CN113025581A

CN113025581A - Hybridoma cell strain secreting gelsemine monoclonal antibody and application thereof

Info

Publication number: CN113025581A
Application number: CN202110588098.7A
Authority: CN
Inventors: 杨术鹏; 周金慧; 李熠
Original assignee: Institute of Apicultural Research of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Apicultural Research of Chinese Academy of Agricultural Sciences
Priority date: 2021-05-28
Filing date: 2021-05-28
Publication date: 2021-06-25

Abstract

The invention relates to the technical field of immunodetection, and particularly relates to a hybridoma cell strain secreting gelsemine monoclonal antibody and application thereof. The monoclonal antibody secreted by the hybridoma cell strain MY-2 has good specificity and sensitivity (IC) to gelsemine₅₀The value can reach 0.66 ng/mL), and the content detection of the gelsemine in samples such as honey, blood plasma, vomit and the like can be realized. The gelsemine belongs to the characteristic of gelsemium which is a highly toxic plantThe compound is used for detecting the content of gelsemine in a sample, and has great practical application value for guaranteeing the quality safety of honey, avoiding poisoning events and diagnosing gelsemium poisoning persons in time.

Description

Hybridoma cell strain secreting gelsemine monoclonal antibody and application thereof

Technical Field

The invention relates to the technical field of immunodetection, and particularly relates to a hybridoma cell strain secreting gelsemine monoclonal antibody and application thereof.

Background

Kiss (Gelsemium elegans) Is a plant of the genus caulis Piperis and belonging to the family Loganiaceae, the whole plant is extremely toxic, commonly called gelsemium elegans, also called a big tea drug. Gelsmium elegans is widely distributed in ChinaMountainous areas of fujian, guangdong, and guangxi, south China; in addition, it is distributed in the southeast Asia countries, India and the central and south region of the United states. The gelsemium elegans is mainly externally used by folks for treating skin diseases due to high toxicity; in addition, gelsemium elegans has an insect repellent effect, and is also used in south China for repelling insects by veterinarians. Modern pharmacological studies show that gelsemium elegans contains rich alkaloids, and the gelsemium elegans has the effects of resisting tumors, resisting inflammation, relieving pain, regulating immunity and the like. As gelsemium elegans belongs to a highly toxic plant, a small amount of gelsemium elegans can cause poisoning and even death of serious patients. The fresh flower of gelsemium elegans becomes bright yellow and gorgeous and dazzling, and people often misunderstand that honeysuckle is eaten to cause poisoning. Moreover, gelsemium elegans is also a toxic honey source plant, and in the full-bloom stage, bees collect their nectar and pollen, toxic alkaloids are migrated and concentrated in honey to form toxic honey, and once a person eats the honey, the bee poisoning event occurs. At present, the types of alkaloids identified and identified from gelsemium are more than 120, and the alkaloids have better physiological activity, and are mainly divided into 6 types according to the difference of chemical structures, namely gelesedine type, gelsemine type, humantenine type, koumine type, sarpagine type and yohimbine type. Among them, gelsedine-type alkaloids have the strongest toxicity, and gelsemine, which is the representative substance, has been shown to have LD in mice₅₀Is 0.165 mg/kg B.W., belonging to a highly toxic compound. The gelsemine and the derivatives thereof in the gelsemium have high content and strong toxicity, so that the gelsemium is one of key compounds for causing gelsemium poisoning. Therefore, the method can be used for guaranteeing food safety and diagnosis and identification after gelsemium poisoning by detecting the existence and the content of gelsemium in a sample, and has a vital effect on avoiding poisoning events and saving the life of a patient. Therefore, the rapid detection and content determination of gelsemium elegans alkaloid in honey, blood plasma, vomit and other samples are particularly important.

