CN114409796B - Hybridoma cell strain of anti-fenfluramine monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain of anti-fenfluramine monoclonal antibody and application thereof Download PDFInfo
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- CN114409796B CN114409796B CN202210105671.9A CN202210105671A CN114409796B CN 114409796 B CN114409796 B CN 114409796B CN 202210105671 A CN202210105671 A CN 202210105671A CN 114409796 B CN114409796 B CN 114409796B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a hybridoma cell strain of an anti-fenfluramine monoclonal antibody and application thereof in the field of food safety immunodetection. The hybridoma cell strain has a preservation number of CGMCC NO.45025, and the anti-fenfluramine monoclonal antibody secreted by the hybridoma cell strain has good detection sensitivity and affinity to fenfluramine, can be used for establishing a fenfluramine enzyme-linked immunosorbent assay method or a colloidal gold immunochromatography test strip rapid detection method, and lays a foundation for research and development and popularization of an indirect competition ELISA kit and a colloidal gold test strip.
Description
Technical Field
The invention belongs to the field of food safety immunodetection, and particularly relates to a hybridoma cell strain of an anti-fenfluramine monoclonal antibody and application thereof.
Background
Fenfluramine is an amphetamine appetite suppressant, has pure white crystals in appearance, is crystal clear, has weaker excitation center function, and can reduce blood pressure; can also enhance the utilization of glucose by surrounding tissues to reduce blood sugar; also has cholesterol and triacylglycerol reducing effect. Clinically used for simple obesity and obesity accompanied by diabetes, hypertension, anxiety and cardiovascular diseases. Fenfluramine is also called ice toxin, has extremely strong stimulation effect on the central nervous system of human body, has stronger toxicity, can make the human body produce strong physiological excitation, can consume a great deal of physical strength and reduce immune function, seriously damages heart and brain tissues and even causes death. Along with the change of aesthetic ideas and the love of people, the demand and the use amount of the weight-losing medicine are rapidly increased, the use trend of the weight-losing medicine containing the fenfluramine is increased, and a plurality of health and social problems are caused, so that a method for detecting and analyzing the fenfluramine content in the health-care food with high accuracy and strong operability is urgently needed to be designed.
Conventional methods for detecting fenfluramine include spectrophotometry, thin layer chromatography, gas chromatography, liquid chromatography, nuclear magnetic resonance, and the like. These methods suffer from several drawbacks to varying degrees: time consuming, expensive instrumentation, and extensive sample pretreatment procedures, etc. Thus, these methods are not suitable for in situ detection of profenofos for high throughput analysis. Because of the need for an analysis system, this means that these methods have limited application in field analysis.
The immunoassay method has the characteristics of low cost, high flux, high sensitivity, low relative requirements on technicians and the like, so that the method is suitable for rapid screening of a large number of samples. The premise of detecting the fenfluramine by using an immunoassay method is that the monoclonal antibody with high specificity and high sensitivity to the fenfluramine is obtained, so that the method for preparing the monoclonal antibody with high specificity and high sensitivity to the fenfluramine is very key.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a hybridoma cell strain of an anti-fenfluramine monoclonal antibody and application thereof.
The first aspect of the invention provides a hybridoma cell strain CGM of an anti-fenfluramine monoclonal antibody, and the preservation number of the hybridoma cell strain is CGMCC NO.45025.
The second aspect of the invention provides an anti-fenfluramine monoclonal antibody which is secreted by the hybridoma cell strain CGM.
The third aspect of the invention provides application of the hybridoma cell strain CGM or an anti-fenfluramine monoclonal antibody secreted by the hybridoma cell strain CGM in fenfluramine detection, application in preparation of fenfluramine immunodetection kit or application in preparation of fenfluramine detection colloidal gold test strips.
The fourth aspect of the invention provides a fenfluramine immunoassay kit comprising the hybridoma cell strain CGM or an anti-fenfluramine monoclonal antibody secreted by the hybridoma cell strain CGM.
