WO2021190587A1 - Broad-spectrum microcystin monoclonal antibody and preparation method therefor - Google Patents

Broad-spectrum microcystin monoclonal antibody and preparation method therefor Download PDF

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WO2021190587A1
WO2021190587A1 PCT/CN2021/082941 CN2021082941W WO2021190587A1 WO 2021190587 A1 WO2021190587 A1 WO 2021190587A1 CN 2021082941 W CN2021082941 W CN 2021082941W WO 2021190587 A1 WO2021190587 A1 WO 2021190587A1
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klh
amino
modified
microcystin
asp3
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周小红
刘金钏
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清华大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to a broad-spectrum monoclonal antibody against multiple structural variants of microcystin and a preparation method thereof.
  • Microcystins are the most common cyanobacteria in toxic cyanobacteria blooms.
  • the microcystins (MCs) produced by them are small molecules with a common cyclic heptapeptide structure. They are composed of 5 non-proteinogenic amino acids (such as Hydro-alanine derivatives and special ⁇ -amino acid Adda, etc.) and 2 proprotein amino acids (variable amino acids at positions 2 and 4), and its chemical structure is shown in formula 1. More than 150 structural variants of microcystin with varying degrees of toxicity have been reported.
  • microcystin-LR MC-LR
  • MC-RR MC-RR
  • MC-YR MC-YR
  • the immunoassay method based on "antigen-antibody” specific recognition and binding reaction has the advantages of high sensitivity, low cost, and high throughput. Aiming at the common structure of microcystin, by preparing antibodies with broad-spectrum specificity, it can theoretically identify all the structural variants of microcystin, and as long as there is no significant difference in the cross-reactivity of each structural variant, there is no need By providing all its structural variant standards, it can directly quantitatively detect total microcystin in water, which is very suitable for rapid screening and early warning monitoring of large-scale samples of microcystin.
  • Microcystin is a small hapten molecule, which needs to be coupled with a carrier protein to transform it into a complete antigen (immunogen), and then immunize animals to obtain antibodies.
  • hapten molecular design is the first key point for the successful establishment of an immunoassay method, and the immunization scheme is the second key point.
  • the present invention proposes a method for preparing a broad-spectrum microcystin monoclonal antibody.
  • the purpose of the present invention is to invent a broad-spectrum monoclonal antibody suitable for immunoassay of the total amount of microcystin in water and establish its preparation method.
  • the present invention claims a monoclonal antibody, the monoclonal antibody is the following a, b, c, d or e:
  • the present invention also claims a composition for preparing a broad-spectrum monoclonal antibody against microcystin, said composition comprising MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC -LW, MC-LY, MC-WR, MC-HtyR, MC-HilR, [D-Asp3]MC-LR and [D-Asp3]MC-RR, the above compounds are individually packaged and used separately.
  • the present invention also claims a composition for preparing a broad-spectrum monoclonal antibody to microcystin, said composition consisting of the following 12 complete antigens: NH 2 -MC-LR-KLH, NH 2 -MC- RR-KLH, NH 2 -MC-YR-KLH, NH 2 -MC-LA-KLH, NH 2 -MC-LF-KLH, NH 2 -MC-LW-KLH,, NH 2 -MC-LY-KLH, NH 2 -MC-WR-KLH, NH 2 -MC-HtyR-KLH, NH 2 -MC-HilR-KLH, NH 2 -[D-Asp3]MC-LR-KLH and NH 2 -[D-Asp3]MC -RR-KLH; wherein the NH 2 -MC-LR-KLH is an amino-modified MC-LR and KLH
  • the amino-modified MC-LR is a compound obtained by introducing an amino group into the seventh amino acid residue N-methyldehydroalanine of MC-LR, that is, in formula 2, R1 is leucine and R2 is refined A compound in which amino acid and R3 are methyl;
  • the amino-modified MC-RR is a compound obtained by introducing an amino group into the seventh amino acid residue N-methyldehydroalanine of MC-RR, which is formula 2
  • the amino-modified MC-YR is on the seventh amino acid residue N-methyldehydroalanine of MC-YR
  • the amino-modified MC-LA is the amino acid residue at the seventh position of MC-LA
  • the general structural formula of the microcystin is shown in formula (1), and the general structural formula of the amino-modified microcystin is shown in formula (2).
  • each of the 12 complete antigens has the same quality.
  • the present invention also claims a reagent for detecting microcystin or a kit containing the reagent, the reagent comprising the monoclonal antibody described in a), b), c), d) and e) At least one of them.
  • the molecular structure variants of the microcystin are MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, MC-LY, MC-WR, MC-HtyR, At least one of MC-HilR, [D-Asp3] MC-LR, [D-Asp3] MC-RR.
  • the present invention also claims a method for preparing a broad-spectrum monoclonal antibody to microcystin, comprising immunizing mice with the complete antigen composition to obtain immunized mice, and taking the highest serum titer among the immunized mice.
  • the spleen cells of the mice were fused with the mouse myeloma cells, and the positive hybridoma cell lines were screened out, and then the positive hybridoma cell lines were injected into the abdominal cavity of the same strains of mice to induce ascites, and the results could be combined with 12 kinds of microcystins.
  • Broad-spectrum monoclonal antibodies that bind with small molecules with high affinity;
  • step 2) Mix the 12 kinds of complete antigens of step 1) with equal quality to obtain a mixed immunogen.
  • the spleen cells of the mice are fused with the mouse myeloma cells, and the positive hybridoma cell lines are screened out.
  • the molecular structure variants of the 12 microcystins in the step 1) are MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, MC-LY, MC-WR , MC-HtyR, MC-HilR, [D-Asp3]MC-LR, and [D-Asp3]MC-RR.
  • the carrier protein in the step 1) can be bovine serum albumin, hemocyanin, human serum albumin, ovalbumin, murine serum albumin, thyroglobulin, or rabbit serum albumin.
  • the immunogen in the step 2) is a mixture of equal masses of 12 kinds of microcystin molecular structure variants respectively coupled with KLH or BSA.
  • the immunized mice in the step 2) are Balb/c mice; the immunization method is: the amount of the immunogen for each immunization is 30-50 ⁇ g/mouse, and the interval between two immunizations is 10-30 days; The immunization method is subcutaneous multi-point injection; the mouse myeloma cells are mouse myeloma cells SP2/0.
  • the method for screening positive hybridoma cell lines in the step 2) is: first screening with the coating antigen, and then screening with the protein monomer used to prepare the immunogen and the coating antigen; the coating antigen is the step
  • the amino-modified microcystin molecule in 1) is obtained by coupling with bovine serum albumin (BSA); the coating antigen is different from the carrier protein in the immunogen.
  • BSA bovine serum albumin
  • the application is the detection of microcystin in environmental samples or food; the molecular structure variants of the microcystin in the application are MC-LR, MC-RR, MC-YR, MC-LA, MC-LF , At least one of MC-LW, MC-LY, MC-WR, MC-HtyR, MC-HilR, [D-Asp3]MC-LR, [D-Asp3]MC-RR.
