A monoclonal antibody against patulin and its application

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

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a patulin monoclonal antibody and application thereof. Background Art

[0002] Patulin (PAT) is a secondary metabolite produced by fungi (Penicillium expansum, Penicillium patulum, Penicillium clavuligerus, etc.). The chemical name of patulin is 4-hydroxy-4H-furo[3,2c]pyran-2[6H]-one, with a molecular formula of C7H6O4. It is easily soluble in water, ethanol, acetone, chloroform and ethyl acetate, slightly soluble in benzene and ether, insoluble in petroleum ether, and relatively stable under acidic conditions. Glister (1941) first discovered and isolated and purified it at Oxford University. Experiments showed that patulin is a broad-spectrum antibiotic that can be used to treat human colds, but subsequent studies have confirmed that it is highly toxic to experimental animals. Since then, the focus of research on patulin has shifted to its toxicity and contamination of food and feed. Toxicological studies on animal experiments or cell tests have shown that patulin has multiple hazards such as acute toxicity, subacute toxicity, reproductive toxicity, and immunotoxicity. Patulin is classified as a Class III suspected carcinogen by the International Agency for Research on Cancer (IARC). Patulin has a detrimental effect on respiration. Patulin ingested into the body changes the permeability of the cell membrane, causing abnormal movement of membrane substances, thereby indirectly causing physiological respiratory abnormalities. Patulin mainly contaminates apples, hawthorns and their products. my country's "National Food Safety Standard Limits of Mycotoxins in Food" (GB 2761-2017) stipulates that the limit of patulin in apple and hawthorn products is 50μg / kg.

[0003] At present, the detection methods of patulin mainly include thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS) and high performance liquid chromatography-mass spectrometry (HPLC-MS). The thin layer chromatography method is not specific, and other fluorescent substances in the sample are easy to interfere with the result determination. The liquid chromatography method is more specific, but the required instruments and equipment are expensive, and the detection activities are subject to certain restrictions. Enzyme-linked immunosorbent assay is one of the more commonly used methods for determining patulin, with strong specificity, high sensitivity, and fast analysis speed. It is more commonly used in actual detection activities. In addition to strong specificity, colloidal gold and fluorescent immunochromatography have high sensitivity, low cost, simple operation, and wide detection scenarios. They are also detection methods with more practical applications. The purpose of the present invention is to provide a monoclonal antibody with high affinity and detection sensitivity for patulin, and the variable regions of the light chain and heavy chain of the monoclonal antibody. Lay the foundation for the research and development and promotion of ELISA, colloidal gold test strips and fluorescent immunoassay strips. Summary of the invention

[0004] To this end, the present invention provides a patulin monoclonal antibody and application thereof.

[0005] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a patulin monoclonal antibody, characterized in that: the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0007] The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1;

[0008] The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 2;

[0009] 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;

[0010] The amino acid sequence of CDR1 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 5;

[0011] The amino acid sequence of CDR2 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No.6;

[0012] The amino acid sequence of CDR3 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No.7;

[0013] The amino acid sequence of CDR1 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.8;

[0014] The amino acid sequence of CDR2 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.9;

[0015] The amino acid sequence of CDR3 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.10.

[0016] In a second aspect, the above-mentioned patulin monoclonal antibody is characterized by:

[0017] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 3;

[0018] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No.4.

[0019] In a third aspect, the present invention provides a use of a monoclonal antibody against patulin in preparing a test strip for detecting patulin.

[0020] Preferably, the test strip is a colloidal gold test strip, or a fluorescent microsphere test strip, or a latex microsphere test strip.

[0021] The present invention has the following advantages:

[0022] The monoclonal antibody of the present invention has the characteristics of strong specificity and high sensitivity, and can be used as a raw material for enzyme-linked immunosorbent assay and colloidal gold immunoassay, laying a foundation for the research, development and promotion of ELISA, colloidal gold test strips and fluorescent immunoassay strips. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0024] Figure 1 This is the result of homology comparison of the heavy chain gene sequence of patulin monoclonal antibody;

[0025] Figure 2 This is the result of homology comparison of amino acid sequence of heavy chain of patulin monoclonal antibody;

[0026] Figure 3 This is the result of homology comparison of the gene sequence of the light chain of the patulin monoclonal antibody;

[0027] Figure 4 This is the result of homology comparison of amino acid sequence of light chain of patulin monoclonal antibody;

[0028] Figure 5 It is a schematic diagram of colloidal gold test strip;

