CN119264260B

CN119264260B - Patulin monoclonal antibody and application thereof

Info

Publication number: CN119264260B
Application number: CN202411726730.XA
Authority: CN
Inventors: 赵荣茂; 巩玉洁; 李浛; 袁婷婷; 石强; 杨晓霞
Original assignee: Beijing Nabai Bio Tech Co ltd
Current assignee: Beijing Nabai Bio Tech Co ltd
Priority date: 2024-11-28
Filing date: 2024-11-28
Publication date: 2025-02-14
Anticipated expiration: 2044-11-28
Also published as: CN119264260A

Abstract

The present invention provides a patulin monoclonal antibody and its application. The monoclonal antibody of patulin comprises a heavy chain and a light chain, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3; the light chain variable region comprises CDR1, CDR2 and CDR3; the amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7 respectively. The amino acid sequences of the light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID No.8, SEQ ID No.9 and SEQ ID No.10 respectively. The monoclonal antibody has a strong binding force with the patulin artificial antigen and has the characteristics of high sensitivity, good specificity and strong stability.

Description

Patulin monoclonal antibody and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a patulin monoclonal antibody and application thereof.

Background

Patulin (Patulin, PAT) is a secondary metabolite produced by fungi (penicillium expansum, patulin, etc.). The chemical name of patulin is 4-hydroxy-4H-furo [3,2C ] pyran-2 [6H ] -ketone, molecular formula C ₇H₆O₄, which is easy to dissolve in water, ethanol, acetone, chloroform and ethyl acetate, slightly soluble in benzene and diethyl ether, insoluble in petroleum ether, and stable under acidic condition. Glister (1941) was first discovered and isolated and purified at oxford university, and the experiment found that patulin is a broad-spectrum antibiotic which can be used for treating human cold, but subsequent studies demonstrated that it has stronger toxicity to experimental animals, from which the study on patulin was focused on its toxicity and pollution to food and feed. Toxicology studies on animal experiments or cell experiments show that patulin has various hazards such as acute toxicity, subacute toxicity, reproductive toxicity, immune toxicity and the like. Patulin is classified by the international agency for research on cancer (IARC) as a third class of suspected carcinogens. Patulin has a respiratory jeopardy effect. The intake of patulin in the body causes abnormality in the movement of substances in the membrane due to the change in permeability of the cell membrane, thereby indirectly causing physiological respiratory abnormality. Patulin mainly pollutes apples, hawthorns and products thereof. The limit of mycotoxin in national Standard food for food safety (GB 2761-2017) standard in China prescribes that the limit standard of patulin in apple and hawthorn products is 50 mug/kg.

Currently, methods for detecting patulin mainly include Thin Layer Chromatography (TLC), high Performance Liquid Chromatography (HPLC), gas Chromatography (GC), mass Spectrometry (MS), high performance liquid-mass spectrometry (HPLC-MS), and the like. The specificity of the thin layer chromatography is not strong, and other fluorescent substances in the sample easily interfere with the result measurement. The liquid chromatography has strong specificity, but the required instruments and equipment are expensive, and the detection activity is limited to a certain extent. The ELISA method is one of the more common methods for measuring the patulin, and has strong specificity, high sensitivity and high analysis speed, and is commonly used in actual detection activities. Besides strong specificity, the colloidal gold and fluorescence immunity chromatography has the characteristics of high sensitivity, low cost, simple and convenient operation, wide detection scene and the like, and is a detection method with more practical application. The invention aims to provide a monoclonal antibody with higher affinity and detection sensitivity to patulin, and a variable region of a light chain and a heavy chain of the monoclonal antibody. Lays a foundation for the research and popularization of ELISA, colloidal gold test strips and fluorescent immune test strips.

Disclosure of Invention

For this reason, the present invention provides a monoclonal antibody against patulin and its use.