  • the application is an application for non-disease diagnosis purposes.
  • the present invention also provides a method for detecting microcystin, which includes the step of using the aforementioned monoclonal antibody, composition, kit or method to detect microcystin in a sample to be tested.
  • the microcystin is MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, MC-LY, MC-WR, MC-HtyR, MC-HilR, [D- At least one of Asp3] MC-LR, [D-Asp3] MC-RR.
  • the present invention also provides a method for purifying microcystin, including the step of using the monoclonal antibody, composition, kit or method to purify the microcystin in the sample to be tested.
  • the microcystin is MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, MC-LY, MC-WR, MC-HtyR, MC-HilR, [D- At least one of Asp3] MC-LR, [D-Asp3] MC-RR.
  • the number of the biological material MCAB1.
  • CGMCC Abbreviation of depository unit
  • the number of the biological material MCAB2.
  • CGMCC Abbreviation of depository unit
  • the number of the biological material MCAB3.
  • CGMCC Abbreviation of depository unit
  • the number of the biological material MCAB4.
  • CGMCC Abbreviation of depository unit
  • the number of the biological material MCAB5.
  • CGMCC Abbreviation of depository unit
  • Figure 1 shows the binding curves of monoclonal antibodies secreted by the positive hybridoma cell line MCAB2 screened in the present invention and different concentrations of the coating antigen BSA-MC-LR.
  • Fig. 2 is a standard curve for detecting MC-LR, MC-RR and MC-YR in water samples by monoclonal antibodies secreted by the positive hybridoma cell line MCAB5 selected by the present invention.
  • Myeloma cell SP2/0 seeds (Beijing Huada Protein Research and Development Center); SPF-grade Balb/c pure-line female mice (Sbeef Beijing Biotechnology Co., Ltd.); newborn calf serum (Nanjing Vicente Biotechnology Co., Ltd.) ; Enzyme-labeled secondary antibody (goat anti-mouse IgG/HRP) (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.); PEG 1500 (Roche); HAT stock solution, HT stock solution, paraffin oil, BSA, hemocyanin (KLH) , Tris-hydroxymethylaminomethane hydrochloride (Tris-HCl), 2.2% methylcellulose, 2-mercaptoethylamine, glutaraldehyde, citric acid, sodium citrate, sodium chloride, disodium hydrogen phosphate, phosphoric acid Sodium dihydrogen, carbonate buffer, tetramethylbenzidine (TMB), hydrogen peroxide and urea (
  • Example 1 Obtainment and identification of positive hybridoma cell lines and preparation of broad-spectrum microcystin monoclonal antibodies
  • the above-mentioned 12 microcystin small molecular structural variants respectively, introduced an amino active group on the seventh amino acid (Mdha) through 2-mercaptoethylamine chemical modification, and purified by solid phase extraction using glutaraldehyde two
  • the modified microcystin molecules were coupled with KLH by step method, and 12 kinds of complete antigens were obtained after gel filtration chromatography, and the immunogen KLH-MCs was obtained after equal mixing.
  • the specific method is as follows:
  • Activation first activate the column with methanol, and flow ultrapure water through the column at a certain flow rate after 15 minutes;
  • Sample loading the sample flows through the column at a certain flow rate for enrichment and concentration, and gravity filtration;
  • Rinse After loading the sample, rinse with 5% by volume methanol aqueous solution to purify the sample;
  • the general structural formulas of the 12 microcystin small molecular structural variants are as shown in formula (1), and the structural general formulas of the 12 modified microcystin small molecular structural variants are as shown in formula (2) As shown, the specific groups of R1, R2 and R3 of the 12 microcystin small molecular structure variants are shown in Table 1, respectively.
  • the immunogen KLH-MCs solution of step 2 was mixed with an equal volume of the immune adjuvant to immunize mice.
  • the method is: subcutaneous multi-point injection, according to the dose of 50 ⁇ g KLH-MCs/mouse, to 5 mice (number: 1, 2, 3, 4, 5) for the first immunization, each mouse is injected with 0.1 mL of liquid each time, wherein the 0.1 mL of liquid is composed of 0.05 mL of immunogen KLH-MCs solution and 0.05 mL of Freund's complete adjuvant; After that, 5 mice were boosted with the amount of 30 ⁇ g KLH-MCs/mouse. The interval of immunization was 2 weeks.
  • a total of 6 immunizations were carried out. Each mouse was injected with 0.06mL liquid each time, of which the 0.06mL liquid It consists of 0.03mL immunogen KLH-MCs solution and 0.03mL Freund's incomplete adjuvant.
  • the coating antigen was an equal mixture of 12 kinds of BSA-MC, and the coating concentration was 2 ⁇ g/mL.
  • an intraperitoneal injection of 50 ⁇ g of immunogen/mouse was performed for impulse immunization.
  • the immunogen used this time was an equal mixture of 12 kinds of BSA-MC (BSA-MCs), 3 days later
  • the titer was determined again, and the mouse spleen cells with the highest serum titer were selected for cell fusion.
  • the synthesis method of the coated antigen BSA-MC is specifically: the synthesis method of the coated antigen is the same as that described in step 1, replace KLH in step 1 with BSA, and finally add 20 ⁇ L to a concentration of 0.2M
  • the lysine solution is reacted at room temperature for 1 to 4 hours to block unreacted aldehyde groups, and the obtained coated antigen is placed in a refrigerator at -20°C for later use.
  • the 12 kinds of BSA-MC were identified by MALDI-TOF/MS by mass spectrometry, and the coupling ratios of the 12 kinds of microcystin small molecules and BSA were all 1:1.
  • mice myeloma cell SP2/0 cells in good condition are gently pipetted off the wall of the culture flask and transferred to a 50mL centrifuge tube; the mouse is put to death by pulling the neck after removing the eyeballs and blood, and putting in 75% alcohol Soak in medium for 5min;
  • Block with blocking solution (2% skimmed milk-PBS), 200 ⁇ L/well, incubate at 37°C for 2 hours, and wash with washing solution 3 times.
  • a 450 value of 2.1 times the negative control is a positive hybridoma cell line.
  • 31 clones that were positive in one screen they were further cultured and then screened for the second time according to the same method to obtain 23 positive hybridoma cell lines.
  • the deposit number of the hybridoma cell line MCAB1 is CGMCC No.19194, the deposit number of the hybridoma cell line MCAB2 is CGMCC No.19195, the hybridoma cell line MCAB3 The deposit number is CGMCC No. 19196, the deposit number of the hybridoma cell line MCAB4 is CGMCC No. 19197, and the deposit number of the hybridoma cell line MCAB5 is CGMCC No. 19198.
  • the above five hybridoma cell lines were taken to prepare monoclonal antibodies according to the method of step (7) and step (8), and the monoclonal antibody secreted by the hybridoma cell line MCAB1 was named antibody MCAB1; the monoclonal antibody secreted by the hybridoma cell line MCAB2
  • the monoclonal antibody is named antibody MCAB2; the monoclonal antibody secreted by the hybridoma cell line MCAB3 is named antibody MCAB3; the monoclonal antibody secreted by the hybridoma cell line MCAB4 is named antibody MCAB4; the monoclonal antibody secreted by the hybridoma cell line MCAB5 is named Antibody MCAB5.
