Monoclonal antibody of alpha, beta, gamma-amanitin and its preparation method and application

Abstract

The application relates to a monoclonal antibody of alpha, beta and gamma amatoxin and a preparation method and application thereof. The monoclonal antibody of alpha amatoxin is prepared from an alpha amatoxin artificial antigen, the monoclonal antibody of beta amatoxin is prepared from a beta amatoxin artificial antigen, and the monoclonal antibody of gamma amatoxin is prepared from a gamma amatoxin artificial antigen; the alpha amatoxin artificial antigen is a conjugate of alpha amatoxin and keyhole limpet hemocyanin, the beta amatoxin artificial antigen is a conjugate of beta amatoxin and bovine serum albumin, and the gamma amatoxin artificial antigen is a conjugate of gamma amatoxin and human serum albumin. The three antibodies have a small cross-reaction rate, can simultaneously detect and distinguish alpha-AMA, beta-AMA and gamma-AMA in a sample, and have high accuracy.

Description

Technical Field

[0001] This application relates to the field of food safety testing technology, and in particular to monoclonal antibodies against α, β, and γ-amanita peptides, their preparation methods, and applications. Background Technology

[0002] Southern my country boasts abundant edible wild mushroom resources due to its terrain and climate. However, because some poisonous mushrooms lack clear morphological differences from edible wild mushrooms, and there is a lack of rapid and reliable methods for identifying poisonous mushrooms, deaths from poisoning due to accidental ingestion of poisonous mushrooms occur frequently. Therefore, mushroom poisoning has always been one of the key food safety issues of concern in my country.

[0003] Statistics show that up to 70% of deaths from mushroom poisoning are caused by ingesting the highly poisonous Amanita mushroom. The lethal toxins of Amanita mushrooms are amatoxins, which can be classified into three categories based on their amino acid composition and structure: amatoxins, phalloidin, and death cap peptides. Among these, amatoxins are bicyclic octapeptides, chemically very stable, and their toxicity cannot be destroyed by ordinary cooking methods. Therefore, the detection of amatoxins is particularly important.

[0004] Currently, the main methods for detecting amatoxins include instrumental analysis and immunological detection. Instrumental analysis methods primarily include high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS / MS), which have advantages such as high sensitivity, low detection limit, and good repeatability. However, these methods also suffer from problems such as long detection time, cumbersome operation, and expensive equipment, limiting their application. Immunological detection methods are mainly based on the principle of specific reaction between antigens and antibodies. They are rapid, simple to operate, and intuitive, making them suitable for preliminary on-site detection.

[0005] When using test strips to detect amatoxins, a single strip typically detects only one type of amatoxin and cannot effectively distinguish between different types. This is because the antibodies have low specificity and exhibit a high cross-reactivity rate. Currently, nine types of amatoxins have been isolated and identified, among which α-amatoxin (α-AMA), β-amatoxin (β-AMA), and γ-amatoxin (γ-AMA) are the main toxins causing death. However, detecting α-AMA, β-AMA, and γ-AMA separately is time-consuming and cumbersome, resulting in low detection efficiency. Therefore, the ability to quickly detect amatoxins and accurately determine their types is crucial for subsequent treatment. Summary of the Invention

[0006] To address the aforementioned technical problems, this application provides monoclonal antibodies against α, β, and γ-amanita peptides, their preparation methods, and applications. The monoclonal antibodies against α-amanita peptides, β-amanita peptides, and γ-amanita peptides of this application exhibit low cross-reactivity rates, enabling simultaneous detection of α-AMA, β-AMA, and γ-AMA. This approach offers advantages such as high detection efficiency, high sensitivity, and low cross-reactivity rates.

[0007] In a first aspect, this application provides a monoclonal antibody against α, β, γ-amanita peptides, employing the following technical solution:

[0008] A monoclonal antibody against α, β, and γ-amatoxins, wherein the monoclonal antibody against α-amatoxins is prepared from an artificial antigen of α-amatoxins, the monoclonal antibody against β-amatoxins is prepared from an artificial antigen of β-amatoxins, and the monoclonal antibody against γ-amatoxins is prepared from an artificial antigen of γ-amatoxins.

[0009] The α-amanitin artificial antigen is a conjugate of α-amanitin and keyhole hemocyanin; the β-amanitin artificial antigen is a conjugate of β-amanitin and bovine serum albumin; and the γ-amanitin artificial antigen is a conjugate of γ-amanitin and human serum albumin.