[0029] Figure 6 This is a schematic diagram of the interpretation of colloidal gold test strip results. DETAILED DESCRIPTION

[0030] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1 Synthesis of Patulin Artificial Antigen

[0032] 1. Synthesis route of patulin-hemiglutaric acid (PAT-HG)

[0033] Patulin, glutaric anhydride and DMAP were dissolved in anhydrous tetrahydrofuran (molar ratio of 1:20:4), shaken at 37℃ for 2 h (TLC monitoring reaction), and distilled water was added to terminate the reaction. The reaction product was spotted onto a preparative thin layer chromatography silica gel plate, developed in a developing cylinder (V (n-hexane): V (ethyl acetate): V (ethanol) = 5:3:1), and the target product patulin hemiglutaric acid (PAT-HG) band was scraped off under 256 nm ultraviolet light, and the scraped silica gel was placed in a centrifuge tube and eluted with methanol three times, and the methanol was vacuum-dried to dry, and the residue was purified PAT-HG.

[0034] 2. Preparation of patulin immune antigen

[0035] The coupling of PAT-HG and carrier protein was carried out by active ester method. PAT-HG, NHS and DCC were dissolved in anhydrous tetrahydrofuran (molar ratio of 1:2:4) and reacted at 15℃ for 6 h (TLC monitoring reaction); the precipitate was removed by centrifugation, the tetrahydrofuran was vacuum-evacuated, and the residue was dissolved in DMSO; 5% bovine serum albumin (BSA) solution dissolved in 0.13 mol / L NaHCO3 was slowly dripped, and the reaction was slowly shaken overnight at 15℃ in the dark; it was dialyzed with PBS buffer (0.1 mol / L, pH 7.4) at 4℃ for 3 days, and freeze-dried in vacuum to obtain patulin immune antigen (PAT-HG-BSA).

[0036] 3. Preparation of patulin detection antigen

[0037] The coupling of PAT-HG and carrier protein was carried out by active ester method. PAT-HG, NHS and DCC were dissolved in anhydrous tetrahydrofuran (molar ratio of 1:2:4) and reacted at 15℃ for 6 h (TLC monitoring reaction); the precipitate was removed by centrifugation, the tetrahydrofuran was vacuum-dried, and the residue was dissolved in DMSO; 5% chicken ovalbumin (OVA) solution dissolved in 0.13 mol / L NaHCO3 was slowly dripped, and the reaction was slowly shaken overnight at 15℃ in the dark; it was dialyzed with PBS buffer (0.1 mol / L, pH 7.4) at 4℃ for 3 days, and freeze-dried in vacuum to obtain patulin detection antigen (PAT-HG-OVA).

[0038] Example 2 Preparation of patulin monoclonal antibody

[0039] 1. Mouse immunization

[0040] The prepared immunogen patulin-BSA was used to immunize mice at a dose of 10 µg per mouse, and the immunogen was diluted to the required concentration with sterile PBS. Equal amounts of adjuvant and immunogen were drawn into two 2.5 mL disposable syringes, which were then inserted into the connecting vessel and pushed 8500 times with an emulsifier. The immunization procedure used one basic immunization and several booster immunizations.

[0041] Select 3 healthy Balb / c female mice aged 6-8 weeks and weighing 18-20 g. For the first immunization, use an equal amount of Freund's complete adjuvant emulsified antigen to subcutaneously inject mice at multiple points. Every 14 days (21 days between the first and second immunizations), use Freund's incomplete adjuvant emulsified antigen for booster immunization. After three immunizations, blood is collected from the tail vein on the 7th to 10th day, diluted 100 times with 1×PBS, and the supernatant is centrifuged to measure the titer and inhibition of mouse serum. When the titer reaches 10W, it can be fused to prepare monoclonal antibodies. Consider the effect of time interval on antibody production. 3 days before fusion, select mice with high serum titers and inject pure antigen diluted with 1×PBS intraperitoneally for booster immunization.

[0042] 2. Hybridoma cell preparation

[0043] Hybridoma cell preparation: SP2 / 0 myeloma cells were adjusted to a cell density of 1×10 6 / mL. Three days after the impact immunization of mice, the feeding spleen cells were taken and the feeding spleen cells were adjusted to 1×10 5 / mL, PEG was added to fuse the two cells, and then the hybridoma cell positive wells were screened. The positive wells were cloned by limiting dilution method to obtain and establish a hybridoma cell line that stably secretes patulin monoclonal antibody.