  • mice The mouse's abdominal cavity was injected with paraffin oil, 500 ⁇ L/Balb/c mice, by intraperitoneal injection one week before inoculation.
  • the cells were inoculated by intraperitoneal injection, and each mouse was injected with 1 mL (cell volume 5 ⁇ 10 5 -9 ⁇ 10 5 cells/mL).
  • Wash impurities Pass the column with 5 times the column volume of coupling buffer and keep the flow rate at 1 mL/min.
  • Elution buffer 0.1M sodium citrate solution, pH 3.5
  • elution buffer 0.1M sodium citrate solution, pH 3.5
  • the obtained antibody was tested for purity by SDS-PAGE (12% separation gel concentration) and concentration by UV spectrophotometer. It was confirmed that the purity of the five antibodies obtained was above 90%, which met the needs of downstream analysis.
  • Example 2 Detect the affinity of monoclonal antibodies secreted by 5 hybridoma cell lines to 12 microcystins
  • Block with blocking solution (2% skimmed milk-PBS), 200 ⁇ L/well, incubate at 37°C for 2 to 4 hours, and wash with washing solution 3 times.
  • the concentration of the coating antigen was 2, 1, 0.5, and 0.25 ⁇ g/mL to determine the antigen-antibody reaction curve.
  • the results are shown in Figure 1.
  • the calculation method is as follows:
  • the total antibody concentration Ab t corresponding to the above dilution factor can be calculated, where the antibody molecular weight is calculated as 150,000 (IgG molecular weight).
  • an affinity constant K 1 can be calculated.
  • the antibody MCAB2 was replaced with the antibody MCAB1 according to the same method as above, and the affinity constants of the antibody MCAB1 and 12 kinds of microcystins and their average affinity constants were obtained. The results are shown in Table 2.
  • the antibody MCAB2 was replaced with the antibody MCAB3 according to the same method as above, and the affinity constants of the antibody MCAB3 and 12 kinds of microcystins and their average affinity constants were obtained. The results are shown in Table 2.
  • the antibody MCAB2 was replaced with the antibody MCAB4 according to the same method as above, and the affinity constants of the antibody MCAB4 and 12 kinds of microcystins and their average affinity constants were obtained. The results are shown in Table 2.
  • the antibody MCAB2 was replaced with the antibody MCAB5 according to the same method described above, and the affinity constants of the antibody MCAB5 and 12 kinds of microcystins and their average affinity constants were obtained. The results are shown in Table 2.
  • the average affinity constant of antibody MCAB1 is 4.10 ⁇ 10 8 L/mol
  • the average affinity constant of antibody MCAB2 and 12 kinds of microcystins is 1.12 ⁇ 10 9 L/mol
  • the average affinity constant of antibody MCAB3 is 5.35 ⁇ 10 8 L/mol
  • the average affinity constant of antibody MCAB4 is 6.64 ⁇ 10 8 L/mol
  • the average affinity constant of antibody MCAB5 is 3.92 ⁇ 10 8 L/mol.
  • Example 3 The monoclonal antibody MCAB5 secreted by the hybridoma cell line CGMCC No. 19198 was used to detect MC-LR, MC-RR and MC-YR in water samples.
  • the MC-LR, MC-RR and MC-YR standards were diluted with ultrapure water in a series of 3-fold gradients respectively, the concentrations were: 1000, 333, 111, 37, 12.34, 4.12, 1.37, 0.46, 0.15 , 0.05 ⁇ g/L, as a water sample.
  • NH 2 -MC-LR-BSA is the replacement of KLH in step 1.1 in Example 1 with BSA, the resulting amino-modified MC-LR and BSA conjugate
  • diluted to 2 ⁇ g/mL with coating solution 100 ⁇ L/well was added to 96-well enzyme-linked plate for coating, coated overnight at 4°C and washed with PBS-T washing solution 3 times.
  • Block with blocking solution (2% skimmed milk-PBS), 200 ⁇ L/well, incubate at 37°C for 2 to 4 hours, wash with washing solution 3 times, and replace the standard water sample solution and blank control (standard solution with PBS) from step 1) Solution instead), respectively add 100 ⁇ L/well to the corresponding wells of the ELISA plate.
  • the 4-parameter Logistic model is used as the basis for data analysis and evaluation of indirect competitive ELISA detection.
  • the model is as follows:
  • x target concentration, independent variable
  • A Absorbance corresponding to x, dependent variable
  • a 2 The asymptotic value of the lower end (x ⁇ ), that is, the minimum absorbance
  • the three standard curves in Figure 2 were fitted with a four-parameter Logistic model.
  • the quantification interval is 0.58 ⁇ g/L-10.33 ⁇ g/L
  • the minimum detection limit is 0.36 ⁇ g/L
  • the quantification interval is 1.06 ⁇ g/L-41.67 ⁇ g /L
  • the minimum detection limit is 0.40 ⁇ g/L
  • the quantitative interval is 1.05 ⁇ g/L-28.62 ⁇ g/L.
  • microcystin in order to obtain highly specific antibodies against the entire cyclic peptide structure of microcystin, 12 kinds of microcystin (MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, MC-LY, MC-WR, MC-HtyR, MC-HilR, [D-Asp3]MC-LR, [D-Asp3]MC-RR) on the seventh amino acid (Mdha) through 2-
  • the chemical modification of mercaptoethylamine introduces an amino active group, and then couples with keyhole limpet hemocyanin (KLH) to obtain 12 kinds of conjugates of microcystin and KLH respectively.
  • KLH keyhole limpet hemocyanin
  • the “mixed immunogen method” is used for immunization. That is, the above-mentioned 12 kinds of microcystins and KLH conjugates (12 kinds of complete antigens) are mixed and used as immunogens to immunize mice; then the above-mentioned 12 kinds of small molecules are respectively combined with or bovine serum albumin (BSA) After being coupled and mixed in equal amounts, the serum titer is determined by indirect ELISA method; the spleen cells of immunized mice whose serum titer meets the requirements are selected and fused with SP2/0 myeloma cells to screen out positive hybridoma cells Strains; culture positive hybridoma cell lines and perform subtype identification; inject the obtained IgG1 subtype positive hybridoma cell lines into the abdominal cavity of the same strain of mice to induce ascites, and obtain broad-spectrum microcystin monoclonal antibodies MCAB1, MCAB2 MCAB3, MCAB4 and MCAB5.
  • the average affinity constant of antibody MCAB1 and 12 kinds of microcystins is 4.10 ⁇ 10 8 L/mol
  • the average affinity constant of antibody MCAB2 and 12 kinds of microcystins is 1.12 ⁇ 10 9 L/mol
  • the antibody MCAB3 has 12 kinds of microcystins.