In order to achieve the above object, the embodiment of the present invention provides the following technical solutions:

in a first aspect, the invention provides a patulin monoclonal antibody, characterized in that the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID No. 1;

the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID No. 2;

The heavy and light chain variable regions are each comprised of a complementarity determining region and a framework region, the complementarity determining regions being comprised of CDR1, CDR2 and CDR 3;

the amino acid sequence of CDR1 of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID No. 5;

the amino acid sequence of CDR2 of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID No. 6;

the amino acid sequence of CDR3 of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID No. 7;

the amino acid sequence of CDR1 of the light chain variable region of the monoclonal antibody is shown as SEQ ID No. 8;

the amino acid sequence of CDR2 of the light chain variable region of the monoclonal antibody is shown as SEQ ID No. 9;

the amino acid sequence of CDR3 of the light chain variable region of the monoclonal antibody is shown as SEQ ID No. 10.

In a second aspect, the patulin monoclonal antibody is characterized in that:

the nucleotide sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID No. 3;

The nucleotide sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID No. 4.

In a third aspect, the invention provides an application of a monoclonal antibody of patulin in preparing a test strip for detecting patulin.

Preferably, the test strip is a colloidal gold test strip, or a fluorescent microsphere test strip, or a latex microsphere test strip.

The invention has the following advantages:

The monoclonal antibody provided by the invention has the characteristics of strong specificity, high sensitivity and the like, can be used as a raw material for enzyme-linked immunosorbent assay and colloidal gold immunoassay, and lays a foundation for development and popularization of ELISA, colloidal gold test strips and fluorescent immune test strips.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.

FIG. 1 shows the result of the alignment of the heavy chain gene sequences of the patulin monoclonal antibody;

FIG. 2 shows the result of alignment of the amino acid sequence homology of the heavy chain of the patulin monoclonal antibody;

FIG. 3 shows the result of the alignment of the light chain gene sequences of the patulin monoclonal antibody;

FIG. 4 shows the result of alignment of the amino acid sequence homology of the light chain of the patulin monoclonal antibody;

FIG. 5 is a schematic diagram of a colloidal gold test strip;

FIG. 6 is a schematic diagram showing the result interpretation of colloidal gold test strips.

Detailed Description

Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

EXAMPLE 1 Synthesis of patulin artificial antigen

1. Synthesis route of patulin-hemi-glutarate (PAT-HG)

Patulin, glutaric anhydride and DMAP were dissolved in anhydrous tetrahydrofuran (molar ratio 1:20:4), reacted by shaking at 37 ℃ for 2h (TLC monitoring reaction), and distilled water was added to terminate the reaction. The reaction product is spotted on a preparative thin layer chromatography silica gel plate, a target product patulin half glutaric acid (PAT-HG) strip is scraped off under 256 nm ultraviolet light in a spreading layer (V (n-hexane): V (ethyl acetate): V (ethanol) =5:3:1), the scraped silica gel is put into a centrifuge tube, eluted 3 times by methanol, the methanol is pumped out in vacuum, and the residue is purified PAT-HG.

2. Preparation of patulin immune antigen

The PAT-HG is coupled with the carrier protein by adopting an active ester method. PAT-HG, NHS, DCC is dissolved in anhydrous tetrahydrofuran (molar ratio is 1:2:4), shaking reaction is carried out at 15 ℃ for 6 h (TLC monitoring reaction), precipitate is removed by centrifugation, tetrahydrofuran is pumped out in vacuum, residues are dissolved in DMSO, 5% Bovine Serum Albumin (BSA) solution dissolved in 0.13 mol/L NaHCO ₃ is slowly dripped in, shaking reaction is carried out slowly under 15 ℃ in dark condition for overnight, PBS buffer solution (0.1 mol/L, pH 7.4) is used for dialysis for 3 days at 4 ℃, and the patulin immune antigen (PAT-HG-BSA) is obtained by freeze vacuum drying.

