Rapid alliin detection method based on immunochromatography

Abstract

The invention provides an immunochromatography tablet for rapidly detecting alliin and a detection method, and more specifically, the method is based on an immunochromatography technology, and alliin is detected through specific binding of an alliin antibody and small molecular alliin on the immunochromatography tablet; according to the method, the content of alliin can be accurately measured, a detection result can be obtained within about 15 minutes, and the method has the advantages of high specificity, strong anti-interference capability, high sensitivity, good precision, high detection speed and simplicity in operation.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunochromatography, and more specifically, relates to a rapid detection method of alliin based on immunochromatography. Background Art

[0002] Alliin (S-allyl-L-cysteine ​​sulfoxide, alliin) is the signature sulfur-containing amino acid of garlic, accounting for 90% of the total sulfur-containing compounds in undamaged garlic. Domestic and foreign studies have shown that alliin and its enzymatic hydrolysis product (allicin) have certain functions in preventing cardiovascular and cerebrovascular diseases and improving immunity.

[0003] At present, the main detection methods for alliin at home and abroad are HPLC and HPLC / UPLC-MS, but the HPLC method requires a complex sample pretreatment process and a long analysis time, usually more than 1 hour.

[0004] In addition, although the HPLC / UPLC-MS method can shorten the analysis time, it still takes about 0.5 hours or more. HPLC / UPLC-MS also has the disadvantages of complex operation, expensive instruments, high detection costs, and high technical requirements for operators.

[0005] Therefore, there is an urgent need in the art to develop an alliin detection method that is simple to operate, cost-effective, highly specific, and has a short detection time. Summary of the invention

[0006] The object of the present invention is to provide a method for quickly detecting alliin.

[0007] In a first aspect of the present invention, an immunochromatographic sheet for detecting alliin is provided, comprising a base plate and a sample pad, an NC membrane and absorbent paper located on the base plate, wherein a detection line T line and a quality control line C line are provided on the NC membrane;

[0008] Wherein, the T line is covalently linked to alliin-carrier protein, and the structural formula is:

[0009]

[0010] Wherein, X is a carrier protein.

[0011] In another preferred embodiment, the carrier protein is hemocyanin (KLH) or bovine serum albumin (BSA).

[0012] In another preferred embodiment, the C line is goat anti-rabbit IgG antibody or rabbit anti-mouse IgG antibody or goat anti-mouse IgG antibody.

[0013] In another preferred embodiment, the C line is 0.1-1 mg / mL goat anti-rabbit IgG, and the T line is 0.1-1 mg / mL BSA-alliin.

[0014] In another preferred embodiment, the NC film is a fluorescent film.

[0015] The second aspect of the present invention provides an immune probe for detecting alliin, wherein the immune probe is a colloidal gold enhancer-alliin antibody.

[0016] In another preferred embodiment, the alliin antibody comprises a heavy chain variable region and a light chain variable region, wherein the complementarity determining region CDR (HCDR) of the heavy chain variable region and the complementarity determining region CDR (LCDR) of the light chain variable region are:

[0017] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 3,

[0018] The amino acid sequence of HCDR2 is shown in SEQ ID NO:4,

[0019] The amino acid sequence of HCDR3 is shown in SEQ ID NO:5,

[0020] LCDR1 having an amino acid sequence as shown in SEQ ID NO: 10,

[0021] LCDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and

[0022] The amino acid sequence of LCDR3 is shown in SEQ ID NO:12.

[0023] In another preferred embodiment, the heavy chain variable region and light chain variable region of the antibody are:

[0024] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0025] In another preferred example, the heavy chain variable region includes the three complementary determining regions HCDR and a framework region of the heavy chain variable region used to connect the heavy chain variable region HCDR; the light chain variable region includes the three light chain complementary determining regions LCDR and a framework region of the light chain variable region used to connect the light chain complementary determining regions LCDR.

[0026] In another preferred embodiment, the alliin antibody is selected from the following group: Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), double scFv, (scFv)2.