  • the average affinity constant of cystins is 5.35 ⁇ 10 8 L/mol
  • the average affinity constant of antibody MCAB4 and 12 kinds of microcystins is 6.64 ⁇ 10 8 L/mol
  • the constant is 3.92 ⁇ 10 8 L/mol.

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Abstract

Disclosed in the present invention are a broad-spectrum microcystin monoclonal antibody and a preparation method therefor. In order to obtain an antibody that has high specificity to the whole cyclic peptide structure of microcystins, an amino active group is introduced into an amino acid (Mdha) at position 7 of each of 12 microcystins by means of chemical modification of 2-mercaptoethyl amine, and then, the microcystins are coupled with KLH to obtain conjugates of the 12 microcystins and KLH, respectively; and then, the conjugates are subjected to a "mixed immunogen method" to obtain the broad-spectrum microcystin monoclonal antibodies MCAB1, MCAB2, MCAB3, MCAB4, and MCAB5. The 5 monoclonal antibodies disclosed by the present invention have relatively high affinity to the 12 microcystins and can be used for detecting microcystins in actual samples.

Description

一种广谱型微囊藻毒素单克隆抗体及其制备方法Broad-spectrum microcystin monoclonal antibody and preparation method thereof 技术领域Technical field
本发明涉及一种针对微囊藻毒素多种结构变异体的广谱型单克隆抗体及其制备方法。The invention relates to a broad-spectrum monoclonal antibody against multiple structural variants of microcystin and a preparation method thereof.
背景技术Background technique
有毒蓝藻水华频繁地爆发，对生态环境和经济带来严重的影响，现已经成为一个全球性的重大环境问题。微囊藻是有毒蓝藻水华中最常见的蓝藻属，其产生的微囊藻毒素(Microcystins，MCs)是一类具有共性环状七肽结构的小分子，由5个非蛋白原氨基酸(如脱氢丙氨酸衍生物和特殊的β-氨基酸Adda等)和2个蛋白原氨基酸(第2位和第4位的可变氨基酸)组成，其化学结构式如式1所示。已报道有150种以上具有不同程度毒性的微囊藻毒素结构变异体，其中微囊藻毒素-LR(MC-LR)、MC-RR和MC-YR是蓝藻水华水体中最常见也是毒性最强的微囊藻毒素类型，其引起的急性毒性会使细胞功能和结构丧失，导致肝出血甚至死亡。因此，开发可靠的水体中微囊藻毒素快速筛查技术，具有重要的现实意义。Toxic cyanobacteria blooms frequently erupt, which have serious impacts on the ecological environment and economy, and have now become a major global environmental problem. Microcystins are the most common cyanobacteria in toxic cyanobacteria blooms. The microcystins (MCs) produced by them are small molecules with a common cyclic heptapeptide structure. They are composed of 5 non-proteinogenic amino acids (such as Hydro-alanine derivatives and special β-amino acid Adda, etc.) and 2 proprotein amino acids (variable amino acids at positions 2 and 4), and its chemical structure is shown in formula 1. More than 150 structural variants of microcystin with varying degrees of toxicity have been reported. Among them, microcystin-LR (MC-LR), MC-RR and MC-YR are the most common and most toxic in cyanobacteria blooms. A strong type of microcystin, the acute toxicity caused by it will cause the loss of cell function and structure, leading to liver hemorrhage and even death. Therefore, the development of a reliable rapid screening technology for microcystins in water bodies has important practical significance.
Figure PCTCN2021082941-appb-000001
Figure PCTCN2021082941-appb-000001
基于“抗原-抗体”特异性识别和结合反应的免疫分析方法，具有灵敏度高、成本低、高通量等优势。针对微囊藻毒素的共性结构，通过制备具有广谱特异性的抗体，理论上可以识别所有的微囊藻毒素结构变异体，且只要每种结构变异体的交叉反应性无明显差异，则无需提供其所有的结构变异体标准品，便可直接定量检测水体中总微囊藻毒素，非常适合于微囊藻毒素的大规模样品快速筛查和预警监测。The immunoassay method based on "antigen-antibody" specific recognition and binding reaction has the advantages of high sensitivity, low cost, and high throughput. Aiming at the common structure of microcystin, by preparing antibodies with broad-spectrum specificity, it can theoretically identify all the structural variants of microcystin, and as long as there is no significant difference in the cross-reactivity of each structural variant, there is no need By providing all its structural variant standards, it can directly quantitatively detect total microcystin in water, which is very suitable for rapid screening and early warning monitoring of large-scale samples of microcystin.
建立微囊藻毒素总量免疫分析方法，获得高质量的广谱型抗体是建立微囊藻毒素免疫筛查技术的关键。微囊藻毒素属于半抗原小分子，需要先将其与载体蛋白偶联，改造成完全抗原(免疫原)，再免疫动物获得抗体。为了制备出高效价的广谱特异性抗体，半抗原分子设计是成功建立免疫分析方法的第一个关键点，而免疫方案是第二个关键点。Establishing an immunoassay method for the total amount of microcystin and obtaining high-quality broad-spectrum antibodies is the key to establishing a microcystin immune screening technology. Microcystin is a small hapten molecule, which needs to be coupled with a carrier protein to transform it into a complete antigen (immunogen), and then immunize animals to obtain antibodies. In order to prepare high-titer, broad-spectrum specific antibodies, hapten molecular design is the first key point for the successful establishment of an immunoassay method, and the immunization scheme is the second key point.
目前有关微囊藻毒素广谱型抗体制备的研究报道并不多，所获抗体的广谱 性未能达到理想要求，测定的交叉反应性数据相对有限，而且结构变异体之间的交叉反应性差异显著。2014年Ingunn A.Samdal课题组采用在可变氨基酸位点存在差异的5种微囊藻毒素分子的混合物与牛血清白蛋白(BSA)偶联制备免疫原，得到的羊多克隆抗体的广谱性和交叉反应性均提升了很多。然而，多克隆抗体在免疫分析批次间误差较大。针对上述问题，本发明提出了一种广谱型微囊藻毒素单克隆抗体的制备方法。At present, there are not many research reports on the preparation of microcystin broad-spectrum antibodies, the broad-spectrum antibodies obtained do not meet the ideal requirements, the measured cross-reactivity data is relatively limited, and the cross-reactivity between structural variants Significant difference. In 2014, Ingunn A. Samdal's research group used a mixture of 5 microcystin molecules with differences in variable amino acid sites to couple with bovine serum albumin (BSA) to prepare immunogens, and obtained a broad spectrum of goat polyclonal antibodies Both sex and cross-reactivity have improved a lot. However, polyclonal antibodies have large errors between batches of immunoassay. In view of the above problems, the present invention proposes a method for preparing a broad-spectrum microcystin monoclonal antibody.
发明公开Invention Disclosure
本发明的目的在于发明一种适用于水体中微囊藻毒素总量免疫分析的广谱型单克隆抗体并建立其制备方法。The purpose of the present invention is to invent a broad-spectrum monoclonal antibody suitable for immunoassay of the total amount of microcystin in water and establish its preparation method.
