CN114409796A

CN114409796A - Hybridoma cell strain of anti-fenfluramine monoclonal antibody and application thereof

Info

Publication number: CN114409796A
Application number: CN202210105671.9A
Authority: CN
Inventors: 刘丽强; 严婕妤; 胥传来; 匡华; 徐丽广; 孙茂忠; 吴晓玲; 马伟; 郝昌龙; 宋珊珊; 吴爱红; 郭玲玲; 胥欣欣; 倪萍; 毕雪威; 郭鹏飞
Original assignee: Wuxi Determine Bio Tech Co ltd
Current assignee: Wuxi Determine Bio Tech Co ltd
Priority date: 2022-01-27
Filing date: 2022-01-27
Publication date: 2022-04-29
Anticipated expiration: 2042-01-27
Also published as: CN114409796B

Abstract

The invention discloses a hybridoma cell strain of an anti-fenfluramine monoclonal antibody and application thereof, belonging to the field of food safety immunodetection. The preservation number of the hybridoma cell strain is CGMCC NO.45025, the anti-fenfluramine monoclonal antibody secreted by the hybridoma cell strain has better detection sensitivity and affinity to fenfluramine, can be used for establishing a fenfluramine enzyme-linked immunosorbent assay method or a colloidal gold immunochromatographic test strip rapid detection method, and lays a foundation for research and development of indirect competition ELISA kits and colloidal gold test strips.

Description

Hybridoma cell strain of anti-fenfluramine monoclonal antibody and application thereof

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 a phenylalanine type appetite suppressant, is pure white crystal in appearance, is glittering and translucent, has a weak function of exciting the center, and can lower blood pressure; can also enhance the utilization of glucose by peripheral tissues to reduce blood sugar; it also has effects in reducing cholesterol, triacylglycerol and plasma total lipid. Is clinically used for simple obesity and obesity accompanied with diabetes, hypertension, anxiety and cardiovascular diseases. Fenfluramine is also called ice toxin, has strong stimulation effect on the central nervous system of a human body and strong toxicity, can generate strong physiological excitation on the human body, can consume a large amount of physical strength and reduce the immune function, and seriously damages heart and brain tissues and even causes death. With the change of aesthetic concepts and the love of people, the demand and the use amount of the weight-reducing drug are increased rapidly, the use trend of the weight-reducing drug containing fenfluramine is also increased, and a plurality of health and social problems are caused, so that a method for detecting and analyzing the content of the fenfluramine 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 have some drawbacks to varying degrees: time consuming, expensive instruments and extensive sample pre-treatment procedures. Therefore, these methods are not suitable for the detection of fenfluramine in situ for high throughput analysis. This means that the use of these methods in field analysis is limited because of the need for an analysis system.

The immunoassay method has the characteristics of low cost, high flux, high sensitivity, low relative requirement on technical personnel and the like, so that the immunoassay method is suitable for rapid screening of a large number of samples. The precondition for detecting fenfluramine by using an immunoassay method is that a monoclonal antibody with high specificity and high sensitivity to fenfluramine is obtained, so that the method for preparing the monoclonal antibody with high specificity and high sensitivity to fenfluramine is very critical.

Disclosure of Invention

In order to overcome 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 invention provides a hybridoma cell strain CGM of an anti-fenfluramine monoclonal antibody in a first aspect, wherein the preservation number of the hybridoma cell strain CGM is CGMCC NO. 45025.

The second aspect of the invention provides an anti-fenfluramine monoclonal antibody which is secreted and produced by the hybridoma cell strain CGM.

The third aspect of the invention provides the application of the hybridoma cell strain CGM or the anti-fenfluramine monoclonal antibody secreted by the hybridoma cell strain CGM in fenfluramine detection, the application in preparing a fenfluramine immunoassay kit or the application in preparing a fenfluramine detection colloidal gold test strip.

The fourth aspect of the invention provides a fenfluramine immunoassay kit, which comprises the hybridoma cell strain CGM or the anti-fenfluramine monoclonal antibody secreted by the hybridoma cell strain CGM.

