A polypeptide that specifically binds to an immune complex of pendimethalin and applications thereof

Abstract

The application discloses a polypeptide specifically combined with an immune complex of pendimethalin antibody, and application of the polypeptide in immune detection of pendimethalin. The application uses an immune complex of a pendimethalin monoclonal antibody and pendimethalin to select a polypeptide specifically combined from a phage display random polypeptide library, and obtains a positive clone and a polypeptide sequence thereof through ELISA and first generation sequencing. A semi-saturation concentration of a non-competitive phage ELISA established based on the best polypeptide is 0.15 ng / mL, which is higher than a pendimethalin immune detection method reported at present. The application also relates to application of the polypeptide to a pendimethalin immune chromatography test strip. A non-competitive immune chromatography test strip established by a phage-free anti-immune complex polypeptide obtained through chemical synthesis has a visual detection limit of 2.5 ng / mL for pendimethalin, and can be used for rapid, sensitive, low-cost and high-specificity detection of pendimethalin residues in environment and agricultural products.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to polypeptides that specifically bind to pendimethalin immune complexes, and their application in enzyme-linked immunosorbent assay (ELISA) and immunochromatographic test strips. Background Technology

[0002] Pendimethalin, a dinitroaniline herbicide, has maintained a significant share of the global herbicide market since its development in 1972 due to its excellent pre-emergence effect and low application cost. Pendimethalin is chemically stable, non-volatile, and has strong soil adsorption capacity, making it prone to environmental residues. Although pendimethalin is classified as a low-toxicity pesticide, studies have shown that it exhibits cytotoxicity and genotoxicity, and it is classified as a Group C compound by the U.S. Environmental Protection Agency (EPA) as a potential carcinogen. my country has clearly stipulated the maximum residue limits (MRLs) for pendimethalin in food (GB2763-2021), such as 0.1, 0.2, and 0.03 mg / kg in corn, cabbage, and citrus fruits, respectively. Therefore, accurate and rapid detection of pendimethalin residues in the environment and food is crucial for ensuring food and environmental safety.

[0003] Compared to instrumental detection methods, immunoassay methods based on antigen-antibody specific reactions offer advantages such as simplicity and low cost, making them more suitable for on-site detection of pesticide residues at the grassroots level. Chemical pesticides like pendimethalin are small molecule compounds (molecular weight less than 1000 Daltons), possessing only one antigenic epitope and binding to only one antibody. Therefore, immunoassay methods for small molecule compounds typically employ competitive analysis. This method is reagent-limited, and its sensitivity depends on the antibody's affinity for the analyte; however, high-affinity antibodies are difficult to obtain. Furthermore, the signal intensity in competitive analysis is negatively correlated with analyte concentration; at lower analyte concentrations, positive and negative signals are difficult to distinguish, resulting in a high detection limit. Conversely, non-competitive analysis typically uses a sandwich method, achieving detection by capturing and labeling antibodies to form a sandwich structure with the antigen. This method can improve sensitivity by increasing reagent dosage. Simultaneously, the reaction signal is positively correlated with analyte concentration, providing more intuitive results and offering advantages in distinguishing trace signals from zero signals, with a higher detection limit.

[0004] Phage display technology allows for the direct expression of exogenous genes onto their coat proteins, making it a powerful tool for screening proteins and peptides. Phage display can be used to obtain anti-immune complex peptides that can bind to pesticide-antibody immune complexes, enabling the construction of non-competitive small-molecule analytical methods. Studies have shown that non-competitive immunoassay methods for pesticides using anti-immune complex peptides outperform competitive analytical methods in terms of sensitivity and specificity. Currently, there are no reports on anti-immune complex peptides for pendimethalin. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a polypeptide that specifically binds to the pendimethalin immune complex, and its application in enzyme-linked immunosorbent assay (ELISA) and immunochromatographic test strips.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A polypeptide that specifically binds to pendimethalin immune complexes, wherein the sequence of the polypeptide is the amino acid sequence shown in SEQ ID NO.4.

[0008] In this process, the cysteine ​​residues at both ends of the polypeptide are cyclized by forming disulfide bonds.