本发明要求保护一种单克隆抗体，所述单克隆抗体为如下a、b、c、d或e：The present invention claims a monoclonal antibody, the monoclonal antibody is the following a, b, c, d or e:
a)由保藏号为CGMCC No.19194的杂交瘤细胞株MCAB1产生的单克隆抗体；a) Monoclonal antibodies produced by the hybridoma cell line MCAB1 with the deposit number CGMCC No. 19194;
b)由保藏号为CGMCC No.19195的杂交瘤细胞株MCAB2产生的单克隆抗体；b) Monoclonal antibodies produced by the hybridoma cell line MCAB2 with the deposit number CGMCC No. 19195;
c)由保藏号为CGMCC No.19196的杂交瘤细胞株MCAB3产生的单克隆抗体；c) Monoclonal antibodies produced by the hybridoma cell line MCAB3 with the deposit number CGMCC No. 19196;
d)由保藏号为CGMCC No.19197的杂交瘤细胞株MCAB4产生的单克隆抗体；d) Monoclonal antibodies produced by the hybridoma cell line MCAB4 with the deposit number CGMCC No. 19197;
e)由保藏号为CGMCC No.19198的杂交瘤细胞株MCAB5产生的单克隆抗体。e) Monoclonal antibodies produced by the hybridoma cell line MCAB5 with the deposit number CGMCC No. 19198.
本发明还要求保护一种用于制备微囊藻毒素广谱型单克隆抗体的组合物，所述组合物包括MC-LR、MC-RR、MC-YR、MC-LA、MC-LF、MC-LW、MC-LY、MC-WR、MC-HtyR、MC-HilR、[D-Asp3]MC-LR和[D-Asp3]MC-RR，上述化合物分别独立包装，分开使用。The present invention also claims a composition for preparing a broad-spectrum monoclonal antibody against microcystin, said composition comprising MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC -LW, MC-LY, MC-WR, MC-HtyR, MC-HilR, [D-Asp3]MC-LR and [D-Asp3]MC-RR, the above compounds are individually packaged and used separately.
本发明还要求保护一种用于制备微囊藻毒素广谱型单克隆抗体的组合物，所述组合物由以下12种完全抗原组成：NH 2-MC-LR-KLH、NH 2-MC-RR-KLH、NH 2-MC-YR-KLH、NH 2-MC-LA-KLH,NH 2-MC-LF-KLH、NH 2-MC-LW-KLH,、NH 2-MC-LY-KLH、NH 2-MC-WR-KLH、NH 2-MC-HtyR-KLH、NH 2-MC-HilR-KLH、NH 2-[D-Asp3]MC-LR-KLH和NH 2-[D-Asp3]MC-RR-KLH；其中所述NH 2-MC-LR-KLH为氨基修饰的MC-LR与KLH偶联物；所述NH 2-MC-RR-KLH为氨基修饰的MC-RR与KLH偶联物；所述NH 2-MC--YR-KLH为氨基修饰的MC--YR与KLH偶联物；所述NH 2-MC-LA-KLH为氨基修饰的MC-LA与KLH偶联物；所述NH 2-MC-LF-KLH为氨基修饰的MC-LF与KLH偶联物；所述NH 2-MC-LW-KLH为氨基修饰的MC-LW与KLH偶联物；所述NH 2-MC-LY-KLH为氨基修饰的MC-LY与KLH偶联物；所述NH 2-MC-WR-KLH为氨基修饰的MC-WR与KLH偶联物；所述NH 2-MC-HtyR-KLH为氨基修饰的MC-HtyR与KLH偶联物；所述NH 2-MC-HilR-KLH为氨基修饰的MC-HilR与KLH偶联物；所述NH 2-[D-Asp3]MC-LR-KLH为氨基修饰的[D-Asp3]MC-LR与KLH 偶联物；所述NH 2-[D-Asp3]MC-RR-KLH为氨基修饰的[D-Asp3]MC-RR与KLH偶联物；其中，KLH为血蓝蛋白。 The present invention also claims a composition for preparing a broad-spectrum monoclonal antibody to microcystin, said composition consisting of the following 12 complete antigens: NH 2 -MC-LR-KLH, NH 2 -MC- RR-KLH, NH 2 -MC-YR-KLH, NH 2 -MC-LA-KLH, NH 2 -MC-LF-KLH, NH 2 -MC-LW-KLH,, NH 2 -MC-LY-KLH, NH 2 -MC-WR-KLH, NH 2 -MC-HtyR-KLH, NH 2 -MC-HilR-KLH, NH 2 -[D-Asp3]MC-LR-KLH and NH 2 -[D-Asp3]MC -RR-KLH; wherein the NH 2 -MC-LR-KLH is an amino-modified MC-LR and KLH conjugate; the NH 2 -MC-RR-KLH is an amino-modified MC-RR and KLH conjugate The NH 2 -MC--YR-KLH is an amino-modified MC-YR and KLH conjugate; the NH 2 -MC-LA-KLH is an amino-modified MC-LA and KLH conjugate; The NH 2 -MC-LF-KLH is an amino-modified MC-LF and KLH conjugate; the NH 2 -MC-LW-KLH is an amino-modified MC-LW and KLH conjugate; the NH 2 -MC-LY-KLH is an amino-modified MC-LY and KLH conjugate; the NH 2 -MC-WR-KLH is an amino-modified MC-WR and KLH conjugate; the NH 2 -MC-HtyR -KLH is a conjugate of amino-modified MC-HtyR and KLH; the NH 2 -MC-HilR-KLH is a conjugate of amino-modified MC-HilR and KLH; the NH 2 -[D-Asp3]MC- LR-KLH is a conjugate of amino-modified [D-Asp3]MC-LR and KLH; the NH 2 -[D-Asp3]MC-RR-KLH is amino-modified [D-Asp3]MC-RR and KLH Conjugate; Among them, KLH is hemocyanin.