Further, the fenfluramine immunodetection kit also comprises an ELISA plate, fenfluramine coating antigen, fenfluramine standard solution, enzyme-labeled secondary antibody and substrate reaction solution;
preferably, the molecular structural formula of the fenfluramine coating antigen is shown as the following formula,
the invention provides a fenfluramine detection colloidal gold test strip, which comprises a sample pad, a colloidal gold binding pad, a nitrocellulose membrane and a water absorption pad, wherein the nitrocellulose membrane is sequentially provided with a detection line and a quality control line, and the colloidal gold binding pad is coated with an anti-fenfluramine monoclonal antibody secreted by a hybridoma cell strain CGM.
Further, the detection line is printed by the fenfluramine coating antigen.
Preferably, the molecular structural formula of the fenfluramine coating antigen is shown as the following formula,
the sixth aspect of the invention provides the fenfluramine immunoassay kit or the application of the fenfluramine detection colloidal gold test strip in fenfluramine detection.
The preparation method of the hybridoma cell strain CGM cell strain comprises the following basic steps:
(1) Preparation and identification of immunogens: the method comprises the steps of taking fenfluramine as a raw material, connecting amino groups of an activated ester method protein carrier, separating complete antigen and unconjugated small molecule hapten through dialysis after the reaction is finished, and identifying the complete antigen through an ultraviolet absorption scanning method;
(2) Immunization of mice: BALB/c mice of 6-8 weeks of age were selected for immunization. After the immunogen and Freund's adjuvant are completely emulsified, the mice are immunized by subcutaneous multipoint injection, freund's complete adjuvant is adopted for primary immunization, freund's incomplete adjuvant is adopted for boosting immunization, the immunization dose is half of the previous immunization dose during sprint immunization, and the mice are directly injected into the abdominal cavity after being uniformly mixed with normal saline; the immunization interval is three weeks; the immunization process comprises 1 primary immunization, 4 booster immunization and 1 sprint immunization; after the third immunization, blood sampling is carried out at intervals of one week to detect serum titer and inhibition; selecting the mice with the best inhibition, performing sprint immunization 18 days after five days, and preparing fusion;
(3) Cell fusion and cell strain establishment: fusing the spleen cells of the mice and myeloma cells of the mice by a polyethylene glycol (PEG 2000) method, culturing by a HAT culture medium, detecting positive cell holes by using an indirect ELISA (enzyme-linked immunosorbent assay), further measuring the inhibition effect of the positive cell holes by using an indirect competition ELISA method, subcloning the positive cell holes with the best inhibition for three times by a limiting dilution method, and finally screening to obtain hybridoma cell strains CGM;
(4) Identification of hybridoma cell line properties: adopting enzyme-labeled secondary antibody suit determination for mouse monoclonal antibody Ig class/subclass identification; IC (integrated circuit) 50 The values, cross-reactivity and affinity were determined by ELISA.
The beneficial effects of the invention are as follows:
the hybridoma cell strain CGM for secreting the anti-fenfluramine monoclonal antibody has good detection sensitivity and affinity for fenfluramine, can be used for establishing a fenfluramine enzyme-linked immunosorbent assay method or a colloidal gold immunochromatography test strip rapid detection method, and lays a foundation for research and development and popularization of indirect competition ELISA kits and colloidal gold test strips.
Drawings
FIG. 1 is a standard inhibition curve of an anti-fenfluramine monoclonal antibody.
Preservation description
The hybridoma cell strain CGM which secretes the anti-fenfluramine monoclonal antibody is named as a monoclonal cell strain, and is preserved in the China general microbiological center of the culture Collection of microorganisms, CGMCC for short, the institute of microbiological study of national academy of sciences of No. 3 of North Chen West Lu 1, beijing, korea, the preservation number of which is CGMCC No.45025, and the preservation date of which is 2021, 12 months and 16 days.