[0010] Using the above-mentioned technical solutions, the conjugate α-Amanita peptides (AMP) and keyhole hemocyanin are conjugates to obtain α-AMA-KLH artificial antigen; the conjugate β-AMP and bovine serum albumin is conjugate β-AMA-HSA artificial antigen; and the conjugate γ-AMP and human serum albumin is conjugate γ-AMA-HAS artificial antigen. After immunizing animals with the obtained artificial antigens, hybridoma cell lines are prepared, and the corresponding antibodies are secreted by the hybridoma cell lines. Testing shows that α-AMA antibodies are effective against... The cross-reactivity rates of β-AMA and γ-AMA are less than 0.2%; the cross-reactivity rates of β-AMA antibody with α-AMA and γ-AMA are less than 0.2%; and the cross-reactivity rates of γ-AMA antibody with α-AMA and β-AMA are less than 0.2%. This indicates that different artificial antigens, through the selection of specific carrier proteins, result in three antibodies with low cross-reactivity rates. Using the three antibodies of this application, α-AMA, β-AMA, and γ-AMA in a sample can be detected and distinguished simultaneously with high accuracy.

[0011] Optionally, the α-amanita peptide artificial antigen is obtained by coupling the amino groups on α-amanita peptide and keyhole hemocyanin with glutaraldehyde.

[0012] By adopting the above technical solution, the action of glutaraldehyde can ensure that α-amanita peptide is not affected by the steric hindrance of protein macromolecules, and can better expose α-amanita peptide to generate an immune response, thereby improving the immunogenicity of the antigen. The monoclonal antibodies prepared in this way have a lower cross-reactivity rate, which can improve the accuracy of sample detection.

[0013] Optionally, the α-amanita peptide artificial antigen is prepared using the following method:

[0014] Keyhole hemocyanin was dissolved in PBS buffer solution, glutaraldehyde was added, and the mixture was stirred for 1-3 hours to remove unreacted glutaraldehyde. Then, α-amanita peptide was added, and the mixture was stirred for 1-3 hours to remove unreacted small molecules and impurities, thus obtaining α-amanita peptide artificial antigen.

[0015] Optionally, the β-amanita peptide artificial antigen is obtained by activating the carboxyl group on the β-amanita peptide with EDC and NHS, and then binding it with the amino group on bovine serum albumin.

[0016] Optionally, the β-amanita peptide artificial antigen is prepared using the following method:

[0017] MES buffer was added to β-amatoxins, EDC, and NHS respectively to prepare β-amatoxins, EDC, and NHS solutions.

[0018] EDC solution and NHS solution were added to β-amatoxins solution and reacted at 0-4℃ in the dark for 14-18 hours to obtain activated β-amatoxins solution.

[0019] Bovine serum albumin was prepared by adding borate buffer to bovine serum albumin solution; activated β-amatoxins solution was added to bovine serum albumin solution and reacted at 0-4℃ for 14-18h. Unreacted small molecules and impurities were removed to obtain β-amatoxins artificial antigen.

[0020] Optionally, the γ-amanita peptide artificial antigen is obtained by coupling the amino group on γ-amanita peptide and the amino group on human serum albumin with glutaraldehyde.

[0021] Secondly, this application provides hybridoma cell lines that secrete monoclonal antibodies against α, β, and γ-amanita peptides.

[0022] A hybridoma cell line secreting a monoclonal antibody against α-amanita peptide, named AMA1, was deposited on October 21, 2022, at the China Center for Type Culture Collection (CCTCC); deposit address: Wuhan University, Wuhan, China; accession number: CCTCC NO: C2022299.

[0023] A hybridoma cell line secreting a monoclonal antibody against β-amanita peptide, named AMA2, was deposited on October 21, 2022, at the China Center for Type Culture Collection (CCTCC); deposit address: Wuhan University, Wuhan, China; accession number: CCTCC NO: C2022328.

[0024] A hybridoma cell line secreting a monoclonal antibody against γ-amanita peptide, named AMA3, was deposited on October 21, 2022, at the China Center for Type Culture Collection (CCTCC); deposit address: Wuhan University, Wuhan, China; accession number: CCTCC NO: C2022329.

[0025] Thirdly, this application provides a method for preparing monoclonal antibodies against α, β, and γ-amanita peptides, employing the following technical solution:

[0026] The method for preparing monoclonal antibodies against α, β, and γ-amatoxins includes the following steps:

[0027] Healthy mice were immunized with artificial antigens of α-amanita peptide, β-amanita peptide, and γ-amanita peptide, respectively. Spleen cells and myeloma cells from the immunized mice were fused, and positive hybridoma cell lines were screened. After antibody production and purification, monoclonal antibodies against α-amanita, β-amanita peptide, and γ-amanita peptide were obtained, respectively.

[0028] Fourthly, this application provides an application of a monoclonal antibody against α, β, γ-amanita peptides, employing the following technical solution:

[0029] Application of monoclonal antibodies against α, β, and γ-amatoxins, wherein the monoclonal antibodies against α-amatoxins, β-amatoxins, and γ-amatoxins are used to prepare test strips or kits for detecting α-amatoxins, β-amatoxins, and γ-amatoxins.