所述氨基修饰的MC-LR为在MC-LR的第七位氨基酸残基N-甲基脱氢丙氨酸上引入一个氨基得到的化合物，即式2中R1为亮氨酸、R2为精氨酸和R3为甲基的化合物；所述氨基修饰的MC-RR为在MC-RR的第七位氨基酸残基N-甲基脱氢丙氨酸上引入一个氨基得到的化合物，即式2中R1为精氨酸、R2为精氨酸和R3为甲基的化合物；所述氨基修饰的MC-YR为在MC-YR的第七位氨基酸残基N-甲基脱氢丙氨酸上引入一个氨基得到的化合物，即式2中R1为酪氨酸、R2为精氨酸和R3为甲基的化合物；所述氨基修饰的MC-LA为在MC-LA的第七位氨基酸残基N-甲基脱氢丙氨酸上引入一个氨基得到的化合物，即式2中R1为亮氨酸、R2为丙氨酸和R3为甲基的化合物；所述氨基修饰的MC-LF为在MC-LF的第七位氨基酸残基N-甲基脱氢丙氨酸上引入一个氨基得到的化合物，即式2中R1为亮氨酸、R2为苯丙氨酸和R3为甲基的化合物；所述氨基修饰的MC-LW为在MC-LW的第七位氨基酸残基N-甲基脱氢丙氨酸上引入一个氨基得到的化合物，即式2中R1为亮氨酸、R2为色氨酸和R3为甲基的化合物；所述氨基修饰的MC-LY为在MC-LY的第七位氨基酸残基N-甲基脱氢丙氨酸上引入一个氨基得到的化合物，即式2中R1为亮氨酸、R2为酪氨酸和R3为甲基的化合物；所述氨基修饰的MC-WR为在MC-WR的第七位氨基酸残基N-甲基脱氢丙氨酸上引入一个氨基得到的化合物，即式2中R1为色氨酸、R2为精氨酸和R3为甲基的化合物；所述氨基修饰的MC-HtyR为在MC-HtyR的第七位氨基酸残基N-甲基脱氢丙氨酸上引入一个氨基得到的化合物，即式2中R1为高酪氨酸、R2为精氨酸和R3为甲基的化合物；所述氨基修饰的MC-HilR为在MC-HilR的第七位氨基酸残基N-甲基脱氢丙氨酸上引入一个氨基得到的化合物，即式2中R1为同异亮氨酸、R2为精氨酸和R3为甲基的化合物；所述氨基修饰的[D-Asp3]MC-LR为在[D-Asp3]MC-LR的第七位氨基酸残基N-甲基脱氢丙氨酸上引入一个氨基得到的化合物，即式2中R1为亮氨酸、R2为精氨酸和R3为氢的化合物；所述氨基修饰的[D-Asp3]MC-RR为在[D-Asp3]MC-RR的第七位氨基酸残基N-甲基脱氢丙氨酸上引入一个氨基得到的化合物，即式2中R1为精氨酸、R2为精氨酸和R3为氢的化合物。The amino-modified MC-LR is a compound obtained by introducing an amino group into the seventh amino acid residue N-methyldehydroalanine of MC-LR, that is, in formula 2, R1 is leucine and R2 is refined A compound in which amino acid and R3 are methyl; the amino-modified MC-RR is a compound obtained by introducing an amino group into the seventh amino acid residue N-methyldehydroalanine of MC-RR, which is formula 2 The compound in which R1 is arginine, R2 is arginine and R3 is methyl; the amino-modified MC-YR is on the seventh amino acid residue N-methyldehydroalanine of MC-YR The compound obtained by introducing an amino group, that is, the compound in formula 2 where R1 is tyrosine, R2 is arginine and R3 is methyl; the amino-modified MC-LA is the amino acid residue at the seventh position of MC-LA The compound obtained by introducing an amino group into N-methyldehydroalanine, that is, the compound in formula 2 where R1 is leucine, R2 is alanine and R3 is methyl; the amino-modified MC-LF is The compound obtained by introducing an amino group into the seventh amino acid residue N-methyldehydroalanine of MC-LF, that is, the compound in formula 2 where R1 is leucine, R2 is phenylalanine and R3 is methyl The amino-modified MC-LW is a compound obtained by introducing an amino group on the seventh amino acid residue N-methyldehydroalanine of MC-LW, that is, in formula 2, R1 is leucine and R2 is Tryptophan and R3 are methyl compounds; the amino-modified MC-LY is a compound obtained by introducing an amino group into the seventh amino acid residue N-methyldehydroalanine of MC-LY, that is, 2 The compound in which R1 is leucine, R2 is tyrosine and R3 is methyl; the amino-modified MC-WR is N-methyldehydroalanine at the seventh amino acid residue of MC-WR The compound obtained by introducing an amino group above, that is, the compound in formula 2 where R1 is tryptophan, R2 is arginine, and R3 is methyl; the amino-modified MC-HtyR is the amino acid residue at the seventh position of MC-HtyR The compound obtained by introducing an amino group into the N-methyldehydroalanine group, that is, the compound in which R1 is high tyrosine, R2 is arginine and R3 is methyl in formula 2; the amino-modified MC-HilR It is a compound obtained by introducing an amino group into the seventh amino acid residue N-methyldehydroalanine of MC-HilR, that is, in formula 2, R1 is homo-isoleucine, R2 is arginine and R3 is methyl The amino-modified [D-Asp3]MC-LR is a compound obtained by introducing an amino group on the seventh amino acid residue N-methyldehydroalanine of [D-Asp3]MC-LR , That is, the compound in formula 2 where R1 is leucine, R2 is arginine and R3 is hydrogen; the amino-modified [D-Asp3]MC-RR is in the seventh position of [D-Asp3]MC-RR The compound obtained by introducing an amino group into the amino acid residue N-methyldehydroalanine, that is, the compound in formula 2 where R1 is arginine, R2 is arginine, and R3 is hydrogen.
所述微囊藻毒素的结构通式如式(1)所示，所述氨基修饰的微囊藻毒素的结构通式如式(2)所示。The general structural formula of the microcystin is shown in formula (1), and the general structural formula of the amino-modified microcystin is shown in formula (2).
Figure PCTCN2021082941-appb-000002
Figure PCTCN2021082941-appb-000002
所述组合物中，所述12种完全抗原中的每种抗原质量相同。In the composition, each of the 12 complete antigens has the same quality.
本发明还要求保护用于检测微囊藻毒素的试剂或含有所述试剂的试剂盒，所述试剂包括所述a)、b)、c)、d)和e)中所述的单克隆抗体中的至少一种。The present invention also claims a reagent for detecting microcystin or a kit containing the reagent, the reagent comprising the monoclonal antibody described in a), b), c), d) and e) At least one of them.
其中，所述微囊藻毒素的分子结构变异体为MC-LR,MC-RR,MC-YR,MC-LA,MC-LF,MC-LW,MC-LY,MC-WR,MC-HtyR,MC-HilR,[D-Asp3]MC-LR,[D-Asp3]MC-RR中的至少一种。Wherein, the molecular structure variants of the microcystin are MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, MC-LY, MC-WR, MC-HtyR, At least one of MC-HilR, [D-Asp3] MC-LR, [D-Asp3] MC-RR.