3. Preparation of patulin detection antigen

The PAT-HG is coupled with the carrier protein by adopting an active ester method. PAT-HG, NHS, DCC is dissolved in anhydrous tetrahydrofuran (molar ratio is 1:2:4), shaking reaction is carried out at 15 ℃ for 6h (TLC monitoring reaction), precipitate is removed by centrifugation, tetrahydrofuran is pumped out in vacuum, residue is dissolved in DMSO, 5% chicken Ovalbumin (OVA) solution dissolved in 0.13 mol/L NaHCO ₃ is slowly dripped in, shaking reaction is carried out slowly under 15 ℃ in dark condition for overnight, PBS buffer solution (0.1 mol/L, pH 7.4) is used for dialysis for 3 days at 4 ℃, and the patulin detection antigen (PAT-HG-OVA) is obtained by freeze vacuum drying.

EXAMPLE 2 preparation of patulin monoclonal antibodies

1. Immunization of mice

The prepared immunogen patulin-BSA was taken and immunized with 10. Mu.g antigen per mouse, the immunogen was diluted to the desired concentration with sterile PBS, the same amount of adjuvant and immunogen were respectively inhaled into two 2.5mL disposable syringes, then inserted into a communicating vessel, and pushed 8500 times with an emulsifier. The immunization procedure employed one basic immunization and several booster immunizations.

3 Healthy Balb/c female mice with the age of 6-8 weeks and the weight of 18-20 g are selected, and the antigen mice emulsified by equivalent Freund's complete adjuvant are subjected to subcutaneous multipoint injection for the first time. Boosting with Freund's incomplete adjuvant emulsified antigen every 14 d (first and second two-way interval 21 d), taking blood from tail vein at 7-10 days after three times of immunization, diluting with 1×PBS 100 times, centrifuging to obtain supernatant, measuring mouse serum titer and inhibiting, and fusing to obtain monoclonal antibody with titer of 10W. Consider the effect of time intervals on antibody production. Mice with high serum titers were selected 3d prior to fusion and boosted by intraperitoneal injection of the diluted neat antigen in 1 XPBS.

2. Hybridoma cell preparation

Preparation of hybridoma cells SP2/0 myeloma cells were conditioned to a cell density of 1X 10 ⁶/mL with RPMI-1640 basal medium. Three days after the mice are subjected to impact immunization, the feeder spleen cells are taken, the feeder spleen cells are regulated to 1X 10 ⁵/mL, PEG is added to fuse the two cells, then hybridoma positive holes are screened, the positive holes are cloned by a limiting dilution method, and a hybridoma cell strain capable of stably secreting the patulin monoclonal antibody is obtained and established.

3. Ascites preparation

Monoclonal antibodies were prepared by in vivo induction. Each mouse was injected with Freund's incomplete adjuvant (0.5 ml) to purge the abdominal cavity, and after 7d, the number of hybridoma cells in the logarithmic growth phase was adjusted to 1X 10 ⁶ cells/ml, and each mouse was inoculated with 0.5ml. The ascites production in mice was observed daily at intervals of about 7 d. When the abdomen of the mice is obviously enlarged, the spirit is worsened and the mice are dying, collecting ascites, centrifuging for 5 min at 12000 r/min, removing surface fat, and carefully sucking the supernatant for ammonium sulfate precipitation.

4. Antibody purification

The name and volume of ascites were recorded. 3mL of pH4.0,0.06M sodium acetate buffer was added, mixed well for 5min, 10ul of octanoic acid was added, and stirred at 4℃for 15min. The mixture was filtered through cotton wool once with a syringe. Centrifuge at 12000rpm for 15min at 4 ℃. The supernatant was taken, added with an equal volume of saturated ammonium sulfate to a final concentration of 50%, stirred while standing at 4℃for 4h. Subsequently, the mixture was centrifuged at 12000rpm at 4℃for 15 minutes, and the supernatant was discarded, and 1.8ml of 0.01M PBS (pH 7.2) was added to the centrifuge tube until the pellet was completely dissolved. The total volume was determined by gun, 1/2 volume of saturated ammonium sulfate was added to a concentration of 33% and stirred at 4 ℃ overnight. Centrifuge at 12000rpm for 15min, discard supernatant, add 0.45ml 0.01M PBS to dissolve precipitate. Treating the dialysis bag (boiling for 5min, cleaning with pure water, and detecting leakage). The dissolved solution was placed in a dialysis bag and dialyzed overnight against 0.01M PBS. Changing the dialysis liquid for 2-3 times, collecting antibody, measuring concentration, and packaging.