[0030] By adopting the above technical solution, the test strips and kits can be used for the rapid detection of α-amatoxins, β-amatoxins, and γ-amatoxins. The test strips have the advantages of simple operation and short detection time.

[0031] Optionally, the test strip includes a base plate and a sample pad, a conjugate pad, a reaction membrane, and an absorbent pad connected sequentially along the chromatography direction on the base plate;

[0032] The reaction membrane is provided with a T3 detection line for detecting γ-amatoxins, a T2 detection line for detecting β-amatoxins, a T1 detection line for detecting α-amatoxins, and a control line in sequence along the chromatography direction.

[0033] The T1 detection line is coated with α-amatoxins artificial antigen, the T2 detection line is coated with β-amatoxins artificial antigen, and the T3 detection line is coated with γ-amatoxins artificial antigen.

[0034] The binding pad is coated with monoclonal antibodies against α-amanita peptide, β-amanita peptide, and γ-amanita peptide.

[0035] By adopting the above technical solution, since the monoclonal antibodies against α-amanita peptide, β-amanita peptide, and γ-amanita peptide of this application have a low cross-reactivity rate, the test strip of this application can be used to simultaneously detect α-amanita peptide, β-amanita peptide, and γ-amanita peptide in a sample. It has the advantages of simple operation, high detection efficiency, and high detection accuracy.

[0036] Optionally, the test strip is a colloidal gold immunochromatographic test strip or a fluorescent microsphere immunochromatographic test strip.

[0037] By adopting the above technical solutions, and after testing, the sensitivity of the colloidal gold immunochromatographic test strip of this application for α-amanita peptide is 1.8 ppb, for β-amanita peptide is 1.1 ppb, and for γ-amanita peptide is 2.1 ppb; the sensitivity of the fluorescent microsphere immunochromatographic test strip for α-amanita peptide is 0.5 ppb, for β-amanita peptide is 0.2 ppb, and for γ-amanita peptide is 0.6 ppb.

[0038] In summary, this application has the following beneficial effects:

[0039] 1. The three antibodies in this application have a small cross-reactivity rate. When using the three antibodies in this application to simultaneously detect and distinguish α-AMA, β-AMA and γ-AMA in a sample, it has the advantages of high detection efficiency and high detection accuracy.

[0040] 2. The colloidal gold immunochromatographic test strip showed a sensitivity of 1.8 ppb for α-amanita peptide, 1.1 ppb for β-amanita peptide, and 2.1 ppb for γ-amanita peptide; the fluorescent microsphere immunochromatographic test strip showed a sensitivity of 0.5 ppb for α-amanita peptide, 0.2 ppb for β-amanita peptide, and 0.6 ppb for γ-amanita peptide. This demonstrates that the test strip of this application has the advantage of high sensitivity. Detailed Implementation

[0041] The present application will be further described below with reference to embodiments.

[0042] Example

[0043] Unless otherwise specified, all raw materials used in the examples are commercially available. α-AMA, β-AMA, and γ-AMA were purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.; the carrier protein keyhole hemocyanin (KLH) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; bovine serum albumin (BSA), human serum albumin (HSA), and chicken ovalbumin (OVA) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the PBS buffer used in the examples was a 10mM buffer with a pH of 7.4; EDC is an abbreviation for 1-ethyl-(3-dimethylaminopropyl)carbodiimide; NHS is an abbreviation for N-hydroxysuccinimide.

[0044] Example 1: Preparation of Artificial Antigen from Amatoxin Peptide

[0045] 1. Preparation of α-AMA-KLH (α-Amatoxins-Keypore Hemocyanin) antigen

[0046] (1) Dissolve 1 mg of carrier protein KLH in 1 mL of PBS buffer, add 10 μL of glutaraldehyde, stir at room temperature for 2 h to obtain KLH solution.

[0047] (2) Dialysis to remove unreacted glutaraldehyde from the KLH solution.

[0048] (3) Add 0.5 mg of α-AMA to the dialyzed KLH solution and stir at room temperature for 2 h to obtain a mixture.

[0049] (4) Remove unreacted α-AMA from the mixture by dialysis with PBS to obtain α-AMA-KLH.

[0050] 2. Preparation of β-AMA-BSA (β-Amanita-Bovine Serum Albumin) antigen

[0051] (1) Add pH 5.0 and 50mM MES buffer to β-AMA, EDC and NHS to prepare β-AMA solution, EDC solution and NHS solution with a concentration of 2mg / mL.

[0052] (2) 0.1 mL of EDC solution and 0.1 mL of NHS solution were added to 0.5 mL of β-AMA solution and reacted at 4 °C in the dark for 16 h to obtain activated β-AMA solution.

[0053] (3) Add 20mM borate buffer solution at pH 8.0 to BSA to prepare a 1mL BSA solution with a concentration of 5mg / mL.

