bath and nitrogen protection, the ice water bath is removed, and stirring is carried out at room temperature for 3 hours. After the reaction, the solvent was evaporated to dryness under reduced pressure in a water bath at 405 ℃ to obtain a pale yellow oily liquid, which was dissolved in 10ml of n-hexane. It can be crystallized in an ice-water bath to a white solid. 239mg (0.6 mmol) of the white solid was washed with n-hexane.

b) Hydrolysis of ester group

To the white solid, 10mL of methanol and 2mL of water were added, and 110mg (4.8 mmol) of lithium hydroxide was added, and the reaction was continued for 20 hours or more after the solution was dissolved by stirring.

At the end of the reaction, the starting material Rf =0.7 and the product Rf =0.3, analyzed by thin-plate chromatography (TLC), adjusted to pH =5 with dilute hydrochloric acid and the solvent was evaporated under reduced pressure in a water bath at 45 ℃. Dissolving in 10ml of absolute ethanol, filtering, and evaporating the solvent to dryness under reduced pressure. Separation by TLC, developing solvent 1: collecting Rf =0.3 product spot under ultraviolet with petroleum ether/methanol = 5: 1 (v/v) as developing agent, repeatedly extracting the collected silica gel with anhydrous ethanol, mixing, and evaporating under reduced pressure to remove anhydrous ethanol to obtain pale yellow oily substance of 256mg (0.54 mmol) of pillared suberic acid.

c) Introduction of carboxyl group by increasing arm length

The above-obtained quebracho was dissolved in 10ml of pyridine, and 111mg (1.08 mmol) of 5-aminopentanoic acid was added, and 207mg (1.08 mmol) of EDC was added thereto, followed by stirring at room temperature for 6 hours or more.

After the reaction was completed, the solvent was distilled off under reduced pressure, 20ml of dichloromethane was added to dissolve the solvent, the mixture was extracted with 20ml _ 3, the lower organic phase was taken out, the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure. Separation by TLC, developing agent 2: a developing solvent is prepared from 95% ethanol, ammonia water, dichloromethane, 1, 4-dioxane = 8: 1: 8: 1 (v/v), and Rf =0.4-0.5 under ultraviolet light, and the collected silica gel is repeatedly extracted with anhydrous ethanol, mixed, and evaporated under reduced pressure to obtain an oily substance, i.e., 280mg (0.5 mmol) of the quebracho hapten.

d) Activated conjugated carrier proteins

100mg of the quebracho hapten was dissolved in 5ml of Dimethylformamide (DMF), and 45mg of N-hydroxysuccinimide (NHS) and 75mg of Dicyclohexylcarbodiimide (DCC) were added and stirred at room temperature overnight. Centrifugation was carried out at 4 ℃ and set at 10000rpm for 5min. And taking the supernatant for later use.

50ml of 10mg/ml BSA/PBS solution were prepared.

The supernatant was added to 10mg/ml BSA/PBS solution at a ratio of 1:3 (V/V), mixed well, stirred at room temperature for 2 hours, and then left to stand at 2-8 ℃ overnight. The reaction solution was dialyzed against 0.5% sodium carbonate solution at pH 12 for three days, and then against PBS buffer solution at pH 7.4 and 0.01M for two days, during which the buffer was changed three times a day. And after the dialysis is finished, centrifuging the reaction solution, and taking supernatant, namely the cap column lignan synthetic antigen with the concentration of 5.2mg/ml.

Preparation of 7-hydroxy-pillarine complete Antigen (Antigen 2):

a) Hydrolysis of ester group

414.5mg (1 mmol) of 7 hydroxycoumarin powder was weighed into a 100mL single-neck flask, 10mL of methanol and 2mL of water were added, 115mg (5 mmol) of lithium hydroxide was added, and after stirring and dissolution, the reaction was continued for 20 hours or more.

At the end of the reaction, the starting material Rf =0.6 and the product Rf =0.25, analyzed by thin-plate chromatography (TLC), adjusted to pH =5 with dilute hydrochloric acid and the solvent was evaporated under reduced pressure in a water bath at 45 ℃. Dissolving in 10ml of absolute ethanol, filtering, and evaporating the solvent to dryness under reduced pressure. Separation by TLC, developing solvent 1: collecting Rf =0.25 product spot under ultraviolet with petroleum ether/methanol = 5: 1 (v/v) as developing agent, repeatedly extracting the collected silica gel with anhydrous ethanol, mixing, and evaporating the anhydrous ethanol under reduced pressure to obtain pale yellow oily 7 hydroxy cap column wood acid 304mg (0.76 mmol) for use.

b) Introduction of carboxyl groups by increasing arm length

The above-obtained pillared aristolic acid was dissolved in 10ml of pyridine, and 156mg (1.52 mmol) of 5-aminopentanoic acid was added thereto, and 392mg (1.52 mmol) of EDC was further added thereto and stirred at room temperature for 6 hours or more.