5. Characterization of monoclonal antibodies to patulin

The sensitivity and the specificity of the patulin monoclonal antibody are detected by adopting an indirect ELISA method. The patulin-OVA artificial antigen is coated according to 1ug/mL, 1mg/mL of monoclonal antibody is subjected to gradient dilution, the sensitivity of the monoclonal antibody is verified, and the result is shown in Table 1. The sensitivity of the monoclonal antibody can reach 1:20000 times dilution, and the inhibition rate can reach 80%. The erythromycin artificial antigen, the chloramphenicol artificial antigen and the bisphenol A artificial antigen are respectively taken for coating according to 1ug/mL, the monoclonal antibody of 1mg/mL is diluted by 1:1000, the specificity of the monoclonal antibody is verified, the result is shown in Table 2, and the monoclonal antibody has no cross reaction with the erythromycin artificial antigen, the chloramphenicol artificial antigen and the bisphenol A artificial antigen, thus showing good specificity.

TABLE 1 monoclonal antibody sensitivity detection

Table 2 monoclonal antibody specific detection

EXAMPLE 3 PCR amplification and sequence identification of the variable region Gene of monoclonal antibody to patulin

The embodiment provides the monoclonal antibody variable region gene PCR amplification and sequence identification of the patulin, which comprises the following specific steps:

1. The hybridoma cells secreting the estriol monoclonal antibody are cultured by using RPMI 1640 complete culture medium under the conditions of 37 ℃ and 5% carbon dioxide, and total RNA in the cells is extracted by using a total RNA extraction kit (purchased from the root of the heaven) when the number of the cells reaches 1X 10 ⁷.

2. The first strand of cDNA was synthesized using the reverse transcription kit (purchased from Tiangen) using the total RNA extracted in step 1 as an amplification template.

3. The downstream primer and upstream universal primer of Lambda strand, kappa strand, heavy strand were designed.

Primer F (SEQ ID No. 11): AAGCAGTGGTATCAACGCAGAG

Rκ(SEQ ID No.12): ACATTGATGTCTTTGGGGTAGAAG

Rλ(SEQ ID No.13): ATCGTACACACCAGTGTGGC

RH(SEQ ID No.14): GGGATCCAGAGTTCCAGGTC

PCR was performed using the first strand of cDNA as a template, and 50. Mu.l of the reaction system was used. 3. Mu.l of template, 2.5. Mu.l of primer (10. Mu.M), 2X Pfu PCR MasterMix. Mu.l, 25. Mu.l, ddH ₂ O17. Mu.l.

The conditions for the drop PCR reaction were 95℃for 5min, 95℃for 30s,63.5℃to 57.5℃for 30s, each drop of 0.5℃until 58℃for 12 cycles, 72℃for 1min, 95℃for 30s,56℃for 30s,72℃for 1min, and 24 cycles, 72℃for 7min to terminate the procedure.

4. The PCR products were subjected to 2% agarose gel electrophoresis, the antibody Kappa chain, lambda chain and Heavy chain amplified fragments were recovered using a PCR product recovery kit (Beijing-day root), the fragments were inserted into pLB vector using a pLB zero background rapid cloning kit (Beijing-day root), transformed into DH 5. Alpha. Competent cells (ampicillin resistance), and screened for sequencing of recombinant positive clones.

5. The variable region gene sequence and amino acid sequence of the monoclonal antibody of patulin of this example are as follows:

(1) The heavy chain variable region gene sequence (SEQ ID No. 3) of the monoclonal antibody of patulin:

ATGAAATGCAGCTGGGTTATCTTCTTCCTGATGGCAGTGGTTACAGGGGTCAATTCAGAGGTTCAACTGCAGCAGTCTGGGGCAGACCTTGTGAAGCCAGGGGCCTCAGTCAAGATGTCCTGCACAGCTTCTGGCTTCAACATTACAGACACCTATATACATTGGGTGAATCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATAGTAAATATGACCCGATGTTCCAGGGCAAGGCCACTATAACAGTAGACACAGCCTACAACACAGTCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGATATGGTAACGGGGGTGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

(2) The light chain variable region gene sequence (SEQ ID No. 4) of the monoclonal antibody to patulin:

ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGATTCCAGGTTCCACAGGTGCCATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCTATAGTTTTATACACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATTGATCCTGTGGAGCCTGGTGATGCTGCGACCTATTACTGTCAGCAAAATAATGAGGATCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA.