Detailed Description
In order that the invention may be more clearly understood, the invention will now be further described with reference to the following examples. The examples are for illustration only and are not intended to limit the invention in any way. In the examples, each of the starting reagent materials is commercially available, and the experimental methods without specifying the specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.
The solution involved in the embodiment of the invention is configured as follows:
carbonate Buffer (CBS): weighing Na 2 CO 3 1.59g，NaHCO 3 2.93g, respectively dissolving in a small amount of double distilled water, mixing, adding double distilled water to about 800mL, mixing, adjusting pH to 9.6, adding double distilled water to 1000mL, and storing at 4deg.C for use;
phosphate Buffer (PBS): 8.00g NaCl,0.2g KCl,0.24g KH 2 PO 4 ，3.62g Na 2 HPO 4 ·12H 2 O is dissolved in 800mL of pure water, pH is regulated to 7.2-7.4 by NaOH or HCl, and volume is regulated to 1000mL;
PBST: PBS containing 0.05% tween 20;
TMB color development liquid: and (3) solution A: na (Na) 2 HPO 4 ·12H 2 18.43g of O, 9.33g of citric acid and pure water to 1000mL; and (2) liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. Mixing the solution B according to the volume ratio of 5:1 to obtain TMB color developing solution, and mixing at present.
The culture medium involved in the embodiment of the invention is as follows:
RPMI-1640 medium (mg/L): l-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23.19, L-valine 20, p-aminobenzoic acid 1, calcium nitrate 100, anhydrous magnesium sulfate 48.84, anhydrous sodium dihydrogen phosphate 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, D-calcium pantothenate 0.25, folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B12.005, sodium bicarbonate 2000.
Example 1: synthesis of fenfluramine complete antigen and coating antigen
1. Fenfluramine hapten
The chemical structural formula of the fenfluramine is shown as the following formula 1, the fenfluramine hapten adopts an analogue of fenfluramine with a similar structure, the structure is shown as the following formula 2, and the CAS number is 61471-64-5.
2. Synthesis of fenfluramine complete antigen (formula 3)
Taking 4.2mg of fenfluramine hapten, adding 5.0mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 3.7mg of NHS (N-hydroxysuccinimide), dissolving by using DMF (N, N-dimethylformamide), stirring at room temperature, and activating for 6 hours; another 15mg BSA (bovine serum albumin) was dissolved in 3mL, 0.05M, CB (carbonate buffer) solution at pH 9.6; the activating solution is added into BSA solution drop by drop, after stirring and reacting at room temperature overnight, the immunogen (namely complete antigen) is taken out and dialyzed for 3 days by PBS, and the subpackaged and stored at minus 20 ℃.
3. Synthesis of Fenfluramine coated antigen (formula 4)
Taking 3.6mg of fenfluramine hapten, adding 5.0mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 3.7mg of NHS (N-hydroxysuccinimide), dissolving the hapten by using DMF (N, N-dimethylformamide), stirring the mixture at room temperature, and activating the mixture for 6 hours; another 15mg of OVA (ovalbumin) was dissolved in 3mL, 0.05M, CB (carbonate buffer) solution at pH 9.6; and (3) dropwise adding the activated solution into the OVA solution, stirring at room temperature for reaction overnight, taking out the immunogen, dialyzing with PBS for 3 days, and subpackaging at-20 ℃.
Example 2: preparation of hybridoma cell strain CGM secreting anti-fenfluramine monoclonal antibody
1. Animal immunization: healthy BALB/c mice of 6-8 weeks of age were selected for immunization. After the complete antigen of fenfluramine (1 mg/mL) was emulsified with an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous multi-point injection (except for sprint immunization). The first immunization adopts Freund's complete adjuvant, and the first immunization dose is 100 mu L of each; boost was performed with Freund's incomplete adjuvant at a dose of 50. Mu.L each; the immunity dose is half of the previous immunity dose when in sprint immunity, and the mixture is uniformly mixed with normal saline and then directly injected into the abdominal cavity; each immunization interval was three weeks. The immunization process involved 1 primary immunization, 4 booster immunizations, and 1 sprint immunization. After the third immunization, blood sampling is carried out at intervals of one week to detect serum titer and inhibition; mice with the best inhibition were selected and were challenged 18 days after five days to prepare for fusion.