本发明还要求保护一种制备微囊藻毒素广谱型单克隆抗体的方法，包括用所述完全抗原组合物免疫小鼠得到被免疫小鼠，取所述被免疫小鼠中血清效价最高的小鼠的脾细胞与小鼠骨髓瘤细胞进行融合，筛选出阳性杂交瘤细胞株，再将所述阳性杂交瘤细胞株注入同系小鼠腹腔诱生腹水，得到可以与12种微囊藻毒素小分子高亲和力结合的广谱型单克隆抗体；The present invention also claims a method for preparing a broad-spectrum monoclonal antibody to microcystin, comprising immunizing mice with the complete antigen composition to obtain immunized mice, and taking the highest serum titer among the immunized mice The spleen cells of the mice were fused with the mouse myeloma cells, and the positive hybridoma cell lines were screened out, and then the positive hybridoma cell lines were injected into the abdominal cavity of the same strains of mice to induce ascites, and the results could be combined with 12 kinds of microcystins. Broad-spectrum monoclonal antibodies that bind with small molecules with high affinity;
具体可以包括以下步骤：Specifically, it can include the following steps:
1)分别在12种微囊藻毒素分子结构变异体的第七位氨基酸残基N-甲基脱氢丙氨酸上引入一个氨基，得到12种经氨基修饰的微囊藻毒分子；再将该12种经氨基修饰的微囊藻毒素分子分别与载体蛋白偶联得到12种完全抗原；1) Introduce an amino group into the seventh amino acid residue N-methyldehydroalanine of 12 kinds of microcystin molecular structure variants to obtain 12 kinds of amino-modified microcystin molecules; The 12 amino-modified microcystin molecules are respectively coupled with carrier proteins to obtain 12 complete antigens;
2)将步骤1)的12种完全抗原等质量混合，得到混合免疫原，对小鼠进行免疫后取小鼠的脾细胞与小鼠骨髓瘤细胞进行融合，筛选出阳性杂交瘤细胞株，得到微囊藻毒素广谱型单克隆抗体；2) Mix the 12 kinds of complete antigens of step 1) with equal quality to obtain a mixed immunogen. After immunizing the mice, the spleen cells of the mice are fused with the mouse myeloma cells, and the positive hybridoma cell lines are screened out. Microcystin broad-spectrum monoclonal antibody;
所述步骤1)中的12种微囊藻毒素的分子结构变异体为MC-LR,MC-RR,MC-YR,MC-LA,MC-LF,MC-LW,MC-LY,MC-WR,MC-HtyR,MC-HilR,[D-Asp3]MC-LR,和[D-Asp3]MC-RR。The molecular structure variants of the 12 microcystins in the step 1) are MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, MC-LY, MC-WR , MC-HtyR, MC-HilR, [D-Asp3]MC-LR, and [D-Asp3]MC-RR.
所述步骤1)中的所述载体蛋白可为牛血清白蛋白、血蓝蛋白、人血清白蛋白、卵清蛋白、鼠血清白蛋白、甲状腺球蛋白或兔血清白蛋白。The carrier protein in the step 1) can be bovine serum albumin, hemocyanin, human serum albumin, ovalbumin, murine serum albumin, thyroglobulin, or rabbit serum albumin.
所述步骤2)中的免疫原为12种微囊藻毒素分子结构变异体分别与KLH或BSA偶联后的等质量混合物。The immunogen in the step 2) is a mixture of equal masses of 12 kinds of microcystin molecular structure variants respectively coupled with KLH or BSA.
所述步骤2)中的免疫小鼠为Balb/c小鼠；免疫方法为：每次免疫的所述免疫原用量为30-50μg/小鼠，两次免疫的间隔时间为10-30天；免疫方式为皮下多点注射；所述小鼠骨髓瘤细胞为小鼠骨髓瘤细胞SP2/0。The immunized mice in the step 2) are Balb/c mice; the immunization method is: the amount of the immunogen for each immunization is 30-50 μg/mouse, and the interval between two immunizations is 10-30 days; The immunization method is subcutaneous multi-point injection; the mouse myeloma cells are mouse myeloma cells SP2/0.
所述步骤2)中的筛选阳性杂交瘤细胞株的方法为：先用包被抗原筛选，再用制备免疫原以及包被抗原所用的蛋白单体筛选；所述包被抗原为将所述步骤1)中的经氨基修饰的微囊藻毒素分子与牛血清白蛋白(BSA)偶联得到；所述包被抗原与所述免疫原中的载体蛋白不相同。The method for screening positive hybridoma cell lines in the step 2) is: first screening with the coating antigen, and then screening with the protein monomer used to prepare the immunogen and the coating antigen; the coating antigen is the step The amino-modified microcystin molecule in 1) is obtained by coupling with bovine serum albumin (BSA); the coating antigen is different from the carrier protein in the immunogen.
所述的单克隆抗体、所述的组合物、所述的免疫原、所述的试剂盒或者所述的方法在检测微囊藻毒素和/或纯化微囊藻毒素中的而应用也应在本发明的保护范围之内。The application of the monoclonal antibody, the composition, the immunogen, the kit or the method in the detection of microcystin and/or purification of microcystin should also be used Within the protection scope of the present invention.
所述应用为检测环境样品或者食品中的微囊藻毒素；所述应用中的微囊藻毒素的分子结构变异体为MC-LR,MC-RR,MC-YR,MC-LA,MC-LF,MC-LW,MC-LY,MC-WR,MC-HtyR,MC-HilR,[D-Asp3]MC-LR,[D-Asp3]MC-RR中的至少一种。The application is the detection of microcystin in environmental samples or food; the molecular structure variants of the microcystin in the application are MC-LR, MC-RR, MC-YR, MC-LA, MC-LF , At least one of MC-LW, MC-LY, MC-WR, MC-HtyR, MC-HilR, [D-Asp3]MC-LR, [D-Asp3]MC-RR.
所述应用为非疾病诊断目的的应用。The application is an application for non-disease diagnosis purposes.
本发明还提供一种检测微囊藻毒素的方法，包括使用上述的单克隆抗体、组合物、试剂盒或者方法进行待测样品中微囊藻毒素的检测的步骤。The present invention also provides a method for detecting microcystin, which includes the step of using the aforementioned monoclonal antibody, composition, kit or method to detect microcystin in a sample to be tested.
所述微囊藻毒素为MC-LR,MC-RR,MC-YR,MC-LA,MC-LF,MC-LW,MC-LY,MC-WR,MC-HtyR,MC-HilR,[D-Asp3]MC-LR,[D-Asp3]MC-RR中的至少一种。The microcystin is MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, MC-LY, MC-WR, MC-HtyR, MC-HilR, [D- At least one of Asp3] MC-LR, [D-Asp3] MC-RR.
本发明还提供一种纯化微囊藻毒素的方法，包括使用所述的单克隆抗体、组合物、试剂盒或者方法纯化待测样品中的微囊藻毒素的步骤。The present invention also provides a method for purifying microcystin, including the step of using the monoclonal antibody, composition, kit or method to purify the microcystin in the sample to be tested.
所述微囊藻毒素为MC-LR,MC-RR,MC-YR,MC-LA,MC-LF,MC-LW,MC-LY,MC-WR,MC-HtyR,MC-HilR,[D-Asp3]MC-LR,[D-Asp3]MC-RR中的至少一种。The microcystin is MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, MC-LY, MC-WR, MC-HtyR, MC-HilR, [D- At least one of Asp3] MC-LR, [D-Asp3] MC-RR.
生物材料保藏说明Biological material preservation instructions
1、生物材料的的分类命名：分泌微囊藻毒素广谱型抗体的阳性小鼠杂交瘤细胞株。1. Classification and naming of biological materials: positive mouse hybridoma cell lines secreting broad-spectrum antibodies to microcystin.