At the end of the reaction, analysis by thin-plate chromatography (TLC) showed starting material Rf =0.6 and product Rf =0.25, PH =5 with dilute hydrochloric acid and evaporation of the solvent under reduced pressure in a water bath at 40-50 ℃. Dissolving in 10ml of absolute ethanol, filtering, and evaporating the solvent under reduced pressure. Separation by TLC, developing solvent 1: the ratio of petroleum ether to methanol = 5: 1 (v/v) was used as a developing solvent, a product spot with Rf =0.25 was collected under ultraviolet, the collected silica gel was repeatedly extracted with absolute ethanol, and after mixing, the absolute ethanol was evaporated under reduced pressure to obtain 335mg (0.69 mmol) of 7 hydroxypillared suberic acid as a pale yellow mono-oil.

c) Activated conjugated carrier proteins

100mg of 7-hydroxypillared-wood-acid was dissolved in 5ml of Dimethylformamide (DMF), and 45mg of N-hydroxysuccinimide (NHS) and 75mg of Dicyclohexylcarbodiimide (DCC) were added thereto, followed by stirring at room temperature overnight. Centrifuging at 4 deg.C, setting 10000rpm, and 5min. And taking the supernatant for later use.

50ml of 10mg/ml BSA/PBS solution were prepared.

The supernatant was added to 10mg/ml BSA/PBS solution at a ratio of 1:3 (V/V), mixed well, stirred at room temperature for 2 hours, and then left to stand at 2-8 ℃ overnight. The reaction solution was dialyzed against 0.5% sodium carbonate solution at pH 12 for three days, and then against PBS buffer solution at pH 7.4 and 0.01M for two days, during which the buffer was changed three times a day. And after dialysis, centrifuging the reaction solution, and taking supernatant, namely the 7-hydroxyl capping column lignan synthetic antigen with the concentration of 5.6mg/ml.

Example 3: preparation of Caragan antigen

Preparation of complete Antigen of quebracho (Antigen 1):

a) Protection of amino groups

398.5mg (1 mmol) of pillared arisin powder is weighed into a150 ml three-neck flask, 60ml of dichloromethane is added, stirring and dissolving are carried out at room temperature, 630mg (3 mmol) of trifluoroacetic anhydride is slowly dropped under the condition of ice water bath and nitrogen protection, the ice water bath is removed, and stirring is carried out at room temperature for 3 hours. After the reaction, the solvent was evaporated to dryness under reduced pressure in a water bath at 45 ℃ to obtain a pale yellow oily liquid, which was dissolved in 10ml of n-hexane. It can be crystallized into white solid in ice-water bath. 391mg (0.8 mmol) of the white solid was washed with n-hexane.

b) Hydrolysis of ester group

To the white solid, 10mL of methanol and 2mL of water were added, and 147mg (6.4 mmol) of lithium hydroxide was added, and after dissolution by stirring, the reaction was continued for 20 hours or more.

At the end of the reaction, analysis by thin-plate chromatography (TLC) showed starting material Rf =0.7 and product Rf =0.3, pH =5 with dilute hydrochloric acid and solvent was evaporated under reduced pressure in a water bath at 40-50 ℃. Dissolving in 10ml of absolute ethanol, filtering, and evaporating the solvent under reduced pressure. Separation by TLC, developing solvent 1: collecting Rf =0.3 product spot under ultraviolet with petroleum ether/methanol = 5: 1 (v/v) as developing agent, repeatedly extracting the collected silica gel with anhydrous ethanol, mixing, and evaporating the anhydrous ethanol under reduced pressure to obtain pale yellow oily substance of cap column wood acid 342mg (0.72 mmol) for use.

c) Introduction of carboxyl groups by increasing arm length

The above-obtained pillared Corynoic acid was dissolved in 10ml of pyridine, 148mg (1.44 mmol) of 5-aminopentanoic acid was added thereto, 276mg (1.44 mmol) of EDC was added thereto, and the mixture was stirred at room temperature for 6 hours or more.

After the reaction, the solvent was evaporated under reduced pressure, dissolved in 20ml of dichloromethane, extracted with 20ml × 3, and the lower organic phase was taken out, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Separation by TLC, developing solvent 2: a developing solvent was prepared by collecting a spot of a product having Rf =0.4 to 0.5 under ultraviolet irradiation at a ratio of 95% ethanol, ammonia water, dichloromethane, 1, 4-dioxane = 8: 1: 8: 1 (v/v), repeatedly extracting the collected silica gel with absolute ethanol, mixing, and evaporating the absolute ethanol under reduced pressure to obtain an oily substance, i.e., cephamine hapten 369mg (0.66 mmol).

d) Activated conjugated carrier proteins

100mg of the carboxyl group-containing derivative was dissolved in 5ml of Dimethylformamide (DMF), and 45mg of N-hydroxysuccinimide (NHS) and 75mg of Dicyclohexylcarbodiimide (DCC) were added thereto, followed by stirring at room temperature overnight. Centrifugation was carried out at 4 ℃ and set at 10000rpm for 5min. And taking the supernatant for later use.