Further, the fenfluramine immunoassay kit further comprises an enzyme label plate, a fenfluramine coating antigen, a fenfluramine standard solution, an enzyme labeled secondary antibody and a substrate reaction solution;

preferably, the structural formula of the fenfluramine-coated antigen molecule is shown as the following formula,

the fifth aspect of the invention provides a fenfluramine detection colloidal gold test strip which comprises a sample pad, a colloidal gold combined 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 combined pad is coated with an anti-fenfluramine monoclonal antibody produced by secretion of a hybridoma cell strain CGM.

Further, the detection line is printed by fenfluramine coated antigen.

the sixth aspect of the invention provides an application of the fenfluramine immunoassay kit or the fenfluramine colloidal gold test strip in fenfluramine detection.

The basic steps of the preparation of the hybridoma cell strain CGM cell strain are as follows:

(1) preparation and identification of immunogen: fenfluramine is taken as a raw material, the fenfluramine is connected with amino of a protein carrier by an activated ester method, after the reaction is finished, a complete antigen and an uncoupled small molecule hapten are separated by dialysis, and the complete antigen is identified by an ultraviolet absorption scanning method;

(2) immunization of mice: and selecting BALB/c mice of 6-8 weeks old for immunization. Emulsifying immunogen and Freund's adjuvant completely, injecting immune mouse via subcutaneous multiple points, adopting Freund's complete adjuvant for first immunization, using Freund's incomplete adjuvant for boosting immunization, mixing immune dose with normal saline uniformly, and injecting into abdominal cavity directly; each immunization interval was three weeks; the immune process comprises 1 first immunity, 4 boosting immunizations and 1 sprint immunity; after the third immunization, blood is collected at intervals of one week to detect serum titer and inhibition; selecting the mouse with the best inhibition, performing thrust immunization 18 days after the five-immunization, and preparing for fusion;

(3) cell fusion and cell line establishment: fusing mouse spleen cells and mouse myeloma cells by a polyethylene glycol (PEG 2000) method, culturing by HAT culture medium, detecting positive cell holes by indirect ELISA, further determining the inhibition effect of the positive cell holes by indirect competition ELISA, carrying out three times of subcloning on the positive cell holes with the best inhibition by a limiting dilution method, and finally screening to obtain a hybridoma cell strain CGM;

(4) and (3) identification of the properties of hybridoma cell strains: determining by using an enzyme-labeled secondary antibody kit for identifying mouse monoclonal antibody Ig class/subclass; IC (integrated circuit)₅₀Values, cross-reactivity and affinity were determined by ELISA.

The invention has the beneficial effects that:

the hybridoma cell strain CGM secreting the anti-fenfluramine monoclonal antibody has good detection sensitivity and affinity to fenfluramine, can be used for establishing a fenfluramine enzyme-linked immunosorbent assay method or a colloidal gold immunochromatographic 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.

Deposit description

The hybridoma cell strain CGM secreting the anti-fenfluramine monoclonal antibody is classified and named as a monoclonal cell strain, is preserved in China general microbiological culture Collection center (CGMCC), China academy of sciences, China institute of microbiology, 3, of Xilu No. 1, North Cheng, the area of the morning of Beijing, and is classified and named as a cloned cell strain with the preservation number of CGMCC No.45025 and the preservation date of 2021 year, 12 months and 16 days.

Detailed Description

In order that the invention may be more clearly understood, it will now be further described with reference to the following examples. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

The solution related in the embodiment of the invention is prepared as follows:

carbonate Buffer (CBS): weighing Na₂CO₃ 1.59g，NaHCO₃2.93g of the mixture is respectively dissolved in a small amount of double distilled water and then mixed, the double distilled water is added to about 800mL and mixed evenly, the pH value is adjusted to 9.6, the double distilled water is added to the volume of 1000mL, and the mixture is stored for later use at 4 ℃;

phosphate Buffered Saline (PBS): 8.00g NaCl, 0.2g KCl, 0.24g KH₂PO₄，3.62g Na₂HPO₄·12H₂Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and fixing the volume to 1000 mL;

PBST: PBS containing 0.05% Tween 20;

TMB color development liquid: solution A: na (Na)₂HPO₄·12H₂O 1843g, 9.33g of citric acid and pure water to reach the volume of 1000 mL; and B, liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. Mixing the liquid B according to the volume ratio of 5: 1 to obtain the TMB color developing solution which is mixed at present.

