Anti-lavender monoclonal antibody and application thereof in detection of lavender honey

Abstract

The invention relates to an anti-lavender monoclonal antibody and application of the anti-lavender monoclonal antibody in detection of lavender honey. The monoclonal antibody is prepared by the following steps: immunizing a Balb / c mouse by using a linalool-KLH conjugate as an immunogen, and carrying out cell fusion, hybridoma screening and ascites purification, so as to obtain the monoclonal antibody. The sequence of a heavy chain variable region is SEQ ID NO: 1, the sequence of a light chain variable region is SEQ ID NO: 5, and the affinity constant (Ka) reaches 1.25 * 10 < 9 > L / mol. The antibody has a linalool sensitivity of 1.05-3.63 [mu] g / mL in competitive ELISA detection, and can specifically distinguish lavender honey (the inhibition rate is greater than or equal to 40%) from other honey source honey (the inhibition rate is less than or equal to 10%). The lavender honey immunodetection method based on the monoclonal antibody is established for the first time, has the advantages of being high in specificity, easy and convenient to operate and low in cost, and is suitable for rapid screening of authenticity of honey products.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biotechnology and food testing, specifically relating to a monoclonal antibody that specifically targets linalool, a characteristic aroma component of lavender, its preparation method, and its application in the identification of the authenticity of lavender honey. In particular, it relates to a competitive ELISA detection system based on this antibody, used for rapid qualitative identification of lavender honey and exclusion of honey from other sources or adulterated honey. Background Technology

[0002] Lavender honey is highly sought after for its unique flavor and health benefits, but its high price has led to significant adulteration issues in the market. Current identification methods primarily rely on chromatographic techniques (such as GC-MS), which require complex pretreatment, expensive equipment, and specialized personnel, making rapid on-site detection difficult. While immunoassays (such as ELISA) have been used in identifying adulterated honey, specific antibodies against linalool, a characteristic component of lavender, have not yet been reported.

[0003] Although immunoassay has potential in identifying adulterated honey, existing antibody-targeted protein markers (such as royal jelly main protein 1) or broad-spectrum sugar components cannot distinguish specific small molecule metabolites of nectar-producing plants. The preparation of antibodies against floral fragrance components (such as linalool) faces three major challenges due to their simple molecular structure and lack of immunodominant epitopes: First, the complexity of hapten design: the characteristic groups of linalool (allyl alcohol structure) must be retained, while an active linker arm (such as a carboxyl group) must be introduced to achieve directional coupling with the carrier protein; otherwise, the antigenic epitope may be destroyed or non-specific antibodies may be induced. Second, the risk of cross-reactivity: linalool is present in various plants (such as mint and coriander), and structural analogs in the honey matrix (such as limonene and α-terpineol) may interfere with antibody recognition, requiring antibodies with strict conformational selectivity. Third, the bottleneck of detection sensitivity: the concentration of linalool in genuine lavender honey is only about 0.8-2.5 mg / kg (equivalent to 1.14-3.55 μg / mL), far below the detection limit of conventional ELISA (usually >10 μg / mL), necessitating the development of ultra-high affinity antibodies.

[0004] Linalool, a key volatile compound in lavender honey, is present in significantly higher concentrations (typically ≥0.8 mg / kg) than in genuine lavender honey compared to other honey sources. However, its small molecular weight (<200 Da) results in weak direct immunogenicity, necessitating the design of hapten-carrier protein conjugates to induce an effective immune response. Current technologies lack high-affinity antibodies against linalool, and no mature immunoassay method supports the identification of genuine lavender honey. Therefore, developing a rapid detection technology based on linalool-specific monoclonal antibodies is crucial for regulating the honey market. Summary of the Invention

[0005] This invention addresses the bottleneck in lavender honey identification technology by providing a highly specific monoclonal antibody against linalool and its immunoassay method, overcoming the limitations of traditional GC-MS methods, which are highly dependent on equipment and complex to operate.