50ml of 10mg/ml BSA/PBS solution were prepared.

The supernatant was added to 10mg/ml BSA/PBS solution at a ratio of 1: 8 (V/V), mixed well, stirred at room temperature for 2 hours, and then left to stand at 2-8 ℃ overnight. The reaction solution was dialyzed against 0.5% sodium carbonate solution at pH 12 for three days and then against PBS buffer at pH 7.4 and 0.01M for two days, during which time the buffer was changed three times a day. And after the dialysis is finished, centrifuging the reaction solution, and taking supernatant, namely the cap column lignan synthetic antigen with the concentration of 8.1mg/ml.

Preparation of 7-hydroxy-pillarine complete Antigen (Antigen 2):

a) Hydrolyzed ester group

414.5mg (1 mmol) of 7 hydroxycoumarin powder was weighed into a 100mL single-neck flask, 10mL of methanol and 2mL of water were added, 230mg (10 mmol) of lithium hydroxide was further added, and after dissolution by stirring, the reaction was continued for 20 hours or more.

At the end of the reaction, analysis by thin-plate chromatography (TLC) showed starting material Rf =0.6 and product Rf =0.25, pH =5 with dilute hydrochloric acid and solvent was evaporated under reduced pressure in a water bath at 45 ℃. Dissolving in 10ml of absolute ethanol, filtering, and evaporating the solvent under reduced pressure. Separation by TLC, developing solvent 1: using the ratio of petroleum ether to methanol = 5: 1 (v/v) as a developing agent, collecting a product spot with Rf =0.25 under ultraviolet, repeatedly extracting the collected silica gel with absolute ethyl alcohol, mixing, and evaporating the absolute ethyl alcohol under reduced pressure to obtain a light yellow oily substance, namely 7-hydroxy-capped lignan acid 328mg (0.82 mmol) for later use.

b) Introduction of carboxyl groups by increasing arm length

The above-obtained pillared aristolic acid was dissolved in 10ml of pyridine, and 169mg (1.64 mmol) of 5-aminopentanoic acid was added thereto, followed by addition of 315mg (1.64 mmol) of EDC and stirring at room temperature for 6 hours or more.

After the reaction was completed, the solvent was distilled off under reduced pressure, 20ml of dichloromethane was added to dissolve the solvent, the mixture was extracted with 20ml _ 3, the lower organic phase was taken out, the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure. Separation by TLC, developing agent 2: using the ratio of 95% ethanol, ammonia water, dichloromethane, 1, 4-dioxane = 8: 1: 8: 1 (v/v) as a developing agent, collecting product points with Rf =0.3-0.4 under ultraviolet, repeatedly extracting the collected silica gel with absolute ethanol, mixing, and evaporating the absolute ethanol under reduced pressure to obtain an oily substance, namely the 7-hydroxy pillared suberin hapten.

c) Activated conjugated carrier proteins

100mg of the carboxyl group-containing derivative was dissolved in 5ml of Dimethylformamide (DMF), and 45mg of N-hydroxysuccinimide (NHS) and 75mg of Dicyclohexylcarbodiimide (DCC) were added thereto, followed by stirring at room temperature overnight. Centrifugation was carried out at 4 ℃ and set at 10000rpm for 5min. And taking the supernatant for later use.

50ml of 10mg/ml BSA/PBS solution were prepared.

The supernatant was added to 10mg/ml BSA/PBS at a ratio of 1: 8 (V/V), mixed well, stirred at room temperature for 2 hours, and then allowed to stand at 2-8 ℃ overnight. The reaction solution was dialyzed against 0.5% sodium carbonate solution at pH 12 for three days, and then against PBS buffer solution at pH 7.4 and 0.01M for two days, during which the buffer was changed three times a day. And after dialysis, centrifuging the reaction solution, and taking supernatant, namely the 7 hydroxyl capping column lignan synthetic antigen with the concentration of 8.2mg/ml.