[0054] (4) Add the activated β-AMA solution from step (2) to the BSA solution from step (3), stir at 4°C for 16 h to obtain a mixture.

[0055] (5) Dialyze the mixture with PBS to remove unreacted small molecules and impurities to obtain β-AMA-BSA antigen.

[0056] 3. Preparation of γ-AMA-HAS (γ-Amatoxins-Human Serum Albumin) antigen

[0057] (1) Dissolve 1 mg of carrier protein HSA in 1 mL of PBS buffer, add 10 μL of glutaraldehyde, stir at room temperature for 2 h to obtain HAS solution;

[0058] (2) Remove unreacted glutaraldehyde from the HAS solution by dialysis;

[0059] (3) Add 0.5 mg of γ-AMA to the dialyzed HAS solution and stir at room temperature for 2 h.

[0060] (4) Remove unreacted γ-AMA by dialysis with PBS to obtain γ-AMA-HSA antigen.

[0061] 4. Preparation of α-AMA-OVA antigen, β-AMA-OVA antigen, and γ-AMA-OVA antigen: α-AMA, β-AMA, and γ-AMA were conjugated with the carrier chicken ovalbumin (OVA) to form antigens α-AMA-OVA, β-AMA-OVA, and γ-AMA-OVA for cell screening and detection.

[0062] (1) Preparation of α-AMA-OVA antigen: The preparation method of α-AMA-OVA antigen is the same as that of α-AMA-KLH antigen, except that KLH is replaced with an equal amount of OVA.

[0063] (2) Preparation of β-AMA-OVA antigen: The preparation method of β-AMA-OVA antigen is the same as that of α-AMA-KLH antigen, except that BSA is replaced with an equal amount of OVA.

[0064] (3) Preparation of γ-AMA-OVA antigen: The preparation method of γ-AMA-OVA antigen is the same as that of α-AMA-KLH antigen, except that HAS is replaced with an equal amount of OVA.

[0065] Example 2: Preparation of Amatoxin Monoclonal Antibody

[0066] Monoclonal antibodies were prepared using the α-AMA-KLH, β-AMA-BSA, and γ-AMA-HSA antigens prepared in Example 1 as specific antigens. The preparation methods were basically the same, except that β-AMA-OVA and γ-AMA-OVA were used to detect cell supernatant during clone formation and screening, and β-AMA and γ-AMA standards were used for cell supernatant inhibition assays. The hybridoma cell line secreting α-AMA was named AMA1; the hybridoma cell line secreting β-AMA was named AMA2; and the hybridoma cell line secreting γ-AMA was named AMA3.

[0067] The following explanation uses the preparation of α-AMA monoclonal antibody as an example.

[0068] 1. Animal immunization

[0069] Healthy BALB / c mice aged 6-8 weeks were selected for immunization. The first immunization involved emulsifying 100 μg of α-AMA-KLH antigen with an equal volume of Freund's complete adjuvant and administering it subcutaneously at multiple sites. A second immunization was performed 28 days later, with the dose halved. This involved emulsifying 50 μg of antigen with an equal volume of Freund's incomplete adjuvant and administering it subcutaneously at multiple sites. A third immunization was performed 14 days later, with the dose again halved. This involved emulsifying 25 μg of antigen with an equal volume of Freund's incomplete adjuvant and administering it intramuscularly in the thigh. One week after the third immunization, tail blood was collected for titer determination. If the titer reached 1:10000 or higher, a pulse immunization was administered 14 days after the third immunization. The pulse immunization dose was 25 μg, and no adjuvant was used. The antigen was diluted with physiological saline and injected intraperitoneally into the mice. Fusion was performed 3 days after the pulse immunization.

[0070] 2. Cell fusion

[0071] (1) Preparation of SP2 / 0 (mouse myeloma cells): SP2 / 0 cells were thawed and passaged to ensure they were in the logarithmic growth phase when used. SP2 / 0 cells were collected and suspended in DMEM culture medium for cell counting.

[0072] (2) Spleen cell preparation: After the mice were immunized, the spleen was taken under sterile conditions, ground through a 70μm sieve, and the red blood cells were lysed with red blood cell lysis buffer to prepare a single cell suspension for cell counting.

[0073] (3) Mix SP2 / 0 cells and spleen cells at a ratio of 1:4, wash twice with electrofusion buffer, and finally resuspend the cell mixture with electrofusion buffer to adjust the cell density to 1×10⁶ cells / year. 7 Add it to the fusion pool, set the parameters of the electrofusion instrument, and perform electrofusion.

[0074] (4) After fusion, the cells were resuspended in HAT complete medium containing 20% ​​FBS (fetal bovine serum) and plated.

[0075] 3. Clonal Formation and Screening

[0076] (1) After fusion for 3 days, the medium was changed with HT medium containing 20% ​​FBS. ELISA screening was performed 7 days after the medium change.

