Monoclonal antibody resistant to phenyllactic acid and application of monoclonal antibody in identification of Nile Xinjiang black bee honey

Abstract

The invention relates to the technical field of immunodetection, and particularly provides an anti-phenyllactic acid monoclonal antibody and application thereof in identification of Nilox Xinjiang black bee honey. The heavy chain variable region sequence of the monoclonal antibody is as shown in SEQ ID NO: 1, and the light chain variable region sequence of the monoclonal antibody is as shown in SEQ ID NO: 5. An immunodetection ELISA method established based on the antibody can realize rapid identification of Nilox Xinjiang black bee honey by specifically combining phenyllactic acid. Experiments show that the content of phenyllactic acid in honey is gt; and when the concentration is 100 mg / kg, the detection signal of the antibody is obviously higher than that of other honey varieties, and the sensitivity reaches 0.42 mu g / mL. The method provided by the invention solves the problems of strong equipment dependence and complex operation of the traditional GC-MS method, and provides an efficient and low-cost solution for detecting the authenticity of the Nilox Xinjiang black bee honey.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to an anti-phenyllactic acid monoclonal antibody and its application in food authenticity identification, and is particularly suitable for rapid identification of Nileke Xinjiang black bee honey. Background Art

[0002] Nileke Xinjiang black bee honey, thanks to its unique nectar source (276 wild plant species from the Ili River Valley in Xinjiang), is rich in highly active ingredients. Its amylase activity and mineral content (such as selenium, potassium, and calcium) significantly exceed national standards, and its market value can be several times that of ordinary honey. However, the high price premium has led to frequent adulteration: unscrupulous merchants often pass off cheap honey (such as vitex honey and acacia honey) or fructose corn syrup as counterfeit, seriously undermining consumer rights and industry order. Due to the lack of authoritative identification standards, traditional methods (such as pollen microscopy) are unable to cope with the complexity of mixed honeys from multiple flower species. There is an urgent need to develop objective identification technologies based on specific markers.

[0003] Authorized patent CN 114002363 B first revealed that phenyllactic acid can be used as a core marker for Nileke honey, with a content >100 mg / kg (compared to <2 mg / kg for ordinary honey) and / or a phenyllactic acid to lactic acid mass ratio >0.05 (compared to <0.05 for ordinary honey). The GC-MS detection method based on this method, while highly accurate, suffers from three major bottlenecks: 1. High equipment dependence: a specialized gas chromatography-mass spectrometer (unit price >2 million yuan) and a skilled operator are required; 2. The process is complex and time-consuming: the sample requires pretreatment with solid-phase extraction, methoxyamination derivatization (40°C for 30 minutes), and silanization derivatization (37°C for 30 minutes), with a single sample testing time exceeding 6 hours; 3. High cost: Derivatization reagents (such as MSTFA), chromatographic column consumables, and equipment maintenance fees bring the cost per sample to over 1,000 yuan, making it difficult to promote in grassroots market supervision.

[0004] Currently, there are no rapid immunological detection solutions for phenyllactic acid. Monoclonal antibody technology, due to its high specificity, low cost, and ease of use, has been widely used for on-site screening of small molecule compounds (such as pesticides and toxins) (e.g., ELISA strips). However, phenyllactic acid has a small molecular weight (166 Da) and requires coupling to a carrier protein (such as BSA) to form an artificial antigen for animal immunization. Antibody development faces challenges such as epitope masking and insufficient affinity. If the bottleneck of antibody preparation technology can be overcome and an antibody-based phenyllactic acid detection method can be established, it would enable "low-cost, high-throughput" identification of Nileke honey, filling a gap in the industry. Summary of the Invention

[0005] The present invention addresses the technical bottleneck of identifying Xinjiang black bee honey in Nileke, provides a highly specific anti-phenyllactic acid monoclonal antibody and an immunoassay method thereof, and breaks through the limitations of the traditional GC-MS method, which is highly dependent on equipment and has complex operations.

[0006] The first object of the present invention is to provide an anti-phenyllactic acid monoclonal antibody, characterized in that it comprises a heavy chain variable region (VH): its complementary 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 complementary 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 certain 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] The second object of the present invention is to provide an immunoassay kit for identifying Nileke Xinjiang black bee honey.

[0009] In certain embodiments, the kit comprises the monoclonal antibody.