Comparative example 1: preparation of Caragan antigen

The molecular structure of the control pillarine hapten is shown below:

preparing a pillared suberin complete antigen:

a) Introduction of carboxyl group by increasing arm length

398.5mg (1 mmol) of pillared suberine powder is weighed into a150 ml three-neck flask, 60ml of dichloromethane and 160mg (1.6 mmol) are added, stirring is carried out at room temperature, after dissolution, 135mg (1 mmol) of anhydrous aluminum trichloride is added under the condition of succinic anhydride ice-water bath and under the protection of nitrogen, the ice-water bath is removed, and stirring is carried out at room temperature for 3 hours. After the reaction is finished, slowly adding ice HCL, adjusting the pH to be about =7, filtering and collecting the precipitate, washing the precipitate with dichloromethane, and drying to obtain 520mg of a product, namely the pillared suberine hemisuccinate derivative;

b) Activated conjugated carrier proteins

100mg of the quebracho hapten was dissolved in 5ml of Dimethylformamide (DMF), and 45mg of N-hydroxysuccinimide (NHS) and 75mg of Dicyclohexylcarbodiimide (DCC) were added and stirred at room temperature overnight. Centrifugation was carried out at 4 ℃ and set at 10000rpm for 5min. And taking the supernatant for later use.

50ml of 10mg/ml BSA/PBS solution were prepared.

The supernatant was added to 10mg/ml BSA/PBS solution at a ratio of 1:3 (V/V), mixed well, stirred at room temperature for 2 hours, and then left to stand at 2-8 ℃ overnight. The reaction solution was dialyzed against 0.5% sodium carbonate solution at pH 12 for three days, and then against PBS buffer solution at pH 7.4 and 0.01M for two days, during which the buffer was changed three times a day. And after dialysis, centrifuging the reaction solution, and taking supernatant, namely the capping column lignan synthetic antigen with the concentration of 6.2mg/ml.

Example 4: ELISA (enzyme-Linked immunosorbent assay) titer detection of quebracho antigen

(1) Wrapping a plate: the antigen to be detected (diluted by CBbuffer) is added into the 96-hole enzyme label plate at the concentration of 1 mu g/ml, each hole is 100 mu l, and the mixture is kept overnight in a refrigerator at the temperature of 2-8 ℃.

(2) And (3) sealing: the wells were decanted, blocked for 2h at 37 ℃ by adding 1% BSA-PBST solution per well, spun off the blocking solution, washed with 0.1MPBST, three times, patted dry, and stored at 4 ℃.

(3) Sample adding: antibodies were diluted to 1mg/ml with PBS and to the corresponding concentrations in the appendix. Taking the well-coated ELISA plate, filling 50 mu l of the sample to be detected in the first hole and the second hole, and diluting each hole by PBS (phosphate buffer solution) in a multiple ratio. 50 μ l of PBS was added to the negative well, the positive control was replaced with CK, the sample was diluted and incubated at 37 ℃ for 30min.

(4) Adding an enzyme-linked secondary antibody: the liquid was spun off, 100. Mu.l of HRP-conjugated goat anti-mouse secondary antibody was added to each well and the plate was mixed well and incubated at 37 ℃ for 30min.

(5) Washing the plate: discarding liquid in the holes, filling the holes with washing liquid, standing for 5 seconds, spin-drying, repeating for 5 times, and then patting dry.

(6) Adding a substrate for color development: adding 100 μ l of TMB into each well, mixing well, sealing, and developing at room temperature for 5min.

(7) And (4) terminating: add stop solution 50. Mu.l to each well and mix well.

(8) And (4) judging a result: and (3) measuring the OD450 value of the sample at 450nm, judging that the sample is positive if the OD value/negative control OD value of the sample is more than or equal to 2.5, and judging that the sample is negative if the sample is not positive. The OD of the negative control is lower than 0.05 and calculated as 0.05, and the OD of the negative control is higher than 0.05 and calculated as the actual OD.

The results show that the antigens synthesized in examples 1, 2 and 3 and comparative example 1 can generate specific reaction with the antibody, wherein the antigen activity of example 1 is the highest, and the synthetic antigen of comparative example 1 has 2 sites capable of coupling with the carrier protein, has low purity and causes low activity.

Example 5: preparation of anti-seizure monoclonal antibody

Step (1) immunization of mice

1-1: preparing immunity: antigen1 and Antigen2 synthesized in example 1 were mixed in a mass ratio of 1: 1 and then diluted to 1mg/ml with 0.01M PBS.

The diluted antigen was mixed with complete adjuvant 1: 1 at day 1, injected subcutaneously into mice at 5 ug/mouse, mixed with incomplete adjuvant at day 6, injected 10 ug/mouse near lymph node at day 11, injected 15 ug/mouse lymph node at day 16, injected 20 ug/mouse lymph node at day 21, injected 25 ug/mouse lymph node at day 24, and injected 5 ug/mouse vein directly at day 24.

The immunization method is a continuous enhanced stimulation method, combines subcutaneous immunization and intravenous immunization, and has the advantages of small antigen dosage, short immunization time, good stability, strong affinity and the like.