[0077] (2) The α-AMA-OVA antigen was coated on an ELISA plate, and the cell supernatant was tested. Positive clones were transferred into 24-well plates and cultured for 3 days before retesting. The cell supernatant inhibition assay was performed using α-AMA standard, and related cross-reactivity was detected. Cell lines that inhibited 1 ng / mL α-AMA but did not inhibit 50 ng / mL β-AMA and 50 ng / mL γ-AMA were screened for subcloning. Subcloning was performed twice consecutively to obtain a positive clone cell line with inhibition. This cell line was named AMA1, and the monoclonal antibody secreted by it was α-AMA-Ab.

[0078] 4. Production of monoclonal antibodies

[0079] Positive clone cell lines were inoculated at a density of 0.5 × 10⁻⁶. 6 Inoculate 200 mL of the culture medium and perform serum-free rolling bottle culture acclimatization. Set the rolling bottle culture incubator speed to 12 r / min and CO2 concentration to 5%. After 7 days of culture, collect the cell supernatant for affinity purification.

[0080] 5. Antibody purification

[0081] (1) Remove the sample to be purified, the protein G purification column, the equilibration buffer (pH 7.4, 10 mM PBS), the elution buffer (pH 3.0, 0.1 M glycine buffer), and the neutralization buffer (pH 9.0, 1 M Tris-HCl buffer) from the refrigerator and allow them to return to room temperature.

[0082] (2) Add 5 mL of equilibration buffer to the purification column and elute the equilibration buffer at a flow rate of about 1 mL / min.

[0083] (3) Load the sample to be purified into the chromatography column at a flow rate of about 1 mL / min.

[0084] (4) Wash the purification column with 30 mL of equilibration buffer at a flow rate of about 2 mL / min until the A280 absorbance of the effluent stabilizes.

[0085] (5) Elute the antibody with 15 mL of elution buffer at a flow rate of about 1 mL / min, collect the eluent containing the target immunoglobulin, and immediately add neutralization buffer to adjust the pH to 7.4.

[0086] (6) Dialyze the antibody with phosphate buffer at pH 7.4, identify the antibody subtype using an antibody subtype identification kit, and finally aliquot the antibody and store it at -20℃.

[0087] Antibody performance testing experiment

[0088] 1. The titer of purified antibodies was detected using an indirect enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows:

[0089] (1) Dilute α-AMA-OVA, β-AMA-OVA and γ-AMA-OVA with coating buffer (pH 9.6, 50mM carbonate buffer) to a concentration of 1μg / mL, add 100μL / well to a 96-well microplate for coating, incubate overnight at 4°C and wash 3 times with PBST washing buffer.

[0090] (2) Block with blocking solution (2% skim milk powder / PBS), 200 μL / well, incubate at 37°C for 2 h, and then wash 3 times with washing solution.

[0091] (3) The three purified antibodies were diluted 2000-fold with PBS, and then serially diluted 2-fold to obtain 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000, for a total of 8 gradients. Primary antibody (the antibodies of each concentration gradient) and PBS solution were added to the corresponding wells, 50 μl / well. The wells were incubated at 37°C for 30 min, and then washed three times with washing buffer.

[0092] (4) Add 100 μL of enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) diluted 4000 times with PBS to each well. Incubate at 37°C for 30 min and then wash three times with washing buffer.

[0093] (5) Add colorimetric solution, 100 μL / well, and develop color for 10 min.

[0094] (6) Add stop solution (2M sulfuric acid), 50 μL / well, to terminate the reaction.

[0095] (7) Measure the absorbance at 450 nm using an ELISA reader and record the data in Table 1.

[0096] Table 1 Antibody titer data (OD450)

[0097] dilution 2000 4000 8000 16000 32000 64000 128000 256000 α-AMA Ab 2.865 2.645 2.456 2.016 1.611 1.135 0.532 0.312 β-AMA Ab 2.778 2.713 2.421 2.115 1.596 1.112 0.516 0.331 γ-AMA Ab 2.798 2.649 2.369 1.996 1.557 1.022 0.523 0.309

[0098] As can be seen from the data in Table 1, the titers of all three antibodies can reach 64,000, which meets the requirements for antibody titer.

[0099] 2. The sensitivity and specificity of antibodies were detected using an indirect competitive enzyme-linked immunosorbent assay (ELISA). The specific operating steps are as follows:

[0100] (1) Dilute α-AMA-OVA, β-AMA-OVA and γ-AMA-OVA with coating buffer to a concentration of 1 μg / mL, add 100 μL / well to a 96-well microplate for coating, incubate overnight at 4°C and wash 3 times with PBST washing buffer.

[0101] (2) Block with blocking solution (2% skim milk powder / PBS), 200 μL / well, incubate at 37°C for 2 h, and then wash with washing solution 3 times.