[0010] The present invention also provides a method for identifying Nileke Xinjiang black bee honey, comprising the following steps: (a) diluting a Xinjiang black bee honey sample 1000 times; (b) using an indirect competitive ELISA method, using the monoclonal antibody described in claims 1-2 as a detection antibody; (c) when the OD450 value is higher than a set threshold, it is determined to be Nileke Xinjiang black bee honey; the threshold corresponds to a phenyllactic acid content >100 mg / kg.

[0011] Finally, the present invention provides an application, which is the application of the monoclonal antibody in preparing a product for identifying Nileke Xinjiang black bee honey.

[0012] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention proposes for the first time that the heavy chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO:1, and the light chain variable region sequence is shown in SEQ ID NO:5. The immunoassay ELISA method established based on this antibody can achieve rapid identification of Nileke Xinjiang black bee honey by specifically binding to phenyllactic acid. Compared with the existing technology, the present invention achieves three major breakthroughs: efficiency improvement: the detection time is shortened from >6 hours of GC-MS method to 2 hours; cost reduction: the cost of single sample detection is reduced from >1,000 yuan to less than 50 yuan; precise matching of industry standards: the detection threshold is fully compatible with the phenyllactic acid >100 mg / kg standard of CN114002363B, meeting industry regulatory requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 SDS-PAGE identification and analysis of phenyllactic acid-bovine serum albumin (PLA-BSA) antigen.

[0014] Figure 2 ELISA was used to screen the monoclonal antibody strains for analysis.

[0015] Figure 3 Affinity testing of monoclonal antibody 3A1 for phenyllactic acid (PLA).

[0016] Figure 4 Sensitivity test of monoclonal antibody 3A1 to phenyllactic acid (PLA) by ic-ELISA.

[0017] Figure 5 Monoclonal antibody 3A1 was identified in different honey samples. DETAILED DESCRIPTION

[0018] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0019] Example 1. Preparation of phenyllactic acid-bovine serum albumin (PLA-BSA) antigen 20 mg of phenyllactic acid (PLA) was dissolved in 1 mL of anhydrous DMSO, and 10 mg of EDC·HCl, 6 mg of NHS, and 1 mg of DMAP were added. The mixture was activated at 25°C in the dark for 2 hours. At the same time, 10 mg of BSA purified by dialysis was dissolved in 0.1 M MES buffer (pH 6.0). The activation solution was added dropwise to the BSA solution at a PLA:BSA molar ratio of 25:1, and the mixture was gently stirred at 25°C for 12 hours. After the reaction was terminated, the PLA-BSA antigen was desalted and purified on a PD-10 column, and then identified and analyzed by SDS-PAGE. Figure 1 .

[0020] Figure 1 The results showed that compared with BSA protein, PLA-BSA antigen showed obvious migration band hysteresis after the molecular weight increased, proving that the artificial antigen coupling was successful.

[0021] Example 2. Preparation, expression and identification of anti-PLA-BSA monoclonal antibodies Immunization was performed subcutaneously and in the dorsal area of ​​female BALB / c mice using PLA-BSA emulsified with Freund's adjuvant. For the first immunization, the immunogen was emulsified with FCA adjuvant and injected subcutaneously and in the dorsal area using a 1 mL syringe, with 100 µL injected into each mouse. Booster immunizations were performed every three weeks using FIA adjuvant emulsified with the immunogen, also injected subcutaneously and in the dorsal area, with 100 µL injected into each mouse. Starting with the fifth immunization, blood was collected from the tail tip seven days after immunization to measure titer and inhibition using an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). The specific procedure was as follows:

[0022] PLA-BSA was diluted to 0.5 μg / mL in pH 9.6 carbonate buffer; 100 μL was added to each well and incubated at 4°C for 12-16 hours; the wells were washed three times with PBST (1 min each); 200 μL of 5% skim milk / PBS was added and the wells were blocked at 37°C; the blocking buffer was discarded and the wells were washed three times with PBST; the PLA standard was diluted to 10 ng / mL in PBS; the mouse serum to be tested was pre-diluted with PBS diluent; competitive incubation treatment: (sample well): 50 μL standard PLA + 50 μL antiserum diluent; (blank control well): 50 μL diluent PBS + 50 μL antiserum diluent, gently mix, and incubate at 37°C for 1 hour; the mixture was transferred to the antigen-coated plate, 100 μL per well; incubated at 37°C for 30 minutes; the solution was discarded and the wells were washed five times with PBST; HRP-goat anti-mouse IgG was diluted in blocking buffer; 100 μL secondary antibody was added to each well and incubated at 37°C for 1 hour. 100 μL of TMB substrate was added to each well and reacted at room temperature for 10–15 minutes in the dark (blue color developed). 50 μL of stop solution was added (color turned yellow) and OD450 nm was measured immediately. The results are shown in Table 1.