[0009] The preparation method of the polypeptide includes the following steps:

[0010] (1) The anti-dimethoprim monoclonal antibody was immobilized on an ELISA plate, and dimethoprim was added to form an immune complex. A phage display peptide library was then added for panning. The eluted phages were amplified and used for the next round of panning. A total of three rounds of panning were performed. In each round, the content of surfactant (Tween-20) in the washing buffer was increased, and the coating concentration of dimethoprim antibody was decreased.

[0011] (2) After panning, phage clones are selected, their DNA sequences are determined, and the sensitivity of different phage peptides is measured. The obtained peptide sequence is the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4. Preferably, the peptide amino acid sequence SEQ ID NO.4 is CNPGWPPIPC. The peptide consists of 10 amino acids and contains a cyclic structure formed by the disulfidation of cysteine ​​residues at both ends;

[0012] (3) An anti-immune complex polypeptide without a phage vector was obtained by solid-phase synthesis. The carbon terminus of the solid-phase synthesized polypeptide was linked to a biotinylated spacer arm (GGGSSK-Biotin) for labeling. The final solid-phase synthesized polypeptide sequence was: CNPGWPPIPCGGGSSK-Biotin.

[0013] The pendimethalin immune complex is a complex formed by the reaction of anti-pendimethalin monoclonal antibody and pendimethalin.

[0014] The anti-dimethoprim monoclonal antibody includes a heavy chain variable region and a light chain variable region.

[0015] The heavy chain variable region has the amino acid sequence of SEQ ID NO.1, and the light chain variable region has the amino acid sequence of SEQ ID NO.2.

[0016] The anti-dimethoprim monoclonal antibody includes the heavy chain constant region and light chain constant region of the murine IgG subtype.

[0017] A second aspect of the invention is to provide a gene encoding the polypeptide that specifically binds to the dimethoprim immune complex, wherein the gene has the nucleotide sequence shown in SEQ ID NO. 5.

[0018] A third aspect of the present invention is to provide a kit comprising the above-described polypeptide that specifically binds to the pendimethalin immune complex.

[0019] A fourth aspect of the present invention is to provide the use of the above-mentioned polypeptide, or gene thereof, which specifically binds to the pendimethalin immune complex, or a kit for the non-diagnostic detection of pendimethalin.

[0020] The application of non-diagnostic testing for pendimethalin includes, but is not limited to, enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay.

[0021] The detection method includes the following steps:

[0022] (1) Prepare anti-immune complex peptide probes using the above-mentioned peptides;

[0023] (2) Immunochromatographic test strips were assembled using anti-pentazine monoclonal antibody and anti-streptavidin polyclonal antibody;

[0024] (3) Mix the sample to be tested and the anti-immune complex polypeptide probe solution, and drop it onto the immunochromatographic test strip. Observe the results after 10 minutes. If two bands appear, the result is positive. If only the C line appears, the result is negative.

[0025] The present invention has the following beneficial effects:

[0026] (1) The first report of a polypeptide that can specifically bind to the pendimethalin antibody immune complex;

[0027] (2) Using the polypeptide provided by the present invention, a non-competitive immunoassay method for pendimethalin can be established;

[0028] (3) Half-saturation concentration (SC) of pendimethalin based on anti-immune complex peptide non-competitive enzyme-linked immunosorbent assay. 50 The sensitivity of the new pendimethalin immunochromatographic assay strip is 0.15 ng / mL, which is 3.5 times higher than the previously reported sensitivity of pendimethalin enzyme-linked immunosorbent assay. The limit of detection of the new pendimethalin immunochromatographic assay strip based on anti-immune complex peptides is 2.5 ng / mL, which is 4 times higher than the limit of detection of existing pendimethalin immunochromatographic assay strips. The cross-reactivity of the new pendimethalin immunochromatographic assay strip based on anti-immune complex peptides is less than 0.25%, and the reaction specificity is higher than that of existing technologies. Attached Figure Description

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0030] Figure 1 The ELISA results are used to verify the ability of the phage clones obtained in the third round of screening to specifically recognize the pendimethalin immune complex. The horizontal axis represents the sequence number of the phage clone, and the vertical axis represents the absorbance value.