[0027] In another preferred example, the light chain of the antibody further includes a light chain constant region.

[0028] In another preferred embodiment, the light chain constant region is of rabbit origin.

[0029] In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

[0030] In another preferred embodiment, the heavy chain constant region is of rabbit origin.

[0031] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0032] In another preferred embodiment, the antibody is a monoclonal antibody.

[0033] In another preferred embodiment, the particle size of the colloidal gold enhancer is 10-50 nm, preferably 30-50 nm.

[0034] In another preferred embodiment, the particle size of the colloidal gold enhancer is 30 nm.

[0035] In another preferred embodiment, in the immune probe, the mass ratio of colloidal gold enhancer to alliin antibody is 1:5-1:10.

[0036] The third aspect of the present invention provides a method for detecting alliin, comprising the steps of:

[0037] (a) providing the immunochromatographic sheet according to the first aspect of the present invention;

[0038] (b) mixing the sample to be tested with the immunoprobe described in the second aspect of the present invention, and loading the mixture onto the sample loading area of ​​the immunochromatographic sheet; and

[0039] (c) observing the T line and the C line of the immunochromatographic sheet to obtain a detection result of whether the sample contains alliin.

[0040] In another preferred example, the detection result includes a qualitative result, a quantitative result, or a combination thereof.

[0041] In another preferred embodiment, the observation includes: observation with the naked eye and observation with instruments.

[0042] In another preferred embodiment, the instrument observation includes: a fluorescent immunoassay analyzer, such as a dry fluorescent immunoassay analyzer.

[0043] In another preferred embodiment, the method is a qualitative or quantitative method.

[0044] In another preferred embodiment, the detection time of the method is 5-30 minutes.

[0045] In another preferred embodiment, the dilution solution in the method is ultrapure water or PBS solution.

[0046] In another preferred embodiment, the samples include: garlic, garlic-related medicines, health foods and health products (disinfection products, foods), cells, and tissue samples.

[0047] A fourth aspect of the present invention provides a kit for rapid detection of alliin, comprising:

[0048] (a) a container and the immunochromatographic sheet as described in the first aspect of the present invention contained in the container; and

[0049] (b) a container and the immunoprobe according to the second aspect of the present invention contained in the container; or

[0050] (c) A container and the container contains the immunochromatographic sheet as described in the first aspect of the present invention, wherein the spotting area of ​​the immunochromatographic sheet contains the immunoprobe as described in the second aspect of the present invention.

[0051] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The detection principle of the background fluorescence quenching-competitive chromatography method in the present invention is shown.

[0053] Figure 2 A schematic diagram showing the immunochromatographic sheet of the present invention is shown.

[0054] Figure 3 A standard curve of alliin with a series of concentrations in one embodiment of the present invention is shown. DETAILED DESCRIPTION

[0055] After extensive and in-depth research, the inventors have invented a rapid detection method for alliin based on immunochromatography. Since alliin has a small molecular weight and is a hapten substance with no immunogenicity, the present invention couples alliin with a carrier protein to obtain a complete alliin antigen; and immunizes animals with the complete antigen to obtain alliin-specific antibodies. The present invention performs alliin detection on a chromatographic sheet based on antigen-antibody reaction, which has high specificity. Compared with chromatography, the detection method of the present invention is simple, the detection equipment is light and easy to carry, and on-site detection can be achieved. The test of the present invention takes less time (about 15 minutes or less) and has high sensitivity. The present invention was completed on this basis.

[0056] the term

[0057] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0058] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value.

[0059] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0060] Immunoprobe

[0061] A probe that is formed by coupling colloidal gold enhancer (CGE) with an antibody and can specifically bind to the corresponding antigen.

[0062] Colloidal gold enhancer is a core-shell structure composite nanomaterial prepared by attaching a silver shell and mixing it with a small molecule compound. It has a strong molar absorption coefficient and can be used as a tracer in immunochromatography.