The culture media involved in the examples of the present invention are 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, calcium D-pantothenate 0.25, Folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B120.005, and sodium bicarbonate 2000.

Example 1: synthesis of fenfluramine complete antigen and envelope antigen

1. Fenfluramine hapten

The chemical structural formula of fenfluramine is shown in the following formula 1, and the fenfluramine hapten of the invention adopts an analogue of fenfluramine with a similar structure, which has the structure shown in the following formula 2, and the CAS number of the fenfluramine hapten is 61471-64-5.

2. Synthesis of fenfluramine complete antigen (formula 3)

Adding 5.0mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 3.7mg of NHS (N-hydroxysuccinimide) into 4.2mg of fenfluramine hapten, dissolving with DMF (N, N-dimethylformamide), stirring at room temperature, and activating for 6 h; dissolving 15mg BSA (bovine serum albumin) in 3mL CB (carbonate buffer solution) solution with pH of 0.05M and 9.6; and dropwise adding the activating solution into a BSA solution, stirring at room temperature for reaction overnight, taking out immunogen (namely complete antigen) PBS, dialyzing for 3 days, and subpackaging and storing at-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 with DMF (N, N-dimethylformamide), stirring at room temperature, and activating for 6 h; dissolving another 15mg of OVA (ovalbumin) in 3mL of CB (carbonate buffer solution) solution with the pH value of 0.05M and 9.6; and dropwise adding the activating solution into an OVA solution, stirring at room temperature for overnight reaction, taking out immunogen PBS, dialyzing for 3 days, and subpackaging and storing at-20 ℃.

Example 2: preparation of hybridoma cell strain CGM secreting monoclonal antibody against fenfluramine

1. Animal immunization: healthy BALB/c mice 6-8 weeks old were selected for immunization. BALB/c mice were immunized by subcutaneous multi-point injection (except for the booster immunization) after the complete fenfluramine antigen (1mg/mL) was emulsified with the same amount of Freund's adjuvant. The first immunization adopts Freund's complete adjuvant, and the dose of the first immunization is 100 mu L each; the booster immunization uses Freund incomplete adjuvant, and the booster immunization dose is 50 mu L each; the immune dose in the spurting immunization is half of the immune dose in the previous time, and the spurting immunization is directly injected into the abdominal cavity after being uniformly mixed with the physiological saline; the intervals between immunizations were three weeks. The immunization process comprises 1 first immunization, 4 booster immunizations and 1 sprint immunization. After the third immunization, blood is collected at intervals of one week to detect serum titer and inhibition; the best-suppressed mice were selected, immunized by boosting 18 days after five immunizations, and prepared for fusion.

2. Cell fusion: after three days of the spurting immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight 2000) method, and the specific steps are as follows:

(1) taking a spleen of a mouse aseptically, grinding the spleen, passing through a 200-mesh cell screen to obtain a spleen cell suspension, and counting cells;

(2) collecting SP2/0 cells, suspending in RPMI-1640 basic culture solution, and counting cells;

(3) mixing splenocytes and SP2/0 cells at a ratio of 2-10: 1, centrifuging, fusing with PEG for 1min, adding RPMI-1640 basic culture medium from slow to fast, centrifuging, suspending in RPMI-1640 screening culture medium containing 20% fetal calf serum and 2% 50 × HAT, adding into 96-well cell culture plate, standing at 37 deg.C and 5% CO₂Cultured in an incubator.