[0031] Figure 2 The standard curve for a non-competitive ELISA based on anti-immune complex peptides for the detection of pendimethalin is shown. The x-axis represents the pendimethalin concentration (ng / mL), and the y-axis represents the absorbance at 450 nm.

[0032] Figure 3 This is a structural diagram of a non-competitive colloidal gold test strip for pendimethalin. In the diagram, 1: sample pad, 2: T line, 3: C line, 4: NC membrane, 5: absorbent pad, 6: PVC base plate.

[0033] Figure 4 This diagram illustrates the interpretation of results from a non-competitive colloidal gold test strip for pendimethalin. In the diagram, A represents the result of a negative sample, and B and C represent the results of positive samples. Detailed Implementation

[0034] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments, but this is not intended to limit the invention. Specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and not intended to limit the invention. Materials of the same or similar type, model, quality, properties, or functions as the reagents and instruments described below can be used to implement the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0035] Example 1: Panning of anti-pentazine immune complex peptides

[0036] Step 1) The pendimethalin monoclonal antibody 6D6 was preserved in our laboratory. The preparation method is described in Chinese Patent ZL2021112980049. The antibody heavy chain variable region sequence is DVKLVESGGGLVKPGGSLKLSCAASGFTFSSYTLSWVRQTPEKRLEWVATISGGGSY IYYVDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMFYCTRDALGLNYYGTPWLIT N, SEQ ID NO.1; the light chain variable region sequence is QIVLTQSPAIMSVSPGEKVTITCSASSSVSYMFWFQQKPGTSPKLWIHDTSNLASGVP GRFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPPTFGGGTTLEIR, SEQ ID NO.2; the antibody constant region is the heavy chain constant region and light chain constant region derived from the mouse IgG subtype. Anti-dimethoprim monoclonal antibody 6D6 was diluted to 10 μg / mL with phosphate buffered saline (PBS, pH=7.4), and 100 μL was added to each well of a 96-well microplate. The plates were coated at 4°C for 12 hours.

[0037] Step 2) Wash the coated wells five times with PBS containing 1‰ Tween-20 (PBST), then add 300μL of 5% skim milk powder to each well and seal for 1.5 hours, then wash five times with 1‰ PBST.

[0038] Step 3) Add 100 μL of 5 μg / mL pendimethalin standard to each well, incubate at 25°C and 300 rpm for 1 hour, and wash 5 times with 1‰ PBST.

[0039] Step 4) Add phage display to each well to display a random circular heptapeptide library (1×10⁻⁶ each) containing heptapeptides, octapeptides, nitopeptides, and decapeptides. 11 Incubate at 25°C and 300 rpm for 1 hour (PFU).

[0040] Step 5) Add 100 μL of elution solution (0.2 M glycine-hydrochloric acid solution containing 10% BSA, pH=2.2) to each well and incubate at 25°C and 300 rpm for 15 minutes.

[0041] Step 6) Add 15 μL of neutralization solution (1 M Tris-HCl, pH = 9.1) to each well, mix well, collect the supernatant, take 1 μL to determine the phage titer, and store the rest at 4℃.

[0042] The method for determining bacteriophage titers is as follows:

[0043] Dilute the phage-containing supernatant to 10⁻⁶. 6 10 8 and 10 10Take 10 μL of each and add it to 100 μL of ER2738 Escherichia coli in the logarithmic growth phase, and infect at 37°C for 30 minutes. Spread the bacterial suspension on LB agar containing 20 μg / mL tetracycline and incubate at 37°C for 12 hours. Calculate the phage titer based on the number of colonies on the plate: Phage titer (pfu / mL) = 100 × dilution factor × colony count.

[0044] Step 7) Add the supernatant collected in Step 6) to 3 mL of ER2738 Escherichia coli in the logarithmic growth phase, infect at 37°C for 1 hour, expand the culture with 50 mL of LB liquid medium, and culture at 37°C and 250 rpm until the logarithmic growth phase. Add helper phage M13KO7 with an infection multiplicity (helper phage number / E. coli number) > 20, and infect at 37°C for 1 hour.