Step (2) fusion of splenocytes with myeloma cells

2-1: preparing feeder cells: killing a mouse by removing eyeballs and bloodletting, soaking the mouse in 75% alcohol for disinfection, tearing the skin outside the abdomen of the mouse, exposing the peritoneum of the mouse, injecting a DMEM serum-free culture medium preheated at 37 ℃, slightly rubbing the abdominal cavity of the mouse for 1-2 minutes, suspending abdominal cavity cells, and sucking out abdominal cavity liquid; cutting the chest cavity of the mouse, taking the thymus, grinding, collecting suspension, combining with the peritoneal fluid, centrifuging, and re-suspending the precipitate with HAT complete culture solution to obtain feeder cell suspension.

2-2: culture of mouse myeloma cells SP 2/0: the mouse myeloma cell SP2/0 was subcultured in DMEM medium containing FBS at a volume fraction of 10%, and passaged one day before cell fusion to ensure that the mouse myeloma cell SP2/0 was suitable for the logarithmic phase of growth and the growth state was good for cell fusion.

2-3: preparation of splenocytes: BALB/c females immunized as above were sacrificed, sterilized in 75% ethanol and dissected open, and spleens were aseptically removed. Placing spleen on stainless steel filter screen connected with 50ml centrifuge tube, immediately adding 5ml serum-free DMEM culture solution, shearing spleen with ophthalmic scissors, lightly grinding spleen with sterile syringe handle to filter spleen cells into centrifuge tube via mesh, adding serum-free culture solution to 30ml, and centrifuging. And discarding the supernatant, and repeatedly centrifuging once by using a serum-free culture solution for later use.

2-4: spleen cells fused with myeloma cells: polyethylene glycol fusion method is adopted. Mixing splenocytes and mouse myeloma cell SP2/0, centrifuging to wash the cells, removing supernatant, placing in water bath at 37 deg.C, adding 0.7m137 deg.C pre-warmed PEG4000, adding the mixture in one minute, and standing for 90 s. Adding 25ml of DMEM serum-free culture solution pre-warmed at 37 ℃ to stop the PEG effect; centrifuging and taking the precipitate; then the pellet was added with a suspension of feeder cells, seeded into a 96-well cell culture plate, and placed at 37 ℃ 5% CO ₂ Culturing in an incubator. After 24 hours, HAT medium was used, and after two weeks of maintenance culture in HAT selection medium, HT medium was used instead, and two weeks of maintenance culture were carried out again in the normal medium instead.

Step (3) screening of hybridoma cells

3-1: primary screening 1: and replacing the fused cells once every 3 days, observing the growth condition of the fused cells in a 96-hole cell culture plate, sucking culture supernatant of the fused cells when the cells grow to cell clusters (the cells are observed under a 16-fold objective lens and a 10-fold ocular lens, and the cell size occupies 1/3 of the visual field), and screening positive clones by adopting an indirect ELISA method.

Cells that were both positive in the ELISA were rescreened using Antigen1 and Antigen2 from example 1 as coating antigens, respectively.

3-2: re-screening: and (3) further carrying out re-screening on the screened positive clones by using a pillarine and 7-hydroxy pillarine standard substance to carry out competitive inhibition ELISA method, and screening out hybridoma cell strains kratom with the highest competitive inhibition rate for clone culture.

3-3: cloning of hybridoma cell line kratom: cloning culture of hybridoma cell strain kratom is carried out according to a limiting dilution method, cells are accurately counted, the cell suspension is diluted into 4/ml cell suspension by using DMEM medium containing 10% FBS, then 200 mu l of diluted cell suspension in each hole is inoculated into a 96-hole cell culture plate, after 7 days, cell growth condition is observed, the antibody level of cell culture supernatant is detected, 3 monoclones with the highest titer are selected for cloning culture, and the monoclonal hole antibody detection positive rate reaches 100 repeatedly; the anti-Katong monoclonal cell capable of stably secreting specific antibodies is named as an anti-Katong hybridoma cell strain Kratom11B7 (preservation unit: china center for type culture Collection; preservation number CCTCCNO: C202146), and the Katong monoclonal antibody is purified after the expanded culture.

Step (4) preparation and purification of anti-cardamine monoclonal antibody

4-1: preparing a Caragan monoclonal antibody: an animal in vivo induction method-ascites preparation method is adopted. The selected monoclonal cell line Kratom11B7 was inoculated into a 24-well cell culture plate using DMEM medium containing 10% by volume fbs, cultured for 2 days and then inoculated into a small cell culture flask, and when the cell size occupied 1/2 field of view by observation under a 16-fold objective lens and a 10-fold eyepiece, it was inoculated into a large cell culture flask, and after 2 days of culture, the cell growth was observed in logarithmic phase, and hybridoma cells were collected by centrifugation. Diluting with 0.9% physiological saline, injecting into abdominal cavity of mice injected with liquid paraffin for 2 weeks, each injection is 1 × 10 ⁶ And (3) hybridoma cells. After about 10 days, the abdomen of the mouse had significantly risen, at which time the abdomen was disinfected with 75% ethanol and ascites was collected with an injection needle.