2. Cell fusion: after three days of sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 2000) method, as follows:
(1) Taking a mouse spleen in a sterile mode, grinding and passing through a 200-mesh cell screen to obtain spleen cell suspension, and performing cell counting;
(2) Collecting SP2/0 cells, suspending in RPMI-1640 basic culture solution, and performing cell count;
(3) Mixing spleen cells and SP2/0 cells according to the counting ratio of 2-10:1, centrifuging, fusing with PEG for 1min, adding RPMI-1640 basic culture solution from slow to fast, centrifuging, suspending in RPMI-1640 screening culture solution containing 20% fetal bovine serum and 2% 50 XHAT, adding into 96-well cell culture plate, placing into 37 ℃ and 5% CO 2 Is cultured in an incubator of (a).
3. Cell screening and cell strain establishment: the cells were subjected to half-replacement of the RPMI-1640 selection medium on day 3 of cell fusion, full-replacement with a 100 XHT-containing RPMI-1640 transition medium containing 20% fetal bovine serum and 1% on day 5, and cell supernatants were collected on day 7 for selection. Screening is carried out in two steps: the first step is to screen out positive cell holes by indirect ELISA, and the second step is to use fenfluramine as a standard substance, and to measure the inhibition effect of positive cells by indirect competition ELISA. Cell holes with better inhibition on fenfluramine are selected, subcloning is carried out by adopting a limiting dilution method, and detection is carried out by adopting the same method. Repeating for three times to obtain cell strain CGM.
Example 3: preparation and identification of anti-Fenfluramine monoclonal antibody
Taking 8-10 week-old BALB/c mice, and injecting paraffin oil into the abdominal cavity of each mouse by 1mL; intraperitoneal injection of 1X 10 per mouse after 7 days 6 Hybridoma cells, collecting ascites from day 7, purifying the ascites by octanoic acid-saturated ammonium sulfate method, and storing the obtained monoclonal antibody at-20deg.C.
Immunoglobulin subtype identification was performed on the monoclonal antibodies obtained by ascites purification using a mouse monoclonal antibody subtype identification kit, the subtype of which was of the IgG2b type, as shown in table l.
TABLE 1 subtype identification of Fenfluramine monoclonal antibodies
| Antibody subclasses | OD value |
| IgA | 0.137 |
| IgG1 | 0.112 |
| IgG2a | 0.231 |
| IgG2b | 2.029 |
| IgG3 | 0.109 |
| IgM | 0.078 |
Determination of monoclonal antibody to fenfluramine IC using indirect competition ELISA method 50 0.67ng/mL, and validated its IC for diclazuril et al 50 And the cross-reactivity is shown in Table 2.