[0045] Step 8) Centrifuge the bacterial culture, collect the precipitate and add it to 100 mL of 2×YT medium containing 25 μg / mL IPTG. Incubate at 37°C and 250 rpm for 12 hours.

[0046] Step 9) After centrifugation, collect the supernatant and add it to 25 mL of 20% PEG2000 and 2.5 M NaCl solution. Let it stand on ice for 4 hours.

[0047] Step 10) Centrifuge at 14000g, 4℃ for 30 minutes, discard the supernatant, resuspend the precipitate in sterile PBS, add an equal volume of glycerol, determine the titer, and store at -20℃.

[0048] Steps 11) and 10) constitute the first round of panning. The second and third rounds of panning are the same as the first round, but the coating concentrations of pendimethalin monoclonal antibody are 5 and 2.5 μg / mL, respectively, and the content of Tween-20 in PBST is 3‰ and 5‰, respectively. The purpose is to obtain anti-immune complex peptides with stronger affinity.

[0049] Step 12) Pick 30 clones from the plates used in the third round of titer determination and transfer them to 1 mL of 2×YT medium containing 50 μg / mL ampicillin and 20 μg / mL tetracycline. Incubate at 37°C and 300 rpm for 4 hours. Store 500 μL of the culture. Add helper phage M13KO7 (multiple infection number of helper phages / E. coli number > 20) to the remaining culture and incubate at 37°C for 1 hour. Add 2 mL of 2×YT medium containing 50 μg / mL ampicillin, 200 μg / mL kanamycin, and 0.5 μg / mL IPTG to the incubated culture. Incubate at 37°C and 300 rpm for 12 hours. Centrifuge and collect the supernatant.

[0050] Step 13) Dilute the pendimethalin standard to 2.5 μg / mL with PBS containing 5% skim milk powder. Add 50 μL of the diluent and 50 μL of the supernatant collected in Step 12) to the wells of an ELISA plate coated with 2.5 μg / mL pendimethalin monoclonal antibody. At the same time, set 50 μL of PBS containing 5% skim milk powder and 50 μL of supernatant for each clone as a control. Incubate at 37°C for 1 hour.

[0051] Step 14) Wash 5 times with 0.5‰ PBST, add 100 μL of HRP-labeled anti-M13 antibody to each well, and incubate at 37°C for 1 hour.

[0052] Step 15) Wash 15 times with 0.5‰ PBST, add 100 μL of TMB colorimetric solution to each well, and react for 15 minutes.

[0053] Step 16) Add 50 μL of 2M H2SO4 to each well to stop the reaction, and measure the absorbance at 450 nm using a microplate reader. The results are shown in the appendix. Figure 1 .

[0054] Step 17) Sequencing was performed on the bacterial culture preserved in step 12) corresponding to the positive clone, using primers 5′-TAGTCCTCAAAGCCTCTGTA-3′. The nucleotide sequence encoding the polypeptide sequence of SEQ ID NO.4 is shown in SEQ ID NO.5.

[0055] The sequencing results are as follows:

[0056] Table 1. Peptide sequences of anti-pentazocine immune complexes

[0057]

[0058] Example 2: Determination of pendimethalin by non-competitive ELISA

[0059] Step 1) Determination of the optimal working concentration: Under different combinations of antibody coating concentration and phage dosage, add 1 μg / mL pendimethalin standard (positive) or 5% skim milk powder (negative), compare the signal-to-noise ratio (positive signal / negative signal), and select the combination with the highest signal-to-noise ratio for subsequent experiments.

[0060] Step 2) Coating: Dilute the pendimethalin monoclonal antibody to the optimal coating concentration, add 100 μL to each well of the enzyme-labeled well, and incubate at 4°C for 12 hours.

[0061] Step 3) Blocking: After washing with 1‰ PBST, add 300 μL of 5% skim milk powder to each well and incubate at 37°C for 2 hours.

[0062] Step 4) Add phage peptide and pendimethalin standard: After washing with 1‰ PBST, add 50 μL of pendimethalin standard at a certain concentration and 50 μL of the optimal amount of phage peptide to each well, and incubate at 37°C for 1 hour.