生物材料的编号：MCAB1。The number of the biological material: MCAB1.
保藏单位全称：中国微生物菌种保藏管理委员会普通微生物中心。Full name of the depository: General Microbiology Center of China Microbial Culture Collection Management Committee.
保藏单位简称：CGMCC。Abbreviation of depository unit: CGMCC.
地址：北京市朝阳区北辰西路1号院3号。Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
保藏日期：2019年12月10日。Date of preservation: December 10, 2019.
保藏编号：CGMCC No.19194。Deposit number: CGMCC No. 19194.
2、生物材料的的分类命名：分泌微囊藻毒素广谱型抗体的阳性小鼠杂交瘤细胞株。2. Classification and naming of biological materials: positive mouse hybridoma cell lines secreting broad-spectrum antibodies to microcystin.
生物材料的编号：MCAB2。The number of the biological material: MCAB2.
保藏单位全称：中国微生物菌种保藏管理委员会普通微生物中心。Full name of the depository: General Microbiology Center of China Microbial Culture Collection Management Committee.
保藏单位简称：CGMCC。Abbreviation of depository unit: CGMCC.
地址：北京市朝阳区北辰西路1号院3号。Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
保藏日期：2019年12月10日。Date of preservation: December 10, 2019.
保藏编号：CGMCC No.19195。Deposit number: CGMCC No. 19195.
3、生物材料的的分类命名：分泌微囊藻毒素广谱型抗体的阳性小鼠杂交瘤细胞株。3. Classification and naming of biological materials: positive mouse hybridoma cell lines secreting broad-spectrum antibodies to microcystin.
生物材料的编号：MCAB3。The number of the biological material: MCAB3.
保藏单位全称：中国微生物菌种保藏管理委员会普通微生物中心。Full name of the depository: General Microbiology Center of China Microbial Culture Collection Management Committee.
保藏单位简称：CGMCC。Abbreviation of depository unit: CGMCC.
地址：北京市朝阳区北辰西路1号院3号。Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
保藏日期：2019年12月10日。Date of preservation: December 10, 2019.
保藏编号：CGMCC No.19196。Deposit number: CGMCC No. 19196.
4、生物材料的的分类命名：分泌微囊藻毒素广谱型抗体的阳性小鼠杂交瘤细胞株。4. Classification and naming of biological materials: positive mouse hybridoma cell lines secreting broad-spectrum antibodies to microcystin.
生物材料的编号：MCAB4。The number of the biological material: MCAB4.
保藏单位全称：中国微生物菌种保藏管理委员会普通微生物中心。Full name of the depository: General Microbiology Center of China Microbial Culture Collection Management Committee.
保藏单位简称：CGMCC。Abbreviation of depository unit: CGMCC.
地址：北京市朝阳区北辰西路1号院3号。Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
保藏日期：2019年12月10日。Date of preservation: December 10, 2019.
保藏编号：CGMCC No.19197。Deposit number: CGMCC No. 19197.
5、生物材料的的分类命名：分泌微囊藻毒素广谱型抗体的阳性小鼠杂交瘤细胞株。5. Classification and naming of biological materials: positive mouse hybridoma cell lines secreting broad-spectrum antibodies to microcystin.
生物材料的编号：MCAB5。The number of the biological material: MCAB5.
保藏单位全称：中国微生物菌种保藏管理委员会普通微生物中心。Full name of the depository: General Microbiology Center of China Microbial Culture Collection Management Committee.
保藏单位简称：CGMCC。Abbreviation of depository unit: CGMCC.
地址：北京市朝阳区北辰西路1号院3号。Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
保藏日期：2019年12月10日。Date of preservation: December 10, 2019.
保藏编号：CGMCC No.19198。Deposit number: CGMCC No. 19198.
附图说明Description of the drawings
图1为本发明筛选出的阳性杂交瘤细胞株MCAB2分泌的单克隆抗体与不同浓度的包被抗原BSA-MC-LR的结合曲线。Figure 1 shows the binding curves of monoclonal antibodies secreted by the positive hybridoma cell line MCAB2 screened in the present invention and different concentrations of the coating antigen BSA-MC-LR.
图2为本发明筛选出的阳性杂交瘤细胞株MCAB5分泌的单克隆抗体分别检测水样中的MC-LR、MC-RR和MC-YR的标准曲线。Fig. 2 is a standard curve for detecting MC-LR, MC-RR and MC-YR in water samples by monoclonal antibodies secreted by the positive hybridoma cell line MCAB5 selected by the present invention.
实施发明的最佳方式The best way to implement the invention
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。The present invention will be further described in detail below in conjunction with specific embodiments, and the examples given are only to illustrate the present invention, not to limit the scope of the present invention. The examples provided below can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute a limitation to the present invention in any way.
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.
骨髓瘤细胞SP2/0种子(北京华大蛋白质研发中心)；SPF级Balb/c纯系雌性小鼠(斯贝福北京生物技术有限公司)；新生牛血清(南京维森特生物技 术有限公司)；酶标二抗(羊抗小鼠IgG/HRP)(北京中杉金桥生物技术有限公司)；PEG 1500(Roche公司)；HAT储液、HT储液、石蜡油、BSA、血蓝蛋白(KLH)、三羟甲基氨基甲烷盐酸盐(Tris-HCl)、2.2％甲基纤维素、2-巯基乙胺、戊二醛、柠檬酸、柠檬酸钠、氯化钠、磷酸氢二钠、磷酸二氢钠、碳酸盐缓冲液、四甲基联苯胺(TMB)、过氧化氢合尿素(CO(NH 2) 2·H 2O 2)、吐温-20、弗氏完全佐剂、弗氏不完全佐剂(Sigma-Aldrich公司)；IMDM培养基(Invitrogen公司)；二甲基亚砜(DMSO)(Amresco公司)；BupH PBS缓冲液(Thermo Fisher Scientific公司)；赖氨酸(上海源叶生物科技有限公司)；无水乙醚、乙酸、异丙醇、甲醇、盐酸、硫酸、氢氧化钠(北京化工厂)；MC-LR、MC-RR、MC-YR、MC-LA、MC-LF、MC-LW、MC-LY、MC-WR、MC-HtyR、MC-HilR、[D-Asp3]MC-LR、[D-Asp3]MC-RR(Enzo Life Sciences公司)。 Myeloma cell SP2/0 seeds (Beijing Huada Protein Research and Development Center); SPF-grade Balb/c pure-line female mice (Sbeef Beijing Biotechnology Co., Ltd.); newborn calf serum (Nanjing Vicente Biotechnology Co., Ltd.) ; Enzyme-labeled secondary antibody (goat anti-mouse IgG/HRP) (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.); PEG 1500 (Roche); HAT stock solution, HT stock solution, paraffin oil, BSA, hemocyanin (KLH) , Tris-hydroxymethylaminomethane hydrochloride (Tris-HCl), 2.2% methylcellulose, 2-mercaptoethylamine, glutaraldehyde, citric acid, sodium citrate, sodium chloride, disodium hydrogen phosphate, phosphoric acid Sodium