[0006] The first objective of this invention is to provide a monoclonal antibody against linalool, comprising a heavy chain variable region (VH): its complementarity-determining region CDRH1 has the sequence shown in SEQ ID NO:2, CDRH2 has the sequence shown in SEQ ID NO:3, and CDRH3 has the sequence shown in SEQ ID NO:4, and a light chain variable region (VL): its complementarity-determining region CDRL1 has the sequence shown in SEQ ID NO:6, CDRL2 has the sequence shown in SEQ ID NO:7, and CDRL3 has the sequence shown in SEQ ID NO:8.

[0007] In some embodiments, the heavy chain variable region sequence of the antibody is shown in SEQ ID NO:1, and the light chain variable region sequence is shown in SEQ ID NO:5.

[0008] A second objective of this invention is to provide an immunoassay kit for identifying lavender honey.

[0009] In some embodiments, the kit contains the monoclonal antibody described above.

[0010] The present invention also provides a method for identifying lavender honey, comprising using the above-mentioned monoclonal antibody as the detection antibody and employing an indirect competitive ELISA method to identify lavender honey.

[0011] Finally, the present invention provides an application in which the monoclonal antibody is used in the preparation of products for identifying lavender honey.

[0012] Compared with the prior art, the present invention has at least the following beneficial effects:

[0013] This invention presents for the first time the heavy chain variable region sequence of monoclonal antibody 6H3 as shown in SEQ ID NO:1, and the light chain variable region sequence as shown in SEQ ID NO:5. An indirect competitive ELISA method based on this antibody can achieve rapid identification of lavender honey through specific binding to linalool. Attached Figure Description

[0014] Figure 1 SDS-PAGE band migration rate assay for antigen conjugation results.

[0015] Figure 2 ELISA was used to screen monoclonal antibody strains.

[0016] Figure 3Monoclonal antibody subtype identification kits are used to identify the subtypes of monoclonal antibodies.

[0017] Figure 4 Detection of affinity of monoclonal antibody 6H3 for linalool.

[0018] Figure 5 Sensitivity detection of monoclonal antibody 6H3 against linalool using ic-ELISA.

[0019] Figure 6 Monoclonal antibody 6H3 was identified in different honey samples. Detailed Implementation

[0020] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0021] Example 1: Preparation of linalool-KLH antigen

[0022] Linalool and succinic anhydride were mixed in an inert organic solvent, dichloromethane or N,N-dimethylformamide, and a catalytic amount of 4-dimethylaminopyridine (DMAP) was added. The mixture was reacted at 40-60°C for 4-12 hours to generate linalool succinate monoester (Linalool-HS). After the reaction, the linalool hapten derivative Linalool-HS containing an active carboxyl group was obtained by extraction, washing, and column chromatography purification. The Linalool-HS was dissolved in anhydrous dimethyl sulfoxide, and EDC and NHS were added sequentially. The mixture was activated at room temperature in the dark for 1-2 hours to obtain an activated ester solution. Keyhole hemocyanin (KLH) was dissolved in pH... 7.4% phosphate buffer was used to slowly add the activated ester solution to the KLH solution under stirring, controlling the organic phase volume ratio to be ≤20%. The coupling reaction was carried out at 4℃ in the dark for 8–16 hours. After the reaction, glycine was added to quench unreacted active sites. The resulting mixture was dialyzed against phosphate buffer for 48–72 hours to remove small molecule impurities. After concentration, it was frozen and stored to obtain linalool-keyhole hemocyanin immunogen (Linalool-KLH). The coupling status was detected by SDS-PAGE band migration rate.

[0023] Figure 1 The results showed that, due to the increased relative molecular mass of Linalool-KLH compared to KLH, the band migration speed of Linalool-KLH was slower within the same time frame, resulting in band lag. This indicates that the linalool-keyhole hemocyanin immunoantigen conjugation was successful.