4-2: purifying the Caragan monoclonal antibody:

carbamine monoclonal antibody purified by A + G-protein affinity chromatography

1. Taking a certain amount of ascites, unfreezing at room temperature, measuring the volume of the ascites, centrifuging at 10000r and 4 ℃ for 20 minutes, and removing larger clots.

2. 10ml of ProteinA-Sepharose CL-4B and 10ml of ProteinG-Sepharose CL-4B were mixed well and loaded onto a chromatography column.

3. The column was loaded with 10 bed volumes of loading buffer at a flow rate of 1ml/min and the effluent pH =7.4 as determined by dipstick.

4. Taking 5ml of pretreated ascites fluid, diluting to 10ml with loading buffer solution, filtering with 0.45 μm filter membrane, loading at flow rate of 1ml/min.

5. Flow washing was performed with loading buffer at 10 bed volumes at a flow rate of 1ml/min, followed by elution of the antibody with 0.02M, pH =4.0 citrate buffer, while monitoring with AKTA150pure, and when a rise in baseline was observed, i.e., an elution peak appeared, a clean 4ml centrifuge tube was collected, and after 3ml of each collection, pH was adjusted to 7.0 with 1M, pH =8.0 Tris-HCl buffer.

6. Collecting eluate until the peak returns to baseline, further leveling with loading buffer and column bed volume of 5-10 times, and adjusting flow rate to 1ml/min. Then, the volume of the column bed is balanced by 10 times by using triple distilled water.

7 filtering the dialyzate. Protein concentration (mg/ml) = OD280 value dilution fold/1.35 = protein concentration).

Performance testing of anti-Caragana monoclonal antibodies

Subtype identification of anti-seizure monoclonal antibody

The anti-katain monoclonal antibody prepared in example 5 was identified as a subtype by referring to a monoclonal antibody typing kit, and the anti-katain monoclonal antibody belongs to 11B7 of IgG2a subtype, 11C1 of IgG1 subtype, 9D2 of IgG2B subtype, 7E3 of IgG2a subtype and 16A5 of IgG1 subtype.

Name of antibody	Heavy chain type	Potency of the drug
			11B7	IgG2a	1∶211～1∶213
11C1	IgG1	1∶211～1∶213
			9D2	IgG2b	1∶211～1∶213
7E3	IgG2a	1∶211～1∶213
			16A5	IgG2a	1∶211～1∶213

Antibody stability assessment

In the preliminary evaluation, the antibodies obtained by 3 strains of cells are all satisfactory, but the gradient of 11B7 is better than that of 11C1, 9D2 and 7E3, the performance is consistent with that of 16A5, but the stability is 16A5, so that 11B7 is confirmed as an original cell strain for quantitative production.

Determination of affinity constant of anti-Caragana monoclonal antibody

Determination of affinity by antibody competition for binding to antigen

(1) Diluting Caragan monoclonal antibody (kratom-BSA) to 1. Mu.g/ml with 0.05M carbonate buffer pH =9.6, adding 100. Mu.l of diluted Caragan artificial antigen to each well of 96-well microplate, and coating overnight at 4 ℃ (2 plates); then wash the plate 3 times with PBS buffer containing 0.05% Tween-20 and pat dry;

(2) Adding 200 μ L of 10% BSA in 0.01mol/L pH =7.2 phosphate buffer per well, setting at 37 ℃ for 60 minutes, washing the plate with 0.05% Tween-20 in PBS buffer for 3 dry strokes for use;

(3) Preparing 5ml of Caragana antigen with the concentration of 40ng/ml and 5ml of Caragana antigen with the concentration of 1000ng/ml from the purified antibody, diluting the Caragana antigen by times with the concentration of 500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.625ng/ml and 7.812ng/ml respectively, mixing the antibody with the same volume with the antigen with the concentration of 8 respectively to obtain 8 samples to be detected, and incubating for 60 minutes at 37 ℃.

(4) Adding 8 samples to be tested to the coated ELISA plate at 100. Mu.l per well, incubating at 37 ℃ for 60 minutes, adding the remaining liquid after incubation to another plate, incubating for 60 minutes, and washing the plate 5 times with PBS buffer containing 0.05% Tween-20;

(5) Adding PBS buffer to goat anti-mouse IgG-HRP, diluting to 4000 times of volume, adding 100 μ l of diluted goat anti-mouse IgG-HRP to each well of a 96-well cell culture plate, reacting at 37 ℃ for 60 minutes, washing the plate with PBS buffer containing 0.05% Tween-20 for 3 times of drying;