[0063] Step 5) is the same as steps 14)-16 in Example 1.

[0064] Step 6) Plotting the concentration of pendimethalin standard on the x-axis and absorbance on the y-axis, fit the nonlinear equation using the logistic function in Origin 2021 software to obtain the half-saturation concentration (the concentration of analyte required to reach half of the maximum signal, SC) of different peptides. 50 The results are shown in Table 2:

[0065] Table 2. Optimal antibody and phage concentration combinations and SCs for different anti-immune complex peptides 50

[0066]

[0067] The results showed that the SC of the non-competitive ELISA based on the peptide CNPGWPPIPC (SEQ ID NO.4) was... 50 The concentration was 0.15 ng / mL, and the standard curve is attached. Figure 2 The sensitivity of this anti-pentetrimazole immunocomplex peptide exceeds that of all currently reported pendimethalin immunoassay methods. Therefore, the prepared anti-pentetrimazole immunocomplex peptide has significant application value in pendimethalin immunoassay and can be used to develop highly sensitive pendimethalin detection methods.

[0068] Example 3: Chemical Synthesis and Identification of Phage-Free Polypeptides

[0069] Based on the amino acid sequence of the anti-immune complex peptide displayed by pendimethalin phage (CNPGWPPIPC), a GGGSSK was added as a spacer arm, and a biotin was added to the end for labeling. The final synthesized phage-free peptide structure is CNPGWPPIPCGGGSSK-biotin. Following the above design, Shanghai ChuTai Biotechnology Co., Ltd. was commissioned to prepare the phage-free anti-immune complex peptide.

[0070] Example 4: Application of phage-free anti-immune complex peptides on immunochromatographic test strips

[0071] Preparation and identification of gold nanoparticles: 100 mL of ultrapure water containing 1% HAuCl4 was heated to boiling, and then 3 mL of 1% sodium citrate solution was rapidly added under vigorous stirring. When the solution color changed from dark blue to wine red, heating and stirring continued for another 5 minutes. After the reaction was complete, the solution was allowed to cool naturally to 25°C for later use or storage.

[0072] Preparation of anti-immune complex peptide probe: The pH of the gold nanoparticle solution was adjusted to 6.0 with 0.1 mol / L potassium carbonate. 10 μg / mL streptavidin was added, and the reaction was carried out with shaking at 25°C for one hour. The gold nanoparticles were then blocked with 1% bovine serum albumin (BSA) for one hour. Next, 10 μg / mL of phage-free anti-immune complex peptide was added to the solution, and the reaction was carried out for one hour. The unbound reagent was removed by centrifugation at 8000 rpm for 15 minutes. The precipitate was resuspended in 0.01 mol / L borate solution (pH = 8.0) and stored at 4°C.

[0073] Immunochromatographic test strip assembly: Anti-pentazocine monoclonal antibody and anti-streptavidin polyclonal antibody were diluted with PBS to 4 mg / mL and 1 mg / mL, respectively, and sprayed onto the NC membrane at a rate of 1 μL / cm using a membrane scrubbing device to serve as the T and C lines. After drying at 37°C for 2 hours, the NC membrane was attached to the center of a PVC base plate, with sample pads and absorbent pads attached to both ends (see attached sample pad). Figure 3 Using a strip cutter, cut the assembled cards into 4mm wide test strips, and store them in a sealed bag to dry.

[0074] Sensitivity determination of non-competitive immunochromatographic test strips: Dilute pendimethalin standard with PBS containing 5% acetonitrile to prepare standard solutions of 20, 10, 5, 2.5, and 1.25 ng / mL. Mix 100 μL of the standard solution with 4 μL of the anti-immune complex peptide probe solution, and add the mixture to the sample pad. Observe the results after 10 minutes. The appearance of two bands indicates a positive result, while the appearance of only the C line indicates a negative result (e.g., ...). Figure 4 (As shown). The results showed that when the concentration of pendimethalin was below 2.5 ng / mL, the result was negative, while when the concentration was greater than or equal to 2.5 ng / mL, the result was positive. The results indicate that the visual limit of detection (vLOD), i.e. the sensitivity, of the immunochromatographic test strip based on phage-free anti-immune complex peptide is 2.5 ng / mL, which is higher than the sensitivity of the pendimethalin test strips reported to date.