[0023]

[0024] Table 1. Serum analysis of mice after immunization

[0025] The results in Table 1 show that the inhibition rate of mouse #3 was the best in the same group, with a higher titer and the best inhibition, so it was selected as the mouse to be fused.

[0026] Mouse #3, which had a high serum titer, was selected for booster immunization. Three days later, spleen cells were harvested and fused with SP2 / 0 cells. Monoclonal antibodies were prepared using hybridoma cell inoculation within the peritoneal cavity of mice, and ascites was collected. Two-month-old female BALB / C mice were injected intraperitoneally with 500 μL of paraffin oil. Within 7–14 days, 300 μL of fused hybridoma SP2 / 0 cells (1×10 6 Cells / mL). After 7 days, ascites fluid was collected and centrifuged at 10,000 rpm for 10 minutes at 4°C to remove impurities. The fluid was then frozen at –20°C until further use. Monoclonal antibody strains were screened by ELISA. Phenyl lactic acid-bovine serum albumin (PLA-BSA) was diluted to 0.5 μg / mL in 0.05 M carbonate buffer (pH 9.6). 100 μL / well of the plate was coated on a 96-well plate and incubated at 4°C overnight (12–16 hours). The coating solution was discarded, the plates were washed three times with PBST (PBS containing 0.05% Tween-20). 200 μL of 5% skim milk / PBS blocking solution was added to each well, and the plates were incubated at 37°C for 2 hours. After washing, the test samples were added: 100 μL of undiluted ascites fluid diluted 1:1000 from the supernatant of the fused hybridoma cell line was added to the experimental wells; 1:100 dilution of non-immunized mouse serum was added to the negative control wells; and PBS was added to the blank control wells. The plates were reacted at 37°C for 1 hour. Wash 5 times with PBST, add 1:8000 diluted HRP-labeled goat anti-mouse IgG secondary antibody (100 μL / well), and incubate at 37°C for 1 hour. After washing, add 100 μL TMB substrate to each well and develop for 10 minutes in the dark. Stop the reaction with 50 μL 2 MH2SO4 and measure the absorbance (OD) at 450 nm on a microplate reader. 450 ). Positive judgment standard: experimental well OD 450 The value must be >0.8, and the P / N value ([experimental well OD 450 - Blank hole OD 450 ] / [Negative well OD 450 - Blank hole OD 450 ]) ≥2.5, all steps were repeated three times, and the data were averaged. Figure 2 .

[0027] Figure 2 The results showed that the 3A1 monoclonal antibody strain had the highest P / N value and the best affinity effect and could be used for subsequent experiments.

[0028] Take 1×10 7Total RNA was extracted from hybridoma cells 3A1 using the TRIzol method, and cDNA was synthesized by reverse transcription using Oligo(dT)18 primers. The heavy chain (VH) and light chain (VL) variable region genes were amplified by nested PCR. The PCR products were purified, ligated into the pGEM-T vector, and transformed into DH5α competent cells. Positive clones were selected and sent for Sanger sequencing. The IMGT / V-QUEST database (http: / / www.imgt.org) was used for analysis to identify V(D) and J gene segments, annotate the complementarity-determining regions (CDRs) 1-3 (according to the Kabat numbering system), and deduced amino acid sequences (see Table 2).

[0029] Table 2. Sequence analysis of the heavy and light chain variable regions of monoclonal antibody 3A1

[0030] Example 3: Affinity and sensitivity testing of monoclonal antibody 3A1 Non-competitive ELISA was used to detect the affinity of monoclonal antibody 3A1 for phenyllactic acid (PLA): phenyllactic acid (PLA) antigen at different concentrations (0.5 and 1.5 μg / mL) was dissolved in carbonate buffer (pH 9.6) and coated onto a 96-well microtiter plate (4°C overnight). After blocking with PBS containing 1% BSA, serially diluted monoclonal antibody 3A1 (concentration range: 1 × 10⁻¹² to 1 × 10⁻) was added. 9 mol / L), incubated at 37°C for 1 hour. After washing, horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (1:5000) was added, TMB was used for color development for 15 minutes, and 2M H2SO4 was used to terminate the reaction. The absorbance at 450 nm (OD) was measured using a microplate reader. 450 By drawing the antibody binding saturation curve at different antigen concentrations, the half-maximal effect concentration (EC 50 ), and the dissociation constant (Ka) was fitted using Scatchard analysis or nonlinear regression to evaluate the antibody-antigen affinity, see Figure 3 .