At present, the content detection of gelsemium alkaloid in a sample mainly depends on instrument analysis, such as large-scale instruments and equipment such as high performance liquid chromatography, liquid phase tandem mass spectrometry and the like, needs professional personnel, is time-consuming, labor-consuming and high in cost, and is difficult to meet the requirements of site, rapidness and simplicity. The immunoassay method developed based on antigen-antibody specific binding has the advantages of simplicity, rapidness, easiness in operation and the like, and is widely applied to the fields of food safety, clinical diagnosis and the like at present. The core of the immunoassay method is an antibody, and once a high-quality antibody is obtained, a plurality of immunoassay modes can be selected to construct different analysis methods. At present, an immunoassay method aiming at gelsemium alkaloids such as gelsemine which is a highly toxic substance is rarely reported, which seriously limits the application of an immunoassay method in analyzing the gelsemium alkaloids in a sample. In order to guarantee life safety of people, quickly identify a gelsemium toxic sample, identify gelsemium poisoning and the like, an immunoassay method of gelsemine needs to be developed urgently.

Disclosure of Invention

In order to solve the technical problems, the invention provides a hybridoma cell strain secreting monoclonal antibodies to gelsemine and application thereof.

Specifically, a hybridoma cell strain MY-2 secreting monoclonal antibody of gelsemine with high sensitivity and high specificity is screened out through long-term tests, and is submitted to a relevant organization for preservation, wherein the preservation number is CGMCC number 20797; the classification is named as: the BalB/C mouse hybridoma cell strain has the preservation unit as follows: china general microbiological culture Collection center (CGMCC) for short; the preservation time is 10 months and 12 days in 2020; and (4) storage address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North; the zip code is 100101.

According to the hybridoma cell strain MY-2 provided by the invention, the invention also provides the gelsemine monoclonal antibody which is obtained by secreting the hybridoma cell strain MY-2.

Preferably, the amino acid sequences of the heavy chain and the light chain of the gelsemine monoclonal antibody are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2.

In some embodiments, the monoclonal antibodies are prepared as follows: and (3) injecting the hybridoma cell strain MY-2 into the abdominal cavity of a mouse pretreated by liquid paraffin, preparing ascites by adopting an in vivo induction method, and purifying by adopting an octanoic acid-ammonium sulfate method to obtain the monoclonal antibody.

The invention further provides a reagent, a test strip or a kit for detecting gelsemine, which contains the gelsemine monoclonal antibody.

In some embodiments, the test strip may be an immunochromatographic test strip or a colloidal gold immunoassay test strip.

In some embodiments, the kit may be an enzyme-linked immunoassay detection kit.

The invention also provides the application of the gelsemine monoclonal antibody or the reagent, the test strip or the kit in the aspect of detecting the gelsemine.

Specifically, the invention also provides a method for detecting gelsemine, which comprises the following steps: the gelsemine monoclonal antibody is used, or the reagent, the test strip or the kit is used.

The detection effect of the gelsemine monoclonal antibody is not interfered by a matrix, so that the gelsemine monoclonal antibody not only can be used for detecting foods (such as honey and the like) possibly containing the gelsemine, but also can be used for detecting blood or vomit samples of patients to determine whether the reason of the gelsemine is the gelsemine.

Based on the characteristics, the invention also provides the application of the gelsemine monoclonal antibody or the reagent, the test strip or the kit in the aspect of detecting toxic food, wherein the toxic food contains the gelsemine.

Further, the present invention provides a method for detecting a toxic food containing gelsemine, the method comprising: the gelsemine monoclonal antibody is used, or the reagent, the test strip or the kit is used.

Based on the technical scheme, the invention at least has the following beneficial effects:

the monoclonal antibody secreted by the hybridoma cell strain MY-2 has good specificity and sensitivity (IC) to gelsemine₅₀The value can reach 0.66 ng/mL), and the content detection of the gelsemine in samples such as honey, blood plasma, vomit and the like can be realized. Since gelsemine belongs to a characteristic compound of gelsemine which is a virulent plant, the content of gelsemine in a sample is detected, and the honey can be ensuredThe quality is safe, the poisoning event is avoided, and the gelsemium poisoning person can be diagnosed in time, so that the method has great practical application value.