[0075] Specificity determination of non-competitive immunochromatographic test strips: Dimethoprim-like compounds (terbuprofen, fluroxypyr, amfluroxypyr, fluroxypyr, and butyrazopyr) were diluted with PBS containing 5% acetonitrile to prepare standard solutions of a certain concentration. The test strips were used to measure the visual detection lines of these compounds. The cross-reactivity rate (%) was calculated according to the formula: cross-reactivity rate (%) = vLOD(dimethoprim) / vLOD(dimethoprim-like compounds) × 100. The cross-reactivity rates of the five dimethoprim-like compounds are shown in Table 3.

[0076] Table 3. Cross-reactivity of non-competitive immunochromatographic test strips to five pendimethalin structural analogues

[0077]

[0078]

[0079] Example 5: Detection of spiked samples by anti-immune complex polypeptide test strips

[0080] Sample preparation and pretreatment: Soil, potato, cabbage, and lettuce blank samples were homogenized and accurately weighed (1 g each). Pendimethalin standard solution was added to the samples to final concentrations of 25, 50, 100, and 200 ng / g, respectively. After standing overnight, 2 mL of PBS containing 40% acetonitrile was added to the samples, vortexed for 5 minutes, sonicated for 15 minutes, and centrifuged at 4000 rpm for 5 minutes. The supernatant was diluted eight-fold to eliminate matrix influence. 100 μL of sample dilution and 4 μL of anti-immune complex peptide probe solution were mixed and added dropwise to the sample pad. The results were observed after 10 minutes. The results are shown in Table 4.

[0081] Table 4. Results of non-competitive immunochromatographic test strips for detecting added samples.

[0082]

[0083]

[0084] a: "-" indicates a negative result;

[0085] b: "+" indicates a positive result.

[0086] The results showed that the non-competitive immunochromatographic test strips had good accuracy in detecting pendimethalin in soil, potato, cabbage and lettuce samples.

[0087] This invention provides a polypeptide that specifically binds to pendimethalin immune complexes, along with its application ideas and methods. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A polypeptide that specifically binds to pendimethalin immune complexes, characterized in that, The polypeptide has the amino acid sequence shown in SEQ ID NO. 4, and the cysteine ​​residues at both ends of the polypeptide are cyclized by forming disulfide bonds.

2. The polypeptide according to claim 1, characterized in that, The aforementioned pendimethalin immune complex is a complex formed by the reaction of anti-pendimethalin monoclonal antibody and pendimethalin.

3. The polypeptide according to claim 2, characterized in that, The anti-dimethoprim monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO. 1 and the light chain variable region has the amino acid sequence of SEQ ID NO.

2.

4. The polypeptide according to claim 3, characterized in that, The anti-dimethoprim monoclonal antibody includes the heavy chain constant region and light chain constant region of the murine IgG subtype.

5. A gene encoding the polypeptide of claim 1.

6. The gene according to claim 5, characterized in that, The gene described herein has the nucleotide sequence shown in SEQ ID NO.

5.

7. A reagent kit, characterized in that, Includes the polypeptide described in claim 1.

8. The use of the polypeptide of claim 1, or the gene of claim 5 or 6, or the kit of claim 7 in the non-diagnostic detection of pendimethalin, wherein the use further comprises an anti-pendimethalin monoclonal antibody, the anti-pendimethalin monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region has the amino acid sequence of SEQ ID NO. 1, and the light chain variable region has the amino acid sequence of SEQ ID NO.

2.

9. The application according to claim 8, characterized in that, The detection method includes the following steps: (1) Using the polypeptide of claim 1 to prepare an anti-immune complex polypeptide probe; (2) Immunochromatographic test strips were assembled using anti-pentazine monoclonal antibody and anti-streptavidin polyclonal antibody; (3) Mix the sample to be tested and the anti-immune complex polypeptide probe solution, add it to the immunochromatographic test strip, and observe the results.

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