[0102] (3) The purified three antibodies were diluted 64,000 times with PBS. The α-AMA, β-AMA and γ-AMA standards were diluted with PBS to concentrations of 0, 0.05, 0.1, 0.2, 0.4 and 0.8 μg / L, respectively. 50 μl of antibody and standard solutions were added to the corresponding wells, and the wells were incubated at 37°C for 30 min, followed by washing three times with washing buffer.

[0103] (4) Add 100 μL of enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) diluted 4000 times with PBS to each well. Incubate at 37°C for 30 min and then wash three times with washing buffer.

[0104] (5) Add colorimetric solution, 100 μL / well, and the color development time is about 15 min.

[0105] (6) Add stop solution (2M concentrated sulfuric acid), 50 μL / well, to terminate the reaction.

[0106] (7) Measure the absorbance at 450 nm using an ELISA reader and record the data in Table 2.

[0107] Table 2. OD values ​​and B / B0 of AMA standards at different concentrations

[0108] AMA concentration(μg / L) 0 0.05 0.1 0.2 0.4 0.8 α-AMA OD450 1.821 1.71 1.513 0.811 0.471 0.236 B / B0(%) 100％ 94％ 83％ 45％ 26％ 13％ β-AMA OD450 1.775 1.462 1.226 0.681 0.339 0.136 B / B0(%) 100％ 82％ 69％ 38％ 19％ 8％ γ-AMA OD450 1.733 1.691 1.386 0.763 0.456 0.222 B / B0(%) 100％ 98％ 80％ 44％ 26％ 13％

[0109] Note: B / B0 refers to the ratio of the OD value of the test wells of standard samples with different concentrations to the OD value of the test wells of standard samples with a content of 0.

[0110] As shown in Table 2, when the α-AMA content is 0.1 μg / L, its B / B0 value is 83%, and its detection OD is significantly different from that of the test well containing 0 μg / L α-AMA. Therefore, the detection limit of this antibody for α-AMA can reach 0.1 μg / L. Similarly, the detection limits for β-AMA and γ-AMA antibodies are 0.05 μg / L and 0.1 μg / L, respectively. This indicates that the α-AMA antibody, β-AMA antibody, and γ-AMA antibody of this application have excellent detection sensitivity for α-AMA, β-AMA, and γ-AMA, respectively.

[0111] 3. The cross-reactivity rate of the three antibodies was detected using an indirect competitive enzyme-linked immunosorbent assay (ELISA).

[0112] The IC50 values ​​of the three antibodies were tested using α-AMA, β-AMA, and γ-AMA, respectively. 50 The cross-reactivity rate was calculated based on the (half-inhibition concentration) value, and the results are shown in Table 3 below.

[0113] Table 3 IC50 of α-AMA antibody, β-AMA antibody, and γ-AMA antibody 50 value

[0114] <![CDATA[IC 50 ]]> a-AMA β-AMA γ-AMA α-AMA antibody 0.183μg / L >100μg / L >100μg / L β-AMA antibody >100μg / L 0.155μg / L >100μg / L γ-AMA antibody >100μg / L >100μg / L 0.189μg / L

[0115] Based on the data in Table 3, the cross-reactivity rate between antibodies was calculated. The calculation method for the cross-reactivity rate is as follows:

[0116] Cross-reactivity rate = (concentration of target substance causing 50% inhibition / concentration of analog substance causing 50% inhibition) / 100%.

[0117] Calculations showed that the cross-reactivity rate of the α-AMA antibody with β-AMA was 0.183 / 100×100 = 0.182%; the cross-reactivity rate of the α-AMA antibody with γ-AMA was also 0.183 / 100×100 = 0.182%. Therefore, the cross-reactivity rate of the α-AMA antibody with both β-AMA and γ-AMA was less than 0.2%. Similarly, the cross-reactivity rate of the β-AMA antibody with both α-AMA and γ-AMA was less than 0.2%; and the cross-reactivity rate of the γ-AMA antibody with both α-AMA and β-AMA was less than 0.2%. This indicates that the three antibodies possess good specificity.

[0118] In summary, the α-AMA antibody, β-AMA antibody, and γ-AMA antibody of this application all achieve a titer of 64,000. The detection limits of the α-AMA antibody, β-AMA antibody, and γ-AMA antibody are 0.1 μg / L, 0.05 μg / L, and 0.1 μg / L, respectively. The cross-reactivity rates among the α-AMA antibody, β-AMA antibody, and γ-AMA antibody are all less than 0.2%, indicating that the monoclonal antibodies of this application have the advantages of high titer, high sensitivity, and low cross-reactivity rate.