TABLE 2 IC of Fenfluramine monoclonal antibody against Fenfluramine, chlorbutamine, sibutramine hydrochloride, bupropion 50 Cross-reactivity ratio
| IC 50 (ng/mL) | Cross reaction rate | |
| Fenfluramine | 0.67 | 100％ |
| Chlorobenzodin butylamine | ＞500 | ＜5％ |
| Sibutramine hydrochloride | ＞500 | ＜5％ |
| Bupropion (bupropion) | ＞500 | ＜5％ |
Example 4: application of anti-fenfluramine monoclonal antibody
The monoclonal antibody prepared from hybridoma cell strain CGM through in vivo ascites is applied to a Fenfluramine ELISA (enzyme-linked immunosorbent assay) additive recovery test, and the specific steps are as follows:
(1) Coating 96-well ELISA plates with 0.1 mug/mL of fenfluramine diluted by Carbonate Buffer (CBS) as coating raw materials, coating 100 mug/well at 37 ℃ for 2 hours, washing the plates three times with PBST washing liquid, 200 mug/well for 3min, and drying;
(2) Blocking with CBS containing 0.2% gelatin, blocking at 37deg.C for 2 hr, washing the plate with PBST wash solution three times, 200 μl each time, 3min each time, and drying;
(3) 0,0.02,0.05,0.1,0.2,0.5,1,2. Mu.g/L of the standard fenfluramine solution was prepared with Phosphate Buffered Saline (PBS). Respectively adding the standard solution and the sample extracting solution to be detected into the sealed ELISA plate, wherein each hole is 50 mu L, each sample is repeated for 3 holes, and then 50 mu L of 1 is added into each hole: after 16000 dilution of the anti-fenfluramine monoclonal antibody and reaction at 37 ℃ for half an hour, washing the plate and beating;
(4) 100 μl of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 with PBS containing 0.1% gelatin was added to each well, reacted at 37deg.C for half an hour, and then washed with a plate and dried;
(5) 100 mu L TMB color developing solution is added into each hole, and after color development is carried out for 15min at 37 ℃, 50 mu L2M H is added into each hole 2 SO 4 Stop solution, measuring light absorption value at 450 nm;
the standard inhibition curve of the anti-fenfluramine monoclonal antibody against fenfluramine is shown in figure 1.
(6) And (3) adding and recycling and sample pretreatment: fresh or warmed (cold stored) milk (5 g) was taken and three different doses of fenfluramine standard were added (5 ng, 10ng, 20ng respectively). The mixture was placed in a 50mL centrifuge tube, 1mL of 50% potassium hydroxide solution was slowly dropped, the mixture was sufficiently shaken on a vortex mixer, 20mL of ethyl acetate was slowly dropped, the mixture was shaken on the vortex mixer for 10 minutes, and the mixture was then placed in a centrifuge and centrifuged at 3000r/min for 5 minutes. 4mL of supernatant was removed from the tube and dried with nitrogen, 1mL of PBS containing 10% methanol was added for reconstitution, and 50. Mu.L was taken for detection. The recovery of the additives was 91.2%, 101.5% and 95.6% respectively by indirect competition ELISA.
Example 5: fenfluramine immunodetection kit
The present example provides a fenfluramine immunoassay kit comprising the anti-fenfluramine monoclonal antibody prepared in example 3, an ELISA plate, fenfluramine coating antigen, fenfluramine standard solution, HRP-labeled goat anti-mouse IgG secondary antibody and TMB color development solution.
The principle of the fenfluramine immunoassay kit for detecting fenfluramine is as follows: and detecting the content of fenfluramine in the sample to be detected by adopting an indirect competition ELISA method. The method comprises the steps of pre-coating a fenfluramine coating antigen in a micropore of an ELISA plate, adding fenfluramine standard solution or a sample to be detected, an anti-fenfluramine monoclonal antibody, an HRP-marked goat anti-mouse IgG secondary antibody and TMB chromogenic solution, preparing a fenfluramine standard inhibition curve, and determining the fenfluramine content in the sample to be detected according to the fenfluramine standard inhibition curve and the absorbance value of the sample to be detected. The detection of fenfluramine can be achieved by operating with methods commonly used in the art.
Example 6: colloidal gold test strip for fenfluramine detection
The embodiment provides a colloidal gold test strip, which comprises a sample pad, a colloidal gold binding pad, a nitrocellulose membrane and a water absorption pad, wherein the nitrocellulose membrane is sequentially provided with a detection line and a quality control line, and the colloidal gold binding pad is coated with the anti-fenfluramine monoclonal antibody prepared in the embodiment 3. The detection line is printed by fenfluramine coating antigen. The quality control line is printed by goat anti-mouse IgG secondary antibody. The colloidal gold test strip can be assembled in a manner commonly used in the art.