[0063] Colloidal gold is formed by the polymerization of chloroauric acid into gold particles of a specific size in the presence of reducing agents such as white phosphorus, ascorbic acid, sodium citrate, tannic acid, etc., and becomes a stable colloidal state due to electrostatic effects. Colloidal gold carries a negative charge in a weakly alkaline environment and can form a strong bond with the positively charged groups of protein molecules. Since this bond is an electrostatic bond, it does not affect the biological properties of the protein.

[0064] The immune probe of the present invention is prepared from alliin antibody and colloidal gold enhancer in a certain ratio.

[0065] Background fluorescence quenching-competitive immunochromatography

[0066] Background fluorescence quenching-immunochromatography (bFQICA) is an analytical technique established on the basis of colloidal gold immunochromatography (GICA). The detection form depends on the structure of the target, and there are usually two forms: sandwich method and competition method. The competition method is suitable for the detection of small molecules. Alliin is a small molecule. Due to its small molecular weight and only a single antigenic determinant, it can only be detected by competition method. This method can not only quickly judge the positive and negative results by observing the color intensity of the T line with the naked eye, but also perform quantitative detection by detecting the relative intensity of the background fluorescence of the membrane strip and the fluorescence of the detection line (T line). And because the relative fluorescence intensity is detected, the interference of factors such as protein binders in the sample on the fluorescence intensity is significantly suppressed, the sensitivity of quantitative detection is significantly improved, and the interference of impurities in the sample is avoided. It has the advantages of strong anti-interference ability, accurate measurement results, high sensitivity, wide linear range, short detection time, simple operation and good precision.

[0067] Complete antigen

[0068] Usually, a hapten needs to be covalently coupled to a macromolecule such as KLH (hemocyanin) or BSA (bovine serum albumin) to become a complete antigen that is both immunoreactive and immunogenic.

[0069] As used herein, the "complete antigen" of the present invention refers to the product of combining the hapten of the present invention with an appropriate protein carrier.

[0070] As used herein, the "protein carrier" in the present invention refers to any immunologically acceptable protein used to form a complete antigen, which may be, for example, hemocyanin, bovine serum albumin, and the like.

[0071] The complete antigen (such as alliin-KLH) prepared by the present invention has good immunogenicity. In one embodiment, it can stimulate New Zealand white rabbits to produce a strong immune response. After one basic immunization and three booster immunizations, the antiserum titer can reach 1:32K; the complete antigen alliin-KLH well retains the immunoreactivity of alliin.

[0072] In a preferred embodiment of the present invention, on the one hand, alliin-BSA complete antigen is used in the T line of the immunochromatographic sheet, and on the other hand, a specific antibody against alliin prepared based on the alliin-KLH complete antigen is used, thereby significantly improving the specificity of antigen-antibody binding.

[0073] Alliin Antibody

[0074] Specific monoclonal antibodies against alliin were produced by immunizing New Zealand white rabbits with alliin-KLH complete antigen.

[0075] In the present invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" are heterotetrameric glycoproteins with the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a constant region, and the heavy chain constant region is composed of three domains CH1, CH2, and CH3. One end of each light chain has a variable region (VL) and the other end has a constant region, and the light chain constant region includes a domain CL; the constant region of the light chain is paired with the CH1 domain of the heavy chain constant region, and the variable region of the light chain is paired with the variable region of the heavy chain. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes κ (Kappa) or λ (Lambda). The heavy chain and light chain of the antibody are covalently linked together by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are covalently linked together by inter-polypeptide disulfide bonds formed between the hinge regions.

[0076] Compared with the prior art, the advantages of the present invention are as follows:

[0077] 1. Short detection time: The detection time is about 15 minutes.

[0078] 2. Simple operation: No complicated sample pretreatment steps are required, just boil, homogenize, filter and dilute the garlic solution.

[0079] 3. High specificity: Based on the combination of alliin and alliin antibody, it has high specificity.