(6) After adding 50. Mu.l of substrate TMB per well for 8 minutes at 37 ℃ reaction, 2M H was added ₂ SO ₄ The reaction was stopped, and the OD was measured at 450nm, then the formula was calculated from the affinity constant: kd (i 0-A0B) = B/(1-B) an affinity Kd value is obtained, where A0 is an antibody initial concentration, i0 is an antigen initial concentration, B is an antibody binding rate, B = (A0-Ai)/A0, and A0 and Ai are D450 values for detection of an initial antibody and an antigen-bound antibody, respectively. The affinity constant of the monoclonal antibody 2B1 is 1.82 × 10 ^-10 mol/L。

Number of times	1	2	3	4	5	6	7	8
									a0(10 -10 mol/l)	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5
i0(10 -7 mol/l)	42	21	10.5	5.25	2.625	1.312	0.656	0
									OD450	0.137	0.270	0.542	1.085	1.559	1.790	2.241	2.748
B	0.910	0.832	0.763	0.575	0.402	0.301	0.168	0

The affinity constant (K) of an antibody, a parameter that characterizes the antigen affinity of an antibody, reflects the strength of the interaction between the antigen-binding site on the antibody molecule and the corresponding epitope. It is generally accepted that the affinity constant is 10 ⁷ ～10 ¹⁰ M ^-1 The affinity of the antibody is high. Affinity constant 10 ⁵ ～10 ⁶ M ^-1 The antibody affinity is low. The monoclonal antibody has an affinity constant of 1.82 × 10 determined by ELISA method ^-10 mol/L, high affinity, and meeting the preparation requirement of the katain saliva detection kit.

anti-Caragana monoclonal antibody reactivity test

The anti-carpain monoclonal antibody prepared in example 1 is detected by indirect ELISA method, OD value is measured by 450nm, and the positive value is OD450 value of anti-carpain monoclonal antibody solution/negative control OD450 value > 2.5. As can be seen from the following table, the reactivity of the prepared anti-katain monoclonal antibody can reach 0.1ng/ml.

OD450 values of the anti-carpain monoclonal antibody prepared in example 1 at different concentrations

Antibody concentration	OD450 value	Antibody concentration	OD450 value
				100ng/ml	2.665	0.008μg/ml	0.641
50ng/ml	2.423	0.78ng/ml	0.535
				25ng/ml	2.011	0.39ng/ml	0.309
12.5ng/ml	1.832	0.195ng/ml	0.158
				6.25ng/ml	1.763	0.1ng/ml	0.1085
3.125ng/ml	1.169	PBS (blank control)	0.018
				1.5625ng/ml	0.906	PBS (blank control)	0.022

Example 6: preparation of kit for detecting katain in saliva

Preparing colloidal gold: 1% chloroauric acid (HAuCl) was taken in 1ml ₄ ) Adding the solution into 100ml of water, heating to boil, adding 1.5ml of 1% trisodium citrate, mixing and boiling for 5 minutes until the color changes from blue to purple and then to purple red again until the color does not change any more. The colloidal gold particles obtained after cooling were 30nm.

Preparing a gold label: taking the prepared colloidal gold, adjusting the pH value to 8.5 by using 1M NaOH, then adding 1ml of the antibody, stirring on a magnetic stirrer, then adding a certain amount of BSA (bovine serum albumin) to the final concentration of 1%, and stirring for later use. And (3) centrifuging the colloidal gold solution coupled with the antibody, discarding the precipitate, and keeping the solution. The precipitate was redissolved with a 20% sucrose-containing gold-labeled antibody diluent, transferred to a brown bottle and stored at 4 ℃ for further use.

Preparing gold label strips: and uniformly spraying the gold marking working solution on a gold marking pad with the thickness of 30cm x 6.5 cm. And (3) putting the dried and cut strips of the gold label pad sprayed with the gold label working solution into a self-sealing bag, then sealing the self-sealing bag into an aluminum foil bag filled with a drying agent, and storing the self-sealing bag at 20 ℃ for later use.

Coating of NC film: loading a certain amount of Caragan-protein complex (hattacrine synthetic antigen) and goat anti-mouse polyclonal antibody (GAM) into T and C columns of a membrane-spotting machine, respectively, and coating the solution of T and C lines on NC membrane.

And (3) glass fiber treatment: cutting the glass cellulose membrane into 17 x 300mm strips, soaking in the glass fiber treatment solution for 1 hour, taking out, drying in a 37 ℃ constant temperature cabinet for 12 hours, taking out, putting into a self-sealing bag, sealing in an aluminum foil bag filled with a drying agent, and storing at 20 ℃ for later use.

Assembling the reagent strip: and respectively sticking the processed NC film, the gold label strip, the glass fiber and the absorbent paper to a PVC plate, and then cutting into 4.0mm strips.

The assembled katain saliva detection reagent is suitable for detecting the katain in saliva.