[0031] Figure 3 The results showed that the Ka value of monoclonal antibody 3A1 for phenyllactic acid (PLA) was calculated based on the S-curve analysis of its affinity test. 10 L / mol.

[0032] Sensitivity test of monoclonal antibody 3A1 against phenyllactic acid (PLA) by ic-ELISA: PLA-BSA was diluted to 0.5 μg / mL in pH 9.6 carbonate buffer; 100 μL was added to each well and incubated at 4°C for 12-16 hours; the plate was washed three times with PBST (1 min each); 200 μL of 5% skim milk / PBS was added and the plate was blocked at 37°C; the blocking buffer was discarded and the plate was washed three times with PBST; PLA standard was diluted to different concentrations in PBS; 50 μL of PLA standard and 50 μL of monoclonal antibody 3A1 were added to each well; the mixture was gently mixed and incubated at 37°C for 1 hour; 100 μL of the mixture was transferred to the antigen-coated plate; the plate was incubated at 37°C for 30 minutes; the solution was discarded and the plate was washed five times with PBST; HRP-goat anti-mouse IgG was diluted in blocking buffer; 100 μL of secondary antibody was added to each well and the plate was incubated at 37°C for 1 hour; the solution was discarded and the plate was washed five times with PBST; 100 μL of secondary antibody was added to each well and the plate was incubated at 37°C for 1 hour; the solution was discarded and the plate was washed five times with PBST; 100 μL of secondary antibody was added to each well TMB substrate, incubate at room temperature in the dark for 10–15 minutes (blue color appears); add 50 µL of stop solution (color changes to yellow), and immediately measure OD450nm. The sensitivity curve of monoclonal antibody 3A1 is shown in the table. Figure 4 .

[0033] Figure 4 The results showed that the standard inhibition curve formula of monoclonal antibody 3A1 for phenyllactic acid detection was: y=0.15+1.65 / [1+(x / 0.54) 1.23 ] established, the fitting degree R 2 =0.998, and the detection range is 0.42~1.70μg / mL. That is, when the concentration of phenyllactic acid in the original honey is 300–1200mg / kg, after diluting it 1000 times according to the density of honey 1.42g / mL, the detection concentration is exactly 0.42–1.70μg / mL, which falls within the quantitative range of the standard curve and can be used for subsequent phenyllactic acid detection in Nileke Xinjiang black bee honey.

[0034] Example 4: Application of Monoclonal Antibody 3A1 in Identification of Nileke Xinjiang Black Bee Honey Honey samples from vitex, acacia, jujube flower, rapeseed, linden, and Nileke Xinjiang black bee were prepared. The honey samples were diluted 1:1000 with PBS and dissolved in carbonate buffer (pH 9.6). The samples were then coated onto a 96-well ELISA plate (4°C overnight). After blocking with PBS containing 1% BSA, monoclonal antibody 3A1 (concentration: 1×10⁻) was added. 10 mol / L), incubate at 37°C for 1 hour. After washing, add horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (1:5000), develop with TMB for 15 minutes, and terminate the reaction with 2M H2SO4. Measure the absorbance at 450nm (OD) using a microplate reader. 450 ),See Figure 5 .

[0035] Figure 5 The results showed that Nileke Xinjiang black bee honey group OD 450 The value is much higher than that of other honey groups, that is, the phenyllactic acid content of Nileke Xinjiang black bee honey is much higher than that of other honey samples, and the result is in line with expectations; therefore, monoclonal antibody 3A1 can be used for qualitative detection of Nileke Xinjiang black bee honey.

[0036] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A monoclonal antibody against phenyllactic acid, characterized in that: It comprises 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.

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 Nileke Xinjiang black bee honey, characterized in that The invention comprises the monoclonal antibody according to any one of claims 1 to 2.

4. A method for identifying Nileke Xinjiang black bee honey, comprising the following steps: (a) diluting a Xinjiang black bee honey sample 1000-fold; (b) employing an indirect competitive ELISA method using the monoclonal antibody described in claims 1-2 as a detection antibody; (c) determining the honey to be Nileke Xinjiang black bee honey when the OD450 value is higher than a set threshold; the threshold corresponds to a phenyllactic acid content greater than 100 mg / kg.

5. Use of the monoclonal antibody according to any one of claims 1-2 in the preparation of a product for identifying Nileke Xinjiang black bee honey.

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