Drawings

FIG. 1 is a chemical structure of gelsemine hapten (HGE-HS) used in an embodiment of the invention.

FIG. 2 is a standard curve of inhibition of gelsemine by the gelsemine monoclonal antibody of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1: synthesis of complete antigen and animal immunization

1. Preparing gelsemine hapten HGE-HS:

in hapten design and synthesis, the chemical structure of gelsemine does not contain sites such as hydroxyl, carboxyl, amino and the like which can be linked by a spacer arm, while the chemical structure of gelsemine is better kept by 14-hydroxyl gelsemine, and a hydroxyl group exists on the carbon 14 site, so that the spacer arm is easy to link. The invention adopts 14-hydroxyl gelsemine as a material for preparing gelsemine hapten, and the synthesis preparation is carried out by the following method.

(1) 14-Hydroxygelsemine (20 mg) and succinic anhydride (10 mg) were dissolved in 6.0 mL of anhydrous pyridine, respectively, and the two solutions were mixed and placed in an oil bath magnetic stirrer to react for 9 h (50 ℃, 240 rpm) in the dark to obtain a reaction product.

(2) The reaction system is quenched by water, extracted by ethyl acetate, washed by 0.1M dilute hydrochloric acid, dried and concentrated by organic phase to obtain a crude hapten product.

(3) After the crude product was separated and purified by preparative chromatography, the white target compound, 14-hydroxygelsemine hapten (11.03 mg, 43% yield), was obtained.Then, the hapten is identified by adopting liquid phase tandem high-resolution mass spectrometry, and the accurate mass number ism/z443.18143 (element composition is C)₂₃H₂₇N₂O₇ ⁺) The structure is shown in figure 1, which indicates the success of hapten synthesis.

2. Preparation of complete gelsemine antigen HGE-HS-BSA:

(1) weighing gelsemine hapten (10 mg),N-hydroxysuccinimide (4.5 mg) and dicyclohexylcarbodiimide (9 mg) in a glass reaction flask, 1 mL DMF was added. The glass bottle containing the reaction solution was placed on a magnetic stirrer and reacted at 400 rpm for 5 hours at room temperature in the dark.

(2) 60 mg BSA (OVA) was dissolved in 10 mL PBS containing 10% (volume percent) DMF to obtain a protein solution.

(3) And (3) dropwise adding the liquid phase which completes the step (1) into the protein solution prepared in the step (2), placing the reaction solution into a magnetic stirrer, and reacting for 5 hours at 4 ℃.

(4) Transferring the protein activating solution obtained in the step (3) into a dialysis bag with the molecular weight cutoff of 7 KDa, and then putting the dialysis bag into PBS buffer for dialysis at 4 ℃ for 3 days (changing every 12 hours).

(5) And (4) after the step (4) is finished, taking out the dialysis bag, centrifuging the liquid phase in the dialysis bag at 3000 rpm for 5min, and collecting supernatant, namely the artificial antigen HGE-HS-BSA (HGE-HS-OVA) solution.

3. Animal immunization: diluting the immunogen to 1 mg/mL (the diluent is 0.01M PBS) during the first immunization, taking the diluted immunogen, mixing the diluted immunogen with Freund's complete adjuvant in the same volume, fully emulsifying, and inoculating Balb/c mice (12 mice are immunized in total) with 8 weeks old subcutaneously and in multiple points on the back of the neck. Complete Freund's adjuvant was used for the first immunization, followed by incomplete Freund's adjuvant. One month is separated between the first immunization and the boosting immunization, and after the last boosting immunization is completed for one week, the antiserum titer and inhibition condition are detected by indirect competition ELISA. The last stroke was with complete immunogen (without adjuvant).