Comparing the katain saliva kit of the invention with the katain urine kit of company A, the color development intensity is determined by a color comparison card shown in figure 1, and the results are as follows:

as can be seen from the table above, the katain saliva kit produced by the invention has higher sensitivity, and the saliva is simpler and more convenient than the urine sampling. The sensitivity of the kit is about 15ng/ml, products of other companies on the market are all urine detection, and the sensitivity is about 300 ng/ml.

The product of the invention has the sensitivity of 15ng/ml except for the cephalomannine and the 7 hydroxyl cephalomannine, and is matched with 100 mu g/ml naltrexone, buprenorphine, naloxone, methamphetamine, ephedrine, pseudoephedrine, diazepam, phenobarbital, ketamine, methadone, tramadol, gatifloxacin, procaine, delta 9-tetrahydrocannabinolic acid, morphine, dolantin, oxycodone, phencyclidine, propoxyphenol, amphetamine, ecstasy, barbiturates, benzodiazepine

The cross reaction does not occur in the samples of drugs or medicines such as the class and the like and negative saliva samples.

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modifications, alterations and equivalent changes made to the above embodiment according to the technical spirit of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims (1)

1. A preparation method of a quebracho total antigen is characterized by comprising the following steps: taking the pillared alkali as a raw material, firstly protecting a secondary amino group by using trifluoroacetic anhydride, then hydrolyzing methyl ester by using lithium hydroxide to obtain a carboxyl group, introducing 5-aminopentanoic acid into a carboxyl site to increase the arm length to obtain a pillared alkali hapten, coupling the pillared alkali hapten with carrier protein by using a carbodiimide method, and then removing a protecting group to obtain a pillared alkali complete antigen;

the method specifically comprises the following steps:

a) Protection of amino group: dissolving the quebracho nut alkali in an organic solvent, stirring and dissolving at room temperature, slowly dropwise adding trifluoroacetic anhydride with the molar weight being 1-3 times that of the quebracho nut alkali in an ice water bath under the protection of inert gas, removing the ice water bath, and stirring and reacting at room temperature; after the reaction is finished, evaporating the solvent to dryness under reduced pressure in a water bath to obtain a light yellow oil liquid, adding n-hexane for dissolving, crystallizing the light yellow oil liquid into a white solid in an ice water bath, and washing the white solid by using the n-hexane;

b) Hydrolysis of the ester group: adding methanol and water into the obtained white solid, adding lithium hydroxide in an amount which is 5 to 10 times of the molar weight of the white solid, stirring and dissolving, and continuing to react; after the reaction is finished, analyzing by using a thin plate chromatography, adjusting the pH of the raw material Rf =0.6-0.8 and the product Rf =0.2-0.4 by using dilute hydrochloric acid, and evaporating the solvent under reduced pressure in a water bath; dissolving in anhydrous alcohol, filtering, and evaporating the solvent under reduced pressure; separating by thin plate chromatography, collecting product spot with Rf =0.2-0.4 under ultraviolet with petroleum ether/methanol mixed solution as developing agent, collecting obtained silica gel, repeatedly extracting with anhydrous ethanol, mixing, and evaporating anhydrous ethanol under reduced pressure to obtain yellowish oily substance cap column wood alkali acid;

c) Increasing arm length introduces carboxyl: dissolving the obtained pillared aristolic acid in pyridine, adding 1-3 times of 5-aminopentanoic acid in molar amount, adding EDC in equimolar amount with 5-aminopentanoic acid, and stirring at room temperature; after the reaction is finished, evaporating the solvent under reduced pressure, adding dichloromethane for dissolution, extracting for multiple times, taking a lower organic phase, drying the organic phase, filtering, and evaporating the solvent under reduced pressure; separating by thin plate chromatography, collecting product spots with Rf =0.4-0.5 under ultraviolet with 95wt% ethanol/ammonia water/dichloromethane/1, 4-dioxane mixed solution as developing agent, repeatedly extracting the collected silica gel with anhydrous ethanol, mixing, and evaporating under reduced pressure to remove anhydrous ethanol to obtain oily substance, namely calyx-shaped column lignan hapten;

d) Activation of coupled carrier protein: dissolving the obtained calophylline hapten in dimethylformamide, adding N-hydroxysuccinimide 0.4-0.5 times of the calophylline hapten and dicyclohexylcarbodiimide 0.7-0.8 times of the calophylline hapten, stirring at room temperature overnight, centrifuging, and taking the supernatant; adding the supernatant into 8-12mg/ml carrier protein/PBS solution according to the volume ratio of 1; putting the reaction solution into a sodium carbonate solution with the pH value of 11-13 for dialysis, and then dialyzing in a PBS buffer solution, wherein the buffer solution is changed every day; and after the dialysis is finished, centrifuging the reaction solution, and taking supernatant, namely the cap column lignan complete antigen.

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