[0119] Application examples

[0120] Application Example 1: A colloidal gold immunochromatographic test strip for detecting α, β, and γ-amatoxins was prepared using the following method:

[0121] 1. Colloidal gold-labeled antibodies

[0122] (1) Preparation of colloidal gold

[0123] Measure 200 mL of ultrapure water and add it to a clean 250 mL Erlenmeyer flask. Place the flask on the heating plate of a magnetic stirrer, add the stir bar, and turn on the stirring knob to start stirring. Use a pipette to add 2 mL of 1% chloroauric acid solution to the 100 mL of ultrapure water. Turn on the heating knob and heat to boiling. Quickly add 1-3 mL of 2% trisodium citrate aqueous solution. The solution will change from gray to black within 2 minutes, and finally turn red. Heat and stir for 10 minutes, then turn off the heating knob and stir until it reaches room temperature. Add ultrapure water to a final volume of 200 mL and store in the dark.

[0124] (2) Preparation of gold-labeled antibodies

[0125] ① Take three clean 1.5mL centrifuge tubes and add 1mL of colloidal gold to each tube.

[0126] ② Add 10 μL of 0.2 M potassium carbonate to each container and mix well.

[0127] ③ Add 10 μg each of α-AMA, β-AMA and γ-AMA antibodies, mix well and react at room temperature for 30 min.

[0128] ④ Add 10 μL of 20% BSA to each container, mix well, and react at room temperature for 20 min.

[0129] ⑤ Centrifuge at 13000 rpm for 30 min, discard the supernatant, and reconstitute the precipitate with 1 / 10 volume of reconstitution solution for later use.

[0130] 2. Test strip assembly

[0131] (1) Cut the nitrocellulose membrane (NC membrane) into 30cm strips, attach them to the PVC board, and equilibrate for 30 minutes at 25℃ and 50% humidity.

[0132] (2) Membrane scratching

[0133] ① Line C: Dilute goat anti-mouse IgG with 10mM, pH 7.4 PBS solution, and spray it onto nitrocellulose membrane using a membrane scrubbing instrument. The line is 5mm away from the absorbent pad. This is the quality control line.

[0134] ②T1 line: Dilute the α-AMA-KLH artificial antigen with 10mM, pH 7.4 PBS solution, apply it to the nitrocellulose membrane using a membrane scrubbing instrument, and place it 9mm away from the absorbent pad. This is the α-AMA detection line.

[0135] ③T2 line: Dilute the β-AMA-BSA artificial antigen with 10mM, pH 7.4 PBS solution, apply it to the nitrocellulose membrane using a membrane scrubbing instrument, and place it 13mm away from the absorbent pad. This is the β-AMA detection line.

[0136] ④T3 line: Dilute the γ-AMA-HSA artificial antigen with 10mM, pH 7.4 PBS solution, apply it to the nitrocellulose membrane using a membrane scrubbing instrument, and place it 17mm away from the absorbent pad. This is the γ-AMA detection line.

[0137] ⑤ Drying: Dry the marked film at 37°C for 24 hours.

[0138] (3) Spraying gold

[0139] ① Gold spraying instrument parameter settings: spray volume 8μl / cm, gold spraying length 295mm;

[0140] ② The antibody-labeled colloidal gold was dried in an oven at 37°C for 24 hours.

[0141] (4) Test strip assembly

[0142] ① Apply absorbent pad: Peel off the film on the PVC board and apply an absorbent pad to the PVC board near the C line of the NC film, pressing the absorbent pad 2mm over the NC film;

[0143] ② Apply colloidal gold pad: Apply colloidal gold pad to the PVC board away from the absorbent pad, and press the colloidal gold pad 2mm over the NC film;

[0144] ③ Apply sample pad: Apply the sample pad to the PVC board near one end of the colloidal gold pad, with the sample pad pressing 2mm over the gold pad;

[0145] ④ Cutting strips: Use a strip cutter to cut the test strips to a width of 3.5mm.

[0146] Application Example 2: A microsphere immunochromatographic test strip for detecting α, β, and γ-amatoxins was prepared using the following method:

[0147] The difference between this application example and Application Example 1 is that the binding pad is a microsphere pad. The microspheres can be carboxylated colored microspheres, time-resolved fluorescent microspheres, or fluorescent quantum dot microspheres. In this example, time-resolved fluorescent microspheres are selected. Specifically, the antibody labeled with the microspheres is prepared using the following method:

[0148] ① Prepare 10 mg / mL NHS solution and 10 mg / mL EDC solution immediately before use:

[0149] Solution A: 10 mg / mL N-hydroxysuccinimide (NHS) / MES solution.

[0150] Solution B: 10 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) / MES solution.

[0151] ② In a 2 mL centrifuge tube, add 100 μL of microspheres, then add 900 μL of 50 mM pH 6.0 MES buffer, and vortex for 5 seconds to mix thoroughly.

[0152] ③ Add 10 μl of solution A to the microsphere suspension, mix for 5 seconds, then add 10 μl of solution B, mix again for 5 seconds, and shake well at room temperature for 60 minutes.