The primary test result of the collected mouse antiserum shows that the immunized mice all obtain better immunization effect,all mice had serum dilutions of 1: serum of more than 8000, 2# mice to obtain IC₅₀Most preferably, the concentration is 1.5 ng/mL. Therefore, 2# mice were selected and subjected to impact immunization with a complete immunogen to prepare monoclonal antibodies.

Example 2: screening and preparation of gelsemine monoclonal antibody cell strain

1. Cell fusion:

three days after the impact immunization, PEG (polyethylene glycol, molecular weight 1450) is used as a fusion agent to perform cell fusion, 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) spleen cells and SP2/0 cells were mixed at a ratio of 1:8, centrifuged, and fused with 1 mL of PEG 1450 at a 50% concentration by mass. Fusion was completed within 1 min. Then, 35 mL of RPMI-1640 basic culture medium was added from slow to fast within 2 min, and the mixture was centrifuged and suspended in a screening culture medium of RPMI-1640 containing 20% fetal bovine serum and 2% 50 XHAT. Adding the cell suspension into 96-well cell culture plate, and placing in 5% CO at 37 deg.C₂Cultured in an incubator.

2. Cell screening and subcloning:

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 a transitional RPMI-1640 medium containing 20% by mass of fetal bovine serum and 1% by mass of 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, gelsemine 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 good inhibition effect on the gelsemine standard substance, performing subcloning by adopting a limiting dilution method, and detecting by using the same method. Repeating the steps for three times until the cell positive rate reaches 100%, and obtaining 5 monoclonal antibody cell strains.

3. Preparation and identification of monoclonal antibody:

taking 12-week-old BALB/c mice, and injecting 1 mL of paraffin oil into the abdominal cavity of each mouse; after 7 days, 1X 10 injections were administered to the abdominal cavity of each mouse⁶And 2, collecting ascites from the seventh day after screening the hybridoma cells obtained in the step 2, purifying the collected ascites by an octanoic acid-ammonium sulfate method to obtain monoclonal antibodies which are respectively numbered as HGE-HS-BSA-1# -5 #, and storing the monoclonal antibodies in a refrigerator at the temperature of-20 ℃.

Example 3: evaluation and application of gelsemine monoclonal antibody

An indirect competitive enzyme-linked immunoassay method for determining gelsemine in a honey sample is established based on the purified monoclonal antibody, and comprises the following specific steps:

1. determination of the potency of monoclonal antibodies

The antibody titer was detected by indirect ELISA. The indirect ELISA procedure was as follows:

(1) coating: the coated HGE-HS-BSA was diluted to 0.25. mu.g/mL with 0.05M carbonate buffer (pH 9.6), added to a 96-well clear plate (100. mu.L/well), incubated at 37 ℃ in an incubator for 2 h, and the plate was washed 3 times with PBST buffer.

(2) And (3) sealing: add blocking solution (5% skim milk) 150. mu.L/well, incubate at 37 ℃ for 1 h, discard blocking solution, wash with PBST buffer 1 time, pat dry.

(3) Adding the antibody to be detected: 50 μ L of 0.01M PBS (pH7.4) was added to each well, and 50 μ L of diluted gelseminal monoclonal antibody was added, starting from 1:2000, and starting with a 2-fold gradient of 0.01M PBS, for a total of 8 gradients. The sample addition was 50. mu.L per well, incubated at 37 ℃ for 30 min, washed 3 times with PBST buffer, and blotted dry.

Meanwhile, an unimmunized mouse monoclonal antibody is set as a negative control.

(4) Adding an enzyme-labeled secondary antibody: adding an HRP-labeled goat anti-mouse IgG antibody diluted by an enzyme-labeled secondary antibody diluent according to the volume ratio of 1:5000, wherein the loading amount is 100 mu L/hole, reacting in an incubator at 37 ℃ for 30 min, washing with PBST buffer solution for 3 times, and patting to dry.