[0024] Example 2: Preparation, expression, and identification of anti-Linalool-KLH monoclonal antibody

[0025] For the initial immunization, Linalool-KLH and Freund's complete adjuvant were mixed in a 1:1 volume ratio. Four 6-8 week old female Balb / c mice were selected and immunized subcutaneously via multiple abdominal injections of the emulsified antigen. A second immunization was performed 21 days later, using Linalool-KLH mixed with Freund's incomplete adjuvant for subcutaneous injection. Third and fourth intraperitoneal immunizations were performed on days 42 and 63, respectively, followed by a pulse immunization on day 77. The immunization dose per mouse was 100 μg. Mouse spleen cells and SP2 / 0 myeloma cells were fused at a 5:1 ratio (number ratio) in 50% PEG and cultured in selective medium. The cell supernatant was collected, and positive hybridoma cells were screened using ELISA. Positive clones were screened from the hybridoma cell culture supernatant 10-14 days after cell fusion, specifically including the following steps: Linalool-BSA was prepared using 0.05M pH... 9.6 Dilute with carbonate buffer to 1-10 μg / mL, add 100 μL / well to a 96-well polystyrene microplate, and coat overnight (12-16 hours) at 4°C. Discard the coating solution, wash three times with PBST (0.05% Tween-20 PBS), add 200 μL of PBST blocking buffer containing 1-5% BSA to each well, and incubate at 37°C for 1-2 hours. Discard the blocking buffer, wash three times with PBST, add 100 μL of hybridoma cell culture supernatant to each well, and set up a negative control: SP2 / 0 cell culture supernatant. Incubate at 37°C for 1 hour. After washing, add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution) to each well, and incubate at 37°C for 1 hour. Wash five times, add 100 μL of TMB chromogenic substrate to each well, and react at room temperature in the dark for 10-15 minutes. Stop the reaction by adding 50 μL of 2M H2SO4. The absorbance (OD) at 450 nm was measured using an ELISA reader. 450 When the OD value of the sample well is ≥ 2.1 times the OD value of the negative control well (i.e., P / N ≥ 2.1), it is determined to be a positive hybridoma cell line. See [link to relevant documentation]. Figure 2 .

[0026] Figure 2 The results showed that the P / N value of the 6H3 monoclonal antibody strain was much higher than that of other monoclonal antibody strains, and it was used for subsequent experiments.

[0027] Two 6-8 week old female Balb / c mice were used. Each mouse was intraperitoneally injected with sterile paraffin oil at a dose of 0.5 mL. Seven days later, the selected monoclonal hybridoma cell line 6H3 was injected. Ascites fluid was collected when the mice's abdomens became distended and the volume stabilized. The ascites fluid was purified using protein G affinity chromatography, and the titer was preliminarily determined. One monoclonal antibody was isolated and named 6H3. The monoclonal antibody was identified using a monoclonal antibody subtype identification kit. The identification results are shown below. Figure 3 .

[0028] Figure 3 The results showed that, based on subtype identification, monoclonal antibody 6H3 was identified as IgG1.

[0029] Total RNA was extracted from hybridoma cell line 6H3, and cDNA was synthesized by reverse transcription. The light chain (VL) and heavy chain (VH) genes were then amplified by PCR using universal primers for the variable region of mouse IgG. The target fragments were cloned into pMD. TM The 19-T vector was used to screen ≥10 single colonies for Sanger sequencing; the sequences were compared with the IMGT database, and the CDR region was defined according to the Kabat standard, as shown in Table 1.

[0030]

[0031]

[0032] Example 3: Detection of the affinity and sensitivity of monoclonal antibody 6H3

[0033] Dissolve 1 μg / mL linalool antigen in carbonate buffer (pH 9.6) and coat 96-well microplates (4°C overnight). After blocking with PBS containing 1% BSA, add serially diluted monoclonal antibody 6H3 at concentrations ranging from 1 × 10⁻⁶. -11 Up to 1×10 -8 Incubate at 37°C for 1 hour with a concentration of mol / L. After washing the plate, add horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (1:5000), incubate with TMB for 15 minutes, and terminate the reaction with 2M H2SO4. Measure the absorbance (OD) at 450 nm using a microplate reader. 450 Plot antibody binding saturation curves and calculate the half-maximal effective concentration (EC50). 50 The antibody-antigen affinity was assessed using Scatchard analysis or nonlinear regression to fit the dissociation constant (Ka). (See attached text.) Figure 4 .

[0034] Figure 4 The results showed that the Ka value of monoclonal antibody 6H3 was 1.25 × 10⁻⁶. 9 L / mol.