3. Cell screening and cell strain establishment: on day 3 of cell fusion, the fused cells were subjected to RPMI-1640 screening medium half-replacement, on day 5, to total-replacement with RPMI-1640 medium containing 20% fetal bovine serum and 1% 100 XHT, and on day 7, cell supernatants were collected and screened. The screening is divided into two steps: in the first step, positive cell holes are screened by indirect ELISA, in the second step, fenfluramine is selected as a standard substance, and the inhibition effect of positive cells is measured by indirect competitive ELISA. And selecting a cell hole with better inhibition on fenfluramine, carrying out subcloning by adopting a limiting dilution method, and detecting by using the same method. Repeating the steps for three times to obtain the cell strain CGM.

Example 3: preparation and identification of anti-fenfluramine monoclonal antibody

Taking BALB/c mice 8-10 weeks old, and injecting 1mL of paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 10⁶And (3) collecting ascites from the 7 th day, purifying the ascites by an octanoic acid-saturated ammonium sulfate method, and storing the obtained monoclonal antibody at-20 ℃.

And (3) carrying out immunoglobulin subtype identification on the monoclonal antibody obtained by ascites purification by using a mouse monoclonal antibody subtype identification kit, wherein the subtype is IgG2b type, and is specifically shown in table l.

TABLE 1 subtype identification of fenfluramine monoclonal antibodies

Subclass of antibody	OD value
		IgA	0.137
IgG1	0.112
		IgG2a	0.231
IgG2b	2.029
		IgG3	0.109
IgM	0.078

Determination of IC of monoclonal antibody to fenfluramine Using Indirect competitive ELISA₅₀0.67ng/mL, and verified its IC for diclazuril, etc₅₀And the cross-reactivity ratio are shown in Table 2.

TABLE 2 IC of Finfluramine monoclonal antibody against fenfluramine, chlobenzamide, sibutramine hydrochloride, bupropion₅₀And cross reaction rate

	IC₅₀(ng/mL)	Rate of cross reaction
			Fenfluramine	0.67	100％
Chlorobenzene butylamine	＞500	＜5％
			Sibutramine hydrochloride	＞500	＜5％
Bupropion derivatives	＞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 fenfluramine ELISA addition recovery test, and the specific steps are as follows:

(1) coating with 0.1 mu g/mL fenfluramine diluted by Carbonate Buffer Solution (CBS) as a coating antigen to coat a 96-well enzyme label plate, wherein each well is 100 mu L, after coating at 37 ℃ for 2h, washing the plate with PBST washing solution three times, each well is 200 mu L, each time is 3min, and then patting to dry;

(2) sealing with CBS containing 0.2% gelatin, sealing at 37 deg.C for 2 hr with 200 μ L per well, washing the plate with PBST lotion for three times, each time with 200 μ L per well, each time for 3min, and drying;

(3) the standard solutions of fenfluramine were prepared in 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2. mu.g/L with Phosphate Buffered Saline (PBS), respectively. Respectively adding the standard solution and the extract of the sample to be detected into the closed enzyme label plate, wherein each hole is 50 mu L, each sample is repeatedly provided with 3 holes, and each hole is added with 50 mu L1: 16000 diluting anti-fenfluramine monoclonal antibody, reacting at 37 deg.C for half an hour, washing and drying;

(4) adding 100 μ L of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1: 3000 with PBS containing 0.1% gelatin into each well, reacting at 37 deg.C for half an hour, washing and drying;

(5) adding 100 μ L of TMB color developing solution into each well, developing at 37 deg.C for 15min, and adding 50 μ L of 2M H into each well₂SO₄Stopping solution, measuring the light absorption value at 450 nm;

the standard inhibition curve of the anti-fenfluramine monoclonal antibody against fenfluramine is shown in figure 1.