(5) Color development: mixing horseradish peroxidase substrate 3,3 ', 5, 5' -tetramethylbenzidine solution and 30% hydrogen peroxide by mass according to a volume ratio of 1:1, adding into a microplate (100 muL/hole), and developing at 37 ℃ for 15 min.

(6) And (4) terminating: 50 μ L of 2mol/L concentrated sulfuric acid was added to each well.

(7) Reading: by OD₄₅₀Wavelength measurement of each well OD value. The negative OD value is not more than 0.15, and the corresponding antibody dilution with the maximum OD value between 1.5 and 1.8 is taken as the antibody titer.

The optimal dilution of the monoclonal antibodies HGE-HS-BSA-1# -5 # is shown in the table 1, the data in the table can be used for obtaining, the dilution of all the monoclonal antibodies is more than 1:8000, and the better immune effect can be obtained by immunizing a mouse after the two synthesized haptens are coupled with the carrier protein. When the immunogen is HGE-HS-BSA, the titer distribution range of the monoclonal antibody is 1:8000-1:64000, wherein the titer of the HGE-HS-BSA-1# is up to 1: 64000.

2. Monoclonal antibody IC₅₀Measurement of (2)

(1) The coating and sealing processes are the same as above.

(2) Adding standard substances and antibodies: mu.L of gelsemine standard solution and 50. mu.L of antibody dilution (diluted according to the antibody titers in Table 1) were added to each well, incubated at 37 ℃ for 30 min, then washed 3 times with PBST solution and patted dry. The solvent of the standard solution is PBS buffer solution. When the monoclonal antibody HGE-HS-BSA-1# is detected, the concentration gradient of the standard substance is as follows: 0. 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, and 72.9 ng/mL of gelsemine solutions, each concentration in triplicate.

(3) Adding enzyme labeled secondary antibody, developing, terminating and reading.

The measured data are plotted on the abscissa against the concentration of gelsemine and on the OD₄₅₀The value is a vertical coordinate, a four-parameter equation of Origin 8.0 is used for fitting, and a standard curve is established to obtain IC₅₀The value is obtained. IC of 5 monoclonal antibodies₅₀The values are shown in Table 1, and it can be seen from the data in Table 1 that the sensitivity of the monoclonal antibody HGE-HS-BSA-1# is optimum and IC is₅₀It was 0.66 ng/mL. Thus, the hapten structure designed in the invention can be used as an antigenic determinant to effectively stimulate the mouse to produce the antibody. HLinear detection range (IC) of GE-HS-BSA-1# for gelsemine₂₀-IC₈₀) Is 0.37-1.18 ng/mL (see FIG. 2).

TABLE 1

Note: IC (integrated circuit)₅₀The detection standard substance during the determination is gelsemine.

3. Specificity and cross-reaction assay of monoclonal antibodies:

determination of IC of monoclonal antibody HGE-HS-BSA-1# for gelsemine using indirect competitive ELISA₅₀Comprises the following steps: 0.66 ng/mL, and moreover, the antibody has better cross-reaction (IC) to 14-hydroxyl gelsemine₅₀0.63 ng/mL), but less than 1% cross-reactivity to gelsemine alkaloids such as gelsemine, gelsemium, caraway, and speranskia herb, with substantially no cross-reactivity. The data show that the antibody has good sensitivity and specificity to gelsemine and 14-hydroxyl gelsemine, and can be used for immunoassay detection of the gelsemine and the 14-hydroxyl gelsemine.

Based on the results, the hybridoma cell strain secreting the monoclonal antibody HGE-HS-BSA-1# is named as hybridoma cell strain MY-2, and the hybridoma cell strain is submitted to a relevant organization for preservation, wherein the preservation number is CGMCC number 20797; the classification is named as: the BalB/C mouse hybridoma cell strain has the preservation unit as follows: china general microbiological culture Collection center (CGMCC) for short; the preservation time is 10 months and 12 days in 2020; and (4) storage address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North; the zip code is 100101.