[0035] Linalool-BSA conjugate (2 μg / mL, 100 μL / well) was coated into a 96-well plate and incubated overnight at 4°C. After washing with PBST, the plate was blocked with 1% BSA-PBS at 37°C for 1 hour. 50 μL of linalool standards at different concentrations and an equal volume of 6H3 monoclonal antibody were added, and the plates were competitively bound at 37°C for 30 minutes. After washing, HRP-labeled goat anti-mouse secondary antibody (1:5000) was added, and the plates were incubated at 37°C for 1 hour. TMB was used for color development for 10 minutes, and the reaction was stopped with 2M H2SO4. OD was then measured. 450 The sensitivity curve for detecting monoclonal antibody 6H3 is shown in the figure. Figure 5 .

[0036] Figure 5 The results showed that the detection range of linalool by monoclonal antibody 6H3 was 1.05–3.63 μg / mL. When the concentration of linalool in raw honey was 0.8–2.5 mg / kg, after direct conversion based on the honey density of 1.42 g / mL, the detection concentration range was 1.14–3.55 μg / mL. The detection range of monoclonal antibody 6H3 covered the concentration range of linalool in raw honey and can be used for subsequent qualitative detection of lavender honey.

[0037] Example 4: Application of monoclonal antibody 6H3 in the identification of lavender honey

[0038] Linalool-BSA conjugate (2 μg / mL, 100 μL / well) was coated into a 96-well plate and incubated overnight at 4°C. After washing with PBST, the plate was blocked with 1% BSA-PBS at 37°C for 1 hour. 50 μL of the test solution + 50 μL of 6H3 monoclonal antibody working solution (1×10⁻⁹ mol / L) were added to each well. The test solutions were prepared according to the following categories: Positive control well: French Provence lavender honey; Negative control 1: acacia honey; Negative control 2: rapeseed honey; Negative control 3: citrus honey; Negative control 4: syrup honey; Negative control 5: buckwheat honey; Blank control: PBST buffer. Competitive binding was performed at 37°C for 30 minutes. After washing with PBST, HRP-labeled goat anti-mouse secondary antibody (1:5000) was added, and the plate was incubated at 37°C for 1 hour. TMB was used for color development for 10 minutes, and the reaction was terminated with 2M H₂SO₄. OD₄⁵⁰ was measured, and the inhibition rate was calculated: Inhibition rate (%) = [1 - (sample OD₄⁵⁰) / (sample OD₄⁵⁰)]. 450 / blank OD 450 [×100%]; Lavender honey judgment criteria: Positive: inhibition rate ≥40% (corresponding to linalool ≥0.8mg / kg); Negative: inhibition rate ≤10% (linalool not detected); see Figure 6 .

[0039] Figure 6 The results showed that the inhibition rate of the lavender honey group was ≥40%, while the inhibition rates of the other groups were ≤10%, which was in line with expectations. Therefore, monoclonal antibody 6H3 can be used for the qualitative detection of lavender honey.

[0040] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against lavender, characterized in that, Contains heavy chain variable region (VH): its complementary determinant region CDRH1 has the sequence shown in SEQ ID NO:2, CDRH2 has the sequence shown in SEQ ID NO:3, and CDRH3 has the sequence shown in SEQ ID NO:4 and light chain variable region (VL): its complementary determinant region CDRL1 has the sequence shown in SEQ ID NO:6, CDRL2 has the sequence shown in SEQ ID NO:7, and CDRL3 has the sequence shown in SEQ ID NO:

8.

2. The monoclonal antibody according to claim 1, characterized in that, The heavy chain variable region sequence is shown in SEQ ID NO:1, and the light chain variable region sequence is shown in SEQ ID NO:

5.

3. An immunoassay kit for identifying lavender honey, characterized in that... It comprises the monoclonal antibody according to any one of claims 1-2.

4. A method for identifying lavender honey, comprising the following steps: An indirect competitive ELISA method was used, with the monoclonal antibody described in claims 1-2 as the detection antibody.

5. The use of the monoclonal antibody according to any one of claims 1-2 in the preparation of products for identifying lavender honey.

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