[0044] 3. Ascites Preparation

[0045] Monoclonal antibodies were prepared by in vivo induction. Each mouse was injected with 0.5 ml of Freund's incomplete adjuvant to purify the peritoneal cavity. After 7 days, the number of hybridoma cells in the logarithmic growth phase was adjusted to 1×10 6 Each mouse was intraperitoneally inoculated with 0.5 ml of the solution. The mice were observed for ascites every 7 days. When the abdomen of the mice was obviously swollen, the spirit became worse, and the mice were immobile on the verge of death, the ascites was collected and centrifuged at 12000 r / min for 5 min to remove the surface fat and carefully aspirate the supernatant for ammonium sulfate precipitation.

[0046] 4. Antibody Purification

[0047] Record the name and volume of ascites. Add 3mL pH4.0, 0.06M sodium acetate buffer, mix for 5min, add 10ul caprylic acid, stir at 4℃ for 15min. Filter once through cotton wool with a syringe. Centrifuge at 4℃, 12000rpm for 15min. Take the supernatant, add an equal volume of saturated ammonium sulfate to a final concentration of 50%, stir while adding, and let stand at 4℃ for 4h. Then centrifuge at 4℃, 12000rpm for 15min, discard the supernatant, and add 1.8ml 0.01M PBS (pH7.2) to the centrifuge tube until the precipitate is completely dissolved. Determine the total volume with a gun, add 1 / 2 volume of saturated ammonium sulfate to a concentration of 33%, stir while adding, and leave at 4℃ overnight. Centrifuge at 12000rpm for 15min, discard the supernatant, and add 0.45ml 0.01M PBS to dissolve the precipitate. Treat the dialysis bag (boil for 5min, wash with pure water to remove leaks). Add the dissolved solution into a dialysis bag and dialyze overnight in 0.01M PBS. Change the dialysis solution, usually 2-3 times, collect the antibody, measure the concentration and package it.

[0048] 5. Characterization of monoclonal antibodies against patulin

[0049] The sensitivity and specificity of patulin monoclonal antibody were detected by indirect ELISA method. Patulin-OVA artificial antigen was coated at 1ug / mL, and 1mg / mL monoclonal antibody was diluted in gradient to verify the sensitivity of monoclonal antibody. The results are shown in Table 1. The sensitivity of monoclonal antibody can reach 1:20000 dilution, and the inhibition rate can reach 80%. Erythromycin artificial antigen, chloramphenicol artificial antigen and bisphenol A artificial antigen were coated at 1ug / mL, and 1mg / mL monoclonal antibody was diluted 1:1000 to verify the specificity of monoclonal antibody. The results are shown in Table 2. It has no cross reaction with erythromycin artificial antigen, chloramphenicol artificial antigen and bisphenol A artificial antigen, indicating good specificity.

[0050] Table 1 Monoclonal antibody sensitivity test

[0051]

[0052] Table 2 Monoclonal antibody specificity detection

[0053]

[0054] Example 3 PCR Amplification and Sequence Identification of the Variable Region Gene of the Monoclonal Antibody to Patulin

[0055] This example provides PCR amplification and sequence identification of the variable region gene of the monoclonal antibody of patulin, and the specific steps are as follows:

[0056] 1. Use RPMI 1640 complete medium to culture hybridoma cells secreting estriol monoclonal antibodies at 37°C and 5% carbon dioxide until the cell number reaches 1×10 7 Total RNA was extracted from cells using a total RNA extraction kit (purchased from Tiangen).

[0057] 2. Use a reverse transcription kit (purchased from Tiangen) to synthesize the first-chain cDNA using the total RNA extracted in step 1 as an amplification template.

[0058] 3. Design downstream primers and upstream universal primers for Lambda chain, Kappa chain, Heavy chain.

[0059] Primer: F (SEQ ID No. 11): AAGCAGTGGTATCAACGCAGAG

[0060] Rκ (SEQ ID No. 12): ACATTGATGTCTTTGGGGTAGAAG

[0061] Rλ (SEQ ID No.13): ATCGTACACACCAGTGTGGC

[0062] RH (SEQ ID No.14): GGGATCCAGAGTTCCAGGTC

[0063] PCR amplification was performed using the first strand of cDNA as a template, with a reaction system of 50 μl, including 3 μl of template, 2.5 μl of primer (10 μM), 25 μl of 2×Pfu PCR MasterMix, and 17 μl of ddH2O.