[0080] 4. Strong anti-interference ability: Detecting relative fluorescence eliminates the interference of other impurities in the sample on the fluorescence intensity.

[0081] 5. High sensitivity: the detection limit is 0.0756μg / ml.

[0082] 6. Wide range of applications: The detection method can be used for the detection of garlic, garlic-related medicines, health foods and health products (disinfection products, foods), cells, tissue samples, etc.

[0083] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0084] Materials and Methods

[0085] 1. Experimental instruments

[0086] Analytical balance (ME204E, Mettler Toledo (Shanghai) Co., Ltd.); low-temperature centrifuge (TGL-20bR, Shanghai Anting Scientific Instrument Factory); S3 vortex vortexer (X3, LARKSCI); dry fluorescent immunoassay analyzer (FIC-Q100N, Suzhou Helmen Precision Instruments Co., Ltd.); LED digital display heating magnetic stirrer (MS-H280-Pro, Dalong)

[0087] 2. Experimental Reagents

[0088] Table 1 Reagents

[0089]

[0090]

[0091] Example 1. Preparation of BSA-alliin

[0092] (1) 1 mg of BSA protein was dialyzed / dissolved into 1 ml of 25 mM 2-(N-morpholino) (MES), and 1 mg of alliin was added and mixed to dissolve;

[0093] (2) Add 0.5 ml of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution to 1 ml of the mixed solution in the previous step;

[0094] (3) Add 0.16 ml of 25 mM, pH 5 MES to the mixture in the previous step;

[0095] (4) Mix and incubate at 4°C for 2 h;

[0096] (5) Dialyze the solution three times at 4°C for 2 h each time to remove unbound small molecules.

[0097] Example 2. Preparation of Antibody 1A10

[0098] (1) Preparation of alliin-KLH complete antigen

[0099] 1 mg KLH protein was dialyzed / dissolved into 1 ml 25 mM 2-(N-morpholino) (MES) ethanesulfonic acid, and 1 mg alliin was added and mixed to dissolve;

[0100] Add 0.5 ml of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution to 1 ml of the previous mixed solution;

[0101] Then add 0.16 ml of 25 mM MES (pH = 5) to the mixed solution in the previous step;

[0102] Mix and incubate at 4°C for 2 h;

[0103] Dialyze three times at 4°C, 2 h each time, to remove the unbound alliin small molecules in a free state.

[0104] (2) Animal immunization

[0105] Alliin-KLH complete antigen was injected into rabbits for immunization. B cells in peripheral blood were collected after immunization, and positive B cells were screened by flow cytometry and antigen specificity.

[0106] (3) Cloning

[0107] The positive B cells after screening were cultured for 15 days, and the supernatant was taken for indirect ELISA, and clones with high positive values ​​(OD values) were selected. After RNA was extracted from the B cells, reverse transcription was performed to amplify the heavy chain and light chain sequences, and sequencing was performed to obtain the sequence of the monoclonal antibody, as shown in Table 2 (antibody 1A10). Subsequently, the target gene (VH and VL coding sequence) of the monoclonal antibody was transfected (transiently transfected) into HEK293 cells (human embryonic kidney 239 cells) for protein expression, and the antibody was collected after purification.

[0108] Table 2 Antibody 1A10 sequence

[0109]

[0110]

[0111]

[0112] Example 3. Preparation of immunochromatographic sheets

[0113] like Figure 2As shown, a YNHS-120B fluorescent film was attached to a PVC base plate, the C line was stripped with 0.5 mg / mL goat anti-rabbit IgG, and the T line was stripped with 0.5 mg / mL BSA-alliin. A 21 mm long T1 sample pad was placed on one end of the film strip, and an 18 mm long absorbent paper was placed on the other end.

[0114] Example 4. Sample detection and calculation method

[0115] Detection method: Take 70 μl of the test solution, add 2 μl of the immune probe solution, mix well, take 50 μl and spot on the sample pad, chromatograph at room temperature for 15 minutes, and use background fluorescence-immunoassay analyzer to measure F1 / F2 (F1 is the fluorescence intensity between the C line and the T line, and F2 is the fluorescence intensity of the T line).