The principle of detecting the fenfluramine by using the fenfluramine detection colloidal gold test strip is as follows: and detecting whether the sample to be detected contains fenfluramine by using an indirect competition method principle. If the sample to be detected contains fenfluramine, the detection line does not develop color, and the quality control line develops color. If the sample to be detected does not contain fenfluramine, the detection line and the quality control line are both developed. The detection of the fenfluramine can be realized by adopting the common method in the field
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (9)
1. The hybridoma cell strain CGM of the anti-fenfluramine monoclonal antibody is characterized in that the preservation number is CGMCC NO.45025.
2. An anti-fenfluramine monoclonal antibody characterized by being secreted by the hybridoma cell strain CGM of claim 1.
3. The hybridoma cell strain CGM of claim 1 or the application of the anti-fenfluramine monoclonal antibody of claim 2 in preparing fenfluramine immunoassay kit or in preparing fenfluramine assay colloidal gold test strip.
4. A fenfluramine immunoassay kit comprising the hybridoma cell strain CGM of claim 1 or the anti-fenfluramine monoclonal antibody of claim 2.
5. The fenfluramine immunoassay kit of claim 4, further comprising an enzyme-labeled plate, fenfluramine-coated antigen, fenfluramine standard solution, enzyme-labeled secondary antibody, and substrate reaction solution.
6. The fenfluramine immunodetection kit of claim 5, characterized in that the fenfluramine coating antigen has a molecular structural formula shown in the following formula,
。
7. the colloidal gold test strip for detecting the fenfluramine is characterized by comprising a sample pad, a colloidal gold binding pad, a nitrocellulose membrane and a water absorption pad, wherein the nitrocellulose membrane is sequentially provided with a detection line and a quality control line, and the colloidal gold binding pad is coated with the anti-fenfluramine monoclonal antibody of claim 2.
8. The fenfluramine-detecting colloidal gold test strip of claim 7, wherein the detection line is printed from fenfluramine-coated antigen.
9. The test strip for detecting colloidal gold according to claim 8, wherein the molecular structural formula of the fenfluramine-coated antigen is shown as follows,
。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06261784A (en) * | 1992-09-18 | 1994-09-20 | Showa Shell Sekiyu Kk | Method for detecting methamphethahamine |
| KR19980027132A (en) * | 1996-10-11 | 1998-07-15 | 티엔 웨이첸 | Monoclonal antibodies specific for methamphetamine, hybridomas producing the antibody, kits containing the antibody and uses thereof |
| CN105385660A (en) * | 2015-11-03 | 2016-03-09 | 杭州隆基生物技术有限公司 | Hybridoma cell strain, antibody excreted by hybridoma cell strain, preparation method and application |
| CN107121552A (en) * | 2017-04-20 | 2017-09-01 | 江南大学 | One plant is secreted the hybridoma cell strain ZY 2G5 3 of 3,4- methylene benzylene chloride propylamine monoclonal antibodies and its applied |
-
2022
- 2022-01-27 CN CN202210105671.9A patent/CN114409796B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06261784A (en) * | 1992-09-18 | 1994-09-20 | Showa Shell Sekiyu Kk | Method for detecting methamphethahamine |
| KR19980027132A (en) * | 1996-10-11 | 1998-07-15 | 티엔 웨이첸 | Monoclonal antibodies specific for methamphetamine, hybridomas producing the antibody, kits containing the antibody and uses thereof |
| CN105385660A (en) * | 2015-11-03 | 2016-03-09 | 杭州隆基生物技术有限公司 | Hybridoma cell strain, antibody excreted by hybridoma cell strain, preparation method and application |
| CN107121552A (en) * | 2017-04-20 | 2017-09-01 | 江南大学 | One plant is secreted the hybridoma cell strain ZY 2G5 3 of 3,4- methylene benzylene chloride propylamine monoclonal antibodies and its applied |
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