[0064] The touchdown PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 63.5°C-57.5°C for 30 s, decreasing by 0.5°C each time until 58°C, for 12 cycles; 72°C for 1 min; 95°C for 30 s, 56°C for 30 s, 72°C for 1 min, for 24 cycles; and 72°C for 7 min to end the program.

[0065] 4. The PCR products were subjected to 2% agarose gel electrophoresis, and the antibody Kappa chain, Lambda chain and Heavy chain amplified fragments were recovered using a PCR product recovery kit (Beijing Tiangen). The fragments were inserted into the pLB vector using a pLB zero background rapid cloning kit (Beijing Tiangen), transformed into DH5α competent cells (ampicillin resistance), and the recombinant positive clones were screened for sequencing.

[0066] 5. The variable region gene sequence and amino acid sequence of the monoclonal antibody to patulin in this example are as follows:

[0067] (1)Monoclonal antibody heavy chain variable region gene sequence of patulin (SEQ ID No.3):

[0068] ATGAAATGCAGCTGGGTTATCTTCTTCCTGATGGCAGTGGTTACAGGGGTCAATTCAGAGGTTCAACTGCAGCAGTCTGGGGCAGACCTTGTGAAGCCAGGGGCCTCAGTCAAGATGTCCTGCACAGCTTCTGGCTTCAACATTACAGACACCTATATACATTGGGTGAATCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATAGTAAATATGACCCGATGTTCCAGGGCAAGGCCACTATAACAGTAGACACAGCCTACAACACAGTCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGATATGGTAACGGGGGTGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA。

[0069] (2)Monoclonal antibody light chain variable region gene sequence of patulin (SEQ ID No.4):

[0070] ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGATTCCAGGTTCCACAGGTGCCATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCTATAGTTTTATACACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATTGATCCTGTGGAGCCTGGTGATGCTGCGACCTATTACTGTCAGCAAAATAATGAGGATCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA。

[0071] (3) Amino acid sequence of the heavy chain variable region of the monoclonal antibody against patulin (SEQ ID No. 1):

[0072] MKCSWVIFFLMAVVTGVNSEVQLQQSGADLVKPGASVKMSCTASGFNITDTYIHWVNQRPEQGLEWIGRIDPANGNSKYDPMFQGKATITVDTAYNTVYLQLSSLTSEDTAVYYCARYGNGGVMDYWGQGTSVTVSS.

[0073] (4) Amino acid sequence of the light chain variable region of the monoclonal antibody against patulin (SEQ ID No. 2):

[0074] METDTLLLWVLLLWIPGSTGAIVLTQSPASLAVSLGQRATISCRASESVDSYGYSFIHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTIDPVEPGDAATYYCQQNNEDPRTFGGGTKLEIK.

[0075] 6. Variable region sequence and homology analysis

[0076] Comparison analysis was performed in the NCBI database. The results showed that the patulin monoclonal antibody heavy chain variable region gene had the highest homology with the mouse immunoglobulin heavy chain variable region (Sequence ID: M13328.1), with a homology of 389 / 417 and a homology percentage of 93%. See below Figure 1 The amino acid sequence of the heavy chain variable region of the patulin monoclonal antibody has the highest homology with the mouse immunoglobulin heavy chain variable region (Sequence ID: AAA38411.1), with a homology of 118 / 139 and a homology percentage of 85%, see below Figure 2 The patulin monoclonal antibody light chain variable region gene has the highest homology with the mouse clone light chain variable region (SequenceID: OQ208881.1), with a homology of 382 / 393 and a homology percentage of 97%, see below Figure 3 The amino acid sequence of the light chain variable region of the patulin monoclonal antibody has the highest homology with the light chain variable region of the mouse immunoglobulin (Sequence ID: WVL05780.1), with a homology of 118 / 131 and a homology percentage of 90%, see below Figure 4 .

[0077] 7. CDR region analysis

[0078] The amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody were analyzed at https: / / www.novopro.cn / tools / cdr.html to obtain their CDR regions.