[0116] Calculation formula:

[0117] Alliin content (%) = C × D × 500 × 100% / m / 1000 / 1000 Formula 1

[0118] Where, C is the calculated concentration of alliin in the sample, μg / ml;

[0119] D is the dilution factor;

[0120] m is the sample weight, g.

[0121] Recovery rate (%) = (M2-M0) / M1 Formula 2

[0122] Where, M3 is the amount of alliin actually measured, μg;

[0123] M0 is the original amount of alliin in the sample, μg;

[0124] M1 is the amount of alliin reference substance added, μg.

[0125] CV(%) = (SD / M)×100% Formula 3

[0126] In the formula, CV is the coefficient of variation;

[0127] SD is standard deviation;

[0128] M is the mean.

[0129] Example 5. Alliin standard concentration curve

[0130] Reference solution stock solution: Dissolve alliin in water to make a 1 mg / ml stock solution.

[0131] Serial concentration reference solution: dilute the alliin stock solution with PBS to solutions of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 μg / ml.

[0132] Take 70 μl of alliin standard solution of series concentration, add 2 μl 001CGE@1A10, mix well, take 50 μl and spot on the sample pad, chromatograph at room temperature for 15 min, and use background fluorescence-immunoassay to measure F1 / F2 (Table 2). With alliin concentration as the horizontal axis and F1 / F2 as the vertical axis, the Logistic model was used for fitting, and the standard curve was obtained as shown below: Figure 3 As shown, r = 0.9994, indicating that F1 / F2 has a good correlation with alliin concentration in the range of 0.1 to 1.0 μg / ml.

[0133] Table 3 F1 / F2 values ​​of alliin reference substances in a series of concentrations (n=3)

[0134]

[0135] Example 6. Repeatability Verification

[0136] Prepare a test solution with an alliin concentration of 0.5090 μg / ml, and perform the determination according to the detection method in Example 1. Determine 6 copies in parallel, record F1 / F2, substitute it into the standard curve, and calculate the alliin concentration in the sample. The results are shown in the following table.

[0137] Table 4 Repeatability test results

[0138]

[0139] The CV of the repeatability test was 1.79%, which was less than 2%, indicating that the method had good repeatability.

[0140] Example 7. Intermediate precision verification

[0141] Sample solutions with three concentrations of alliin, low (0.3024 μg / ml), medium (0.5090 μg / ml), and high (0.7695 μg / ml), were prepared within 3 days, and three parallel samples were prepared for each concentration. The test was performed according to the detection method in Example 1, and F1 / F2 was recorded and substituted into the standard curve to calculate the alliin concentration in the sample. The results are shown in the following table.

[0142] Table 5 Precision test results (n=3)

[0143]

[0144] The inter-group and intra-group CV% of the three concentrations were all less than 8.00%, indicating that the method had good precision.

[0145] Example 8. Recovery test

[0146] Prepare a sample solution with a known alliin concentration of 0.2545 μg / ml, add reference substances equivalent to 80%, 100%, and 120% of the alliin in the original sample, prepare 5 samples of each concentration, and measure according to the detection method in Example 1, record F1 / F2, substitute it into the standard curve, and calculate the alliin concentration in the sample. The results are shown in the following table.

[0147] Table 6 Recovery test results (n = 5)

[0148]

[0149] As shown in the table, the spiked recoveries of the three concentration samples were between 97.21% and 98.61%, which was in line with the requirement of 90.0% to 108.0% recovery, indicating that the method had a qualified accuracy and could be used for the detection of alliin in garlic.

[0150] Example 9. Detection Limit

[0151] Take a blank solution (PBS solution) and measure it according to the detection method in Example 1. Measure it 10 times continuously, record F1 / F2, calculate the mean value (M) and standard deviation (SD), and obtain the value of M-2SD. Substitute it into the concentration value calculated by the standard curve, which is the detection limit.