[0153] ④ Centrifuge at 14000 r / min for 30 min, discard the supernatant, add 1 mL of borate buffer, sonicate to disperse evenly, and set aside for later use.

[0154] ⑤ After ultrasonic dispersion of the above 1 mL activated microsphere suspension, add 25 μg of antibody, vortex for 5 seconds to mix evenly, and shake at room temperature for 60 min to react.

[0155] ⑥ Add 100 μL of 10% BSA solution, shake well at room temperature, and block the reaction for 60 min;

[0156] ⑦ Centrifuge at 13000 r / min for 15 min. Add 1 mL of microsphere preservation solution to the centrifuged microspheres, mix well, and store in the refrigerator protected from light until use.

[0157] Performance testing of test strips

[0158] The principle of the test strip in this application is competitive inhibition.

[0159] Using the test strips prepared in Application Examples 1 and 2 as samples, 100 μL of sample was added to the sample application area of ​​the test strip and incubated at room temperature for 10 min. When the T line and C line are the same color intensity, the concentration of this standard is the sensitivity of the test strip. When the T line is darker than the C line, the result is negative; when the T line is lighter than the C line, the result is positive. The lighter the T line, the higher the concentration of AMA in the sample.

[0160] After testing, the colloidal gold immunochromatographic test strip used in Example 1 showed a sensitivity of 1.8 ppb for α-AMA, 1.1 ppb for β-AMA, and 2.1 ppb for γ-AMA.

[0161] The fluorescent microsphere immunochromatographic test strip used in Example 2 has a sensitivity of 0.5 ppb for α-AMA, 0.2 ppb for β-AMA, and 0.6 ppb for γ-AMA.

[0162] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A monoclonal antibody against α-amanita peptide, characterized in that, The artificial antigen of α-amanita peptide is obtained by conjugating the amino group on α-amanita peptide and the amino group on keyhole hemocyanin with glutaraldehyde; the monoclonal antibody of α-amanita peptide is secreted by the hybridoma cell line with accession number CCTCC NO: C2022299.

2. A monoclonal antibody against β-amanita peptide, characterized in that, The artificial antigen of β-amanita peptide is obtained by activating the carboxyl group on β-amanita peptide through EDC and NHS, and then binding it to the amino group on bovine serum albumin; the monoclonal antibody of β-amanita peptide is secreted by the hybridoma cell line with accession number CCTCCNO:C2022328.

3. A monoclonal antibody against γ-amanita peptide, characterized in that, The artificial antigen of γ-amanita peptide is obtained by conjugating the amino group on γ-amanita peptide and the amino group on human serum albumin with glutaraldehyde; the monoclonal antibody of γ-amanita peptide is secreted by the hybridoma cell line with accession number CCTCCNO:C2022329.

4. A hybridoma cell line that secretes the α-amanita peptide monoclonal antibody as described in claim 1, characterized in that, The hybridoma cell line was named AMA1 and was deposited at the China Center for Type Culture Collection on October 21, 2022; deposit address: Wuhan University, Wuhan, China; accession number: CCTCCNO:C2022299.

5. A hybridoma cell line secreting a monoclonal antibody against β-amanita peptide as described in claim 2, characterized in that, The hybridoma cell line was named AMA2 and was deposited at the China Center for Type Culture Collection on October 21, 2022; deposit address: Wuhan University, Wuhan, China; accession number: CCTCCNO:C2022328.

6. A hybridoma cell line that secretes a monoclonal antibody against γ-amanita peptide as described in claim 3, characterized in that, The hybridoma cell line was named AMA3 and was deposited at the China Center for Type Culture Collection on October 21, 2022; deposit address: Wuhan University, Wuhan, China; accession number: CCTCCNO:C2022329.

7. The application of a monoclonal antibody against α-amanita peptide as described in claim 1, a monoclonal antibody against β-amanita peptide as described in claim 2, or a monoclonal antibody against γ-amanita peptide as described in claim 3, characterized in that, The monoclonal antibodies against α-amanita peptide, β-amanita peptide, and γ-amanita peptide are used to prepare test strips or kits for detecting α-amanita peptide, β-amanita peptide, and γ-amanita peptide.

8. The application according to claim 7, characterized in that, The test strip includes a base plate and a sample pad, a conjugate pad, a reaction membrane, and an absorbent pad connected sequentially along the chromatography direction on the base plate; The reaction membrane is provided with a T3 detection line for detecting γ-amatoxins, a T2 detection line for detecting β-amatoxins, a T1 detection line for detecting α-amatoxins, and a control line in sequence along the chromatography direction. The T1 detection line is coated with α-amatoxins artificial antigen, the T2 detection line is coated with β-amatoxins artificial antigen, and the T3 detection line is coated with γ-amatoxins artificial antigen. The binding pad is coated with monoclonal antibodies against α-amanita peptide, β-amanita peptide, and γ-amanita peptide.