(3) The heavy chain variable region amino acid sequence of the monoclonal antibody of patulin (SEQ ID No. 1):

MKCSWVIFFLMAVVTGVNSEVQLQQSGADLVKPGASVKMSCTASGFNITDTYIHWVNQRPEQGLEWIGRIDPANGNSKYDPMFQGKATITVDTAYNTVYLQLSSLTSEDTAVYYCARYGNGGVMDYWGQGTSVTVSS.

(4) The light chain variable region amino acid sequence of the monoclonal antibody of patulin (SEQ ID No. 2):

METDTLLLWVLLLWIPGSTGAIVLTQSPASLAVSLGQRATISCRASESVDSYGYSFIHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTIDPVEPGDAATYYCQQNNEDPRTFGGGTKLEIK.

6. variable region sequence and homology analysis

The analysis in NCBI database shows that the homology of the patulin monoclonal antibody heavy chain variable region gene and the mouse immunoglobulin heavy chain variable region (Sequence ID: M13328.1) is highest, the homology percentage is 389/417 and 93%, see figure 1, and the homology of patulin monoclonal antibody heavy chain variable region amino acid and the mouse immunoglobulin heavy chain variable region (Sequence ID: AAA 38411.1) is highest, the homology percentage is 118/139 and 85%, see figure 2. The genes of the light chain variable region of the patulin monoclonal antibody have the highest homology with the mouse clone light chain variable region (Sequence ID: OQ 208881.1) with the homology percentage of 382/393 being 97 percent, see figure 3, and the amino acids of the light chain variable region of the patulin monoclonal antibody have the highest homology with the mouse immunoglobulin light chain variable region (Sequence ID: WVL 05780.1) with the homology percentage of 118/131 being 90 percent, see figure 4.

7. CDR analysis

The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are analyzed at https:// www.novopro.cn/tools/CDR.

Antibody heavy chain CDR regions:

CDR-H1(SEQ ID No.5):DTYIH

CDR-H2(SEQ ID No.6):RIDPANGNSKYDPMFQG

CDR-H3(SEQ ID No.7):YGNGGVMDY

antibody light chain CDR regions:

CDR-L1(SEQ ID No.8):RASESVDSYGYSFIH

CDR-L2(SEQ ID No.9):LASNLES

CDR-L3(SEQ ID No.10):QQNNEDPRT

Example 4 preparation of patulin test strip

The colloidal gold test strip is composed of water absorption paper, a nitrocellulose membrane (NC membrane), a sample pad and a bottom plate, wherein a schematic diagram of the colloidal gold test strip is shown in figure 5, two parallel strips are marked on the nitrocellulose membrane (NC membrane) by a film marking instrument, the spacing of the strips is 6.5mm, and the width of the strips is about 1mm. The first one is a quality control line and is close to the water absorption paper end, and the second one is a detection line and is close to the sample pad end. And spraying goat anti-mouse (IgG) and one-ten-thousandth blue pigment on the quality control line, wherein the concentration is 0.5-1 mg/ml, and the concentration of PAT-MOVA antigen sprayed on the second strip is 0.1-0.5 mg/ml. And (3) obtaining a chromatographic membrane, drying at 40 ℃ for 16 hours, sealing, and preserving at normal temperature. Sequentially adhering water absorbing paper, NC film and sample pad on the bottom plate, wherein the initial end of the sample pad is connected with the tail end of the NC film, the initial end of the NC film is connected with the water absorbing pad, the tail end of the sample pad is aligned with the tail end of the bottom plate, and the initial end of the water absorbing pad is aligned with the initial end of the bottom plate. The glued plastic plate was cut longitudinally into test strips 4.5mm wide by a slitter. The microporous reagent is provided with a microporous plug, and the patulin monoclonal antibody-colloidal gold marker is freeze-dried in the microporous reagent. 8 cut test strips and 1 microporous reagent are taken and put into a reagent bucket for sealing, and the test strips and 1 microporous reagent are preserved at 2-8 ℃.