[0152] Table 7 Detection limit results

[0153]

[0154] Substituting M-2SD (5.95482) into the standard curve, the detection limit was calculated to be 0.0756 μg / ml.

[0155] Example 10. Determination of alliin samples

[0156] Weigh about 6g of garlic into a 500ml beaker, add 200ml of water, boil for 10min, let it cool to room temperature, homogenize for 1min, transfer the homogenate to a 500ml volumetric flask, dilute to scale with water, shake well, filter with a 0.22μm filter membrane, and dilute 100,000 times with PBS. Take 70μl of garlic sample dilution, add 2μl of immune probe, mix well, take 50μl and apply it on the sample pad, chromatograph at room temperature for 15min, and determine F1 / F2. Substitute F1 / F2 into the standard curve to obtain the concentration of alliin in the sample and calculate the content of alliin in the sample.

[0157] Table 8 Alliin extract measurement results (n=3)

[0158]

[0159] Example 11. Kit

[0160] The kit of this embodiment includes:

[0161] (a) a container and the immunochromatographic sheet of the present invention in the container; and

[0162] (b) a container and the alliin immunoprobe of the present invention in the container; or

[0163] (c) A container and the immunochromatographic sheet of the present invention in the container, wherein the spotting area of ​​the immunochromatographic sheet contains the immunoprobe of the present invention.

[0164] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An immunochromatographic sheet for detecting alliin, It is characterized in that It comprises a base plate, a sample pad, an NC membrane and absorbent paper located on the base plate, wherein a detection line T line and a quality control line C line are arranged on the NC membrane; Wherein, the T line is a covalently linked alliin-carrier protein, and its structural formula is: Wherein, X is a carrier protein.

2. The immunochromatographic sheet according to claim 1, It is characterized in that The carrier protein is hemocyanin or bovine serum albumin.

3. An immunoprobe for detecting alliin, It is characterized in that The immune probe is colloidal gold enhancer-alliin antibody.

4. The immunoprobe according to claim 3, It is characterized in that The alliin antibody comprises a heavy chain variable region and a light chain variable region, wherein the complementarity determining region CDR (HCDR) of the heavy chain variable region and the complementarity determining region CDR (LCDR) of the light chain variable region are: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 3, The amino acid sequence of HCDR2 is shown in SEQ ID NO:4, The amino acid sequence of HCDR3 is shown in SEQ ID NO:5, LCDR1 having an amino acid sequence as shown in SEQ ID NO: 10, LCDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and The amino acid sequence of LCDR3 is shown in SEQ ID NO:

12.

5. The immunoprobe according to claim 4, It is characterized in that The heavy chain variable region and light chain variable region of the antibody are: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

6. The immunoprobe according to claim 3, It is characterized in that The particle size of the colloidal gold enhancer is 10-50nm.

7. The immunoprobe according to claim 3, It is characterized in that In the immune probe, the mass ratio of the colloidal gold enhancer to the alliin antibody is 1:5-1:

10.

8. A method for detecting alliin, It is characterized in that Includes steps: (a) providing the immunochromatographic sheet according to claim 1; (b) mixing the sample to be tested with the immunoprobe according to claim 3, and loading the mixture onto the sample loading area of ​​the immunochromatographic sheet; and (c) observing the T line and the C line of the immunochromatographic sheet to obtain a detection result of whether the sample contains alliin.

9. The method according to claim 8, It is characterized in that The methods are either qualitative or quantitative.

10. A kit for detecting alliin, It is characterized in that include: (a) a container and the immunochromatographic sheet as claimed in claim 1 contained in the container; and (b) a container and the immunoprobe according to claim 3 contained in the container; or (c) A container and the immunochromatographic sheet according to claim 1 contained in the container, wherein the spotting area of ​​the immunochromatographic sheet contains the immunoprobe according to claim 3.

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