(6) Adding and recovering and sample pretreatment: taking 5g of fresh or rewarming (refrigerated preservation) milk, and adding three fenfluramine standard substances with different dosages, wherein the dosage is 5ng, 10ng and 20ng respectively. Placing the mixture into a 50mL centrifuge tube, slowly dropping 1mL of 50% potassium hydroxide solution, fully oscillating on a vortex mixer, slowly dropping 20mL of ethyl acetate, oscillating on the vortex mixer for 10min, and then placing the mixture into a centrifuge to centrifuge for 5min at 3000 r/min. 4mL of the supernatant was removed from the other centrifuge tube, blown dry with nitrogen, and 1mL of 10% methanol in PBS was added for reconstitution, and 50. mu.L was used for detection. The additive recovery tests were performed by indirect competitive ELISA with recovery rates of 91.2%, 101.5%, 95.6%, respectively.

Example 5: fenfluramine immunoassay kit

The present example provides a fenfluramine immunoassay kit, which comprises the anti-fenfluramine monoclonal antibody prepared in example 3, an elisa plate, a fenfluramine-coated antigen, a fenfluramine standard solution, an HRP-labeled goat anti-mouse IgG secondary antibody, and a 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 coating fenfluramine coating antigen in micropores of an enzyme label plate in advance, adding fenfluramine standard solution or a sample to be detected, an anti-fenfluramine monoclonal antibody, an HRP-labeled goat anti-mouse IgG secondary antibody and TMB color development solution, making 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 fenfluramine detection can be realized by adopting a method commonly used in the field to operate.

Example 6: colloidal gold test strip for fenfluramine detection

The embodiment provides a colloidal gold test strip, and it includes sample pad, colloidal gold conjugate pad, nitrocellulose membrane and absorbent pad, be equipped with detection line and quality control line on the nitrocellulose membrane in proper order, the colloidal gold conjugate pad coats the anti-fenfluramine monoclonal antibody that embodiment 3 prepared. The detection line is printed by fenfluramine coated antigen. The quality control line is obtained by printing a goat anti-mouse IgG secondary antibody. The colloidal gold test strip can be assembled in a way commonly used in the field.

The principle of detecting fenfluramine by the fenfluramine detection colloidal gold test strip is as follows: and detecting whether the sample to be detected contains fenfluramine or not by utilizing the principle of an indirect competition method. If the sample to be detected contains fenfluramine, the detection line is not colored, and the quality control line is colored. And if the sample to be detected does not contain fenfluramine, the detection line and the quality control line are colored. The fenfluramine detection can be realized by adopting a common method in the field to operate

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. A hybridoma cell strain CGM of the fenfluramine-resistant monoclonal antibody is characterized in that the preservation number is CGMCC NO. 45025.

2. An anti-fenfluramine monoclonal antibody secreted by the hybridoma cell line CGM according to claim 1.

3. The hybridoma cell strain CGM according to claim 1 or the anti-fenfluramine monoclonal antibody according to claim 2, wherein the hybridoma cell strain CGM is used for detecting fenfluramine, the anti-fenfluramine monoclonal antibody is used for preparing a fenfluramine immunoassay kit or a fenfluramine colloidal gold test strip.

4. A fenfluramine immunoassay kit, wherein the fenfluramine immunoassay kit comprises the hybridoma cell line CGM according to claim 1 or the anti-fenfluramine monoclonal antibody according to claim 2.

5. The fenfluramine immunoassay kit according to claim 4, characterized in that the fenfluramine immunoassay kit further comprises an enzyme label plate, a fenfluramine-coated antigen, a fenfluramine standard solution, an enzyme-labeled secondary antibody and a substrate reaction solution.

6. The fenfluramine immunoassay kit according to claim 5, wherein the molecular structural formula of the fenfluramine-coated antigen is shown as the following formula,

7. a fenfluramine detection colloidal gold test strip is characterized in that the colloidal gold test strip 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 fenfluramine-resistant monoclonal antibody of claim 2.

8. The fenfluramine colloidal gold test strip of claim 7, wherein the test line is printed with fenfluramine-coated antigen.

9. The fenfluramine colloidal gold test strip for detecting according to claim 8, wherein the molecular structural formula of the fenfluramine-coated antigen is shown as the following formula,

10. use of the fenfluramine immunoassay kit of claim 4 or the fenfluramine colloidal gold test strip of claim 7 for fenfluramine detection.