4. Hybridoma cell transcriptome assay:

expanding and culturing hybridoma cell strain MY-2 to 5 multiplied by 10 cell number⁷Fully cleaved 5X 10 Using TRIzol Reagent⁷More than one hybridoma cell freshly harvested. Total RNA concentration is not lower than 100 ng/muL, the volume is more than 20 muL, and an RNA band is clear and has no obvious degradation. Samples were placed in dry ice and sequenced. Obtaining antibody heavy chain and light chain sequences by performing genome comparison, transcriptome comparison and transcriptome assembly on the detected sequencesAs shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

5. Application of gelsemine monoclonal antibody:

based on the constructed competitive ELISA method, the invention carries out detection and analysis on 30 suspected gelsemium toxic honey and 5 honey samples artificially mixed into gelsemium collected in Guangdong and Guangxi areas. Fully extracting samples such as honey by using ethyl acetate, drying by using nitrogen, dissolving and diluting the samples by using PBS, redissolving, and detecting and analyzing to be spotted. Test results show that 13 parts of suspected gelsemium toxic honey are positive samples and contain gelsemine with the content of 1.43-342.52 ng/mL; in addition, 10 parts of the gelsemine manually mixed into gelsemium honey were all detected to have a high content of gelsemine.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> bee institute of Chinese academy of agricultural sciences

<120> hybridoma cell strain secreting monoclonal antibody of gelsemine and application thereof

<130> KHP201117414.1YS

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 441

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Thr Phe Ser Ala Ser Glu Asp

20 25 30

Ser Leu Gly Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Pro Ile Tyr Gly Thr Asn Thr Asp Pro Ser Asp Tyr Thr Pro Gly

50 55 60

Tyr Gly Gln Ala Thr Leu Thr Ala Asp Arg Ser Ser Ser Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Thr Ser Ser Thr Arg Val Ala Leu Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr

115 120 125

Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu

130 135 140

Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp

145 150 155 160

Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser

180 185 190

Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser

195 200 205

Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys

210 215 220

Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr

245 250 255

Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser

260 265 270

Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile

290 295 300

Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn

305 310 315 320

Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu

340 345 350

Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe

355 360 365

Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala

370 375 380

Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr

385 390 395 400

Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly

405 410 415

Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His

420 425 430

Thr Glu Lys Ser Leu Ser His Ser Pro

435 440

<210> 2

<211> 220

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Glu Lys Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Val Arg Ala Asn Asp Glu Ser Ala Ser Thr

20 25 30

Gly Ala Thr Ile Asn His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ser Ser Ala Arg Glu Ser Leu Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Pro Asp Phe Thr Leu Ser Ile Asn

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Trp Thr Gly

85 90 95

Gly His Thr Gly Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Lys Ala Asp Ala Ala Pro Thr Val Ile Ser Ile Phe Pro Pro Ser Ser

115 120 125

Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn

130 135 140

Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu

145 150 155 160

Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr

180 185 190

Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr

195 200 205

Ser Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

210 215 220

Claims

1. Hybridoma cell strain MY-2 secreting monoclonal antibody of gelsemine, the preservation number is CGMCC number 20797.

2. The gelsemine monoclonal antibody secreted by the hybridoma cell strain MY-2 of claim 1.

3. A reagent, test strip or kit for detecting gelsemine comprising the gelsemine monoclonal antibody of claim 2.

4. Use of the gelsenicine monoclonal antibody of claim 2 or the reagent, test strip or kit of claim 3 to detect gelsenicine for non-diagnostic purposes.

5. A method for detecting gelsolin for non-diagnostic purposes, comprising: use of the gelsemine monoclonal antibody of claim 2, or use of the reagent, test strip or kit of claim 3.

6. Use of the gelsemine monoclonal antibody of claim 2 or the reagent, test strip or kit of claim 3 for detecting toxic food containing gelsemine.

7. A method of detecting a toxic food comprising gelsemine, the method comprising: use of the gelsemine monoclonal antibody of claim 2, or use of the reagent, test strip or kit of claim 3.