Example 5 application of test strip for detecting patulin colloidal gold

1. Test strip detection

And the required test strip and sample diluent are restored to room temperature (20-25 ℃). 200 mu L of sample solution to be detected is sucked into the microwells by a micropipette, and the sample solution is slowly sucked and fully mixed with the reagents in the microwells. The marked test strip is inserted into the micropore, fully immersed into the solution, incubated for 5min at room temperature, taken out and judged to be invalid according to the schematic diagram (figure 6) and the other time.

And judging a detection result:

negative (-) the color of both the C line and the T line is stronger than that of the C line, indicating that the concentration of the patulin in the sample is lower than the detection limit.

Positive (+). Color development of C line, color development of T line is the same as that of C line, color development of T line is weaker than that of C line or color development of T line is not developed, and the concentration of patulin in the sample is equal to or higher than the detection limit.

And (3) invalidating, wherein no C line appears, which indicates that the incorrect operation process or the test strip is degenerated and fails. In this case, the instructions should be read again carefully and retested with a new test strip.

If the test paper strip needs to be archived, the lower-end sponge cushion needs to be cut off immediately after interpretation, and the test paper strip is dried and archived.

And NBReader can be selected for result interpretation besides naked eye interpretation.

2. Sensitivity detection of patulin colloidal gold detection test strip

The concentration of the patulin standard substance is diluted to be 1ppb and 2ppb, the milk sample is subjected to standard addition detection according to a test strip detection method, the lowest detection limit of the product is verified, and the result is shown in Table 3. The test strip provided by the invention has the detection sensitivity of 2ppb for the patulin standard in milk.

TABLE 3 sensitivity of test strips for detecting patulin colloidal gold

3. Specific detection of patulin colloidal gold detection test strip

The detection of 200ppb of erythromycin, chloramphenicol, bisphenol A was performed according to the detection method, and the results are shown in Table 4. The results are negative, which indicates that the test strip provided by the invention does not have cross reaction with other antibiotic medicines, and has good specificity.

TABLE 4 specificity of test strips for detecting patulin colloidal gold

4. Stability detection of patulin colloidal gold detection test strip

The prepared test strips were placed in an environment of 4 ℃ and 37 ℃ for acceleration test, and used for detecting patulin standard (2 ppb) at 0 d, 7 d, 14 d and 28 d respectively, and error analysis was carried out on the measured concentration and the actual sample concentration values, and the results are shown in table 5.CV is less than 10%, which indicates that the test strip provided by the invention has good stability.

TABLE 5 stability of test strips for detecting patulin colloidal gold

While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. A monoclonal antibody to patulin, characterized in that: the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1;

The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 2;

The heavy chain variable region and the light chain variable region are both composed of complementarity determining regions and framework regions, and the complementarity determining regions are both composed of CDR1, CDR2 and CDR3;

The amino acid sequence of CDR1 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 5;

The amino acid sequence of CDR2 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No.6;

The amino acid sequence of CDR3 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No.7;

The amino acid sequence of CDR1 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.8;

The amino acid sequence of CDR2 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.9;

The amino acid sequence of CDR3 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.10.

2. The patulin monoclonal antibody according to claim 1, characterized in that:

The gene sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 3;

The gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No.4.

3. Use of the patulin monoclonal antibody according to claim 1 in the preparation of patulin detection products.

4. A patulin test strip, characterized in that the test strip is a colloidal gold test strip, and the microporous reagent of the colloidal gold test strip is a monoclonal antibody of patulin according to any one of claims 1 to 2 labeled with colloidal gold.