[0079] Antibody heavy chain CDR region:

[0080] CDR-H1 (SEQ ID No.5): DTYIH

[0081] CDR-H2 (SEQ ID No.6): RIDPANGNSKYDPMFQG

[0082] CDR-H3 (SEQ ID No.7): YGNGGVMDY

[0083] Antibody light chain CDR region:

[0084] CDR-L1 (SEQ ID No.8):RASESVDSYGYSFIH

[0085] CDR-L2 (SEQ ID No.9): LASNLES

[0086] CDR-L3 (SEQ ID No.10): QQNNEDPRT

[0087] Example 4. Preparation of Patulin Test Strips

[0088] The test strip is composed of absorbent paper, nitrocellulose membrane (NC membrane), sample pad, and bottom plate. The schematic diagram of the colloidal gold test strip is as follows Figure 5 As shown; two parallel strips are drawn on the nitrocellulose membrane (NC membrane) with a film drawer, the strip interval is 6.5mm, and the strip width is about 1mm. The first one is the quality control line, close to the end of the absorbent paper, and the second one is the test line, close to the end of the sample pad. The quality control line is sprayed with sheep anti-mouse (IgG) and one ten-thousandth of blue pigment, with a concentration of 0.5~1mg / ml; the second strip is sprayed with PAT-MOVA antigen with a concentration of 0.1~0.5mg / ml. The chromatographic membrane is obtained, dried at 40℃ for 16h, sealed, and stored at room temperature. The absorbent paper, NC membrane and sample pad are pasted on the bottom plate in sequence, the starting end of the sample pad is connected to the end of the NC membrane, the starting end of the NC membrane is connected to the absorbent pad, the end of the sample pad is aligned with the end of the bottom plate, and the starting end of the absorbent pad is aligned with the starting end of the bottom plate. Use a strip cutter to cut the pasted plastic plate into 4.5mm wide test strips longitudinally. The microporous reagent has a microporous plug, and the microporous reagent contains patulin monoclonal antibody-colloidal gold markers in a freeze-dried state. Take 8 cut test strips and 1 microporous reagent and put them into a reagent barrel, seal it, and store it at 2-8°C.

[0089] Example 5: Application of Patulin Colloidal Gold Test Strips

[0090] 1. Test strip detection

[0091] The required test strips and sample diluents are returned to room temperature (20-25°C). Use a micropipette to draw 200µL of the sample solution to be tested into the microwell, slowly aspirate and fully mix with the reagent in the microwell. Insert the marked test strip into the microwell so that it is fully immersed in the solution. After incubating at room temperature for 5 minutes, remove the test strip and follow the schematic diagram ( Figure 6 ) judgment result, and the judgment at other times is invalid.

[0092] Test result interpretation:

[0093] Negative (-): Both the C line and the T line are colored and the color of the T line is stronger than that of the C line, indicating that the concentration of patulin in the sample is lower than the detection limit.

[0094] Positive (+): The C line is colored, and the T line is the same color as the C line, the T line is weaker than the C line, or the T line is not colored, indicating that the concentration of patulin in the sample is equal to or higher than the detection limit.

[0095] Invalid: No C line appears, indicating incorrect operation or the test strip has deteriorated and become invalid. In this case, you should read the instructions carefully again and retest with a new test strip.

[0096] If the test strip needs to be archived, the sponge pad at the bottom must be cut off immediately after reading, and the strip must be dried and archived.

[0097] In addition to naked eye interpretation, NBReader can be used for result interpretation.

[0098] 2. Sensitivity test of patulin colloidal gold test strips

[0099] The concentration of patulin standard was diluted to 1 ppb and 2 ppb, and the milk samples were spiked and tested according to the test strip detection method to verify the minimum detection limit of the product. The results are shown in Table 3. The test strip provided by the present invention has a detection sensitivity of 2 ppb for patulin standard in milk.

[0100] Table 3 Sensitivity of patulin colloidal gold test strips

[0101]

[0102] 3. Specificity detection of patulin colloidal gold test strips

[0103] According to the detection method, 200 ppb of erythromycin, chloramphenicol and bisphenol A were detected, and the results are shown in Table 4. The results were all negative, indicating that the test strip provided by the present invention did not cross-react with other antibiotic drugs and had good specificity.

[0104] Table 4 Specificity of patulin colloidal gold test strips

[0105]

[0106] 4. Stability test of patulin colloidal gold test strips

[0107] The prepared test strips were placed in 4°C and 37°C environments for accelerated testing, and the patulin standard (2 ppb) was detected at 0 d, 7 d, 14 d, and 28 d, respectively. The error analysis of the measured concentration and the actual sample concentration was performed, and the results are shown in Table 5. The CVs were all <10%, indicating that the test strips provided by the present invention have good stability.

[0108] Table 5 Stability of patulin colloidal gold test strips

[0109]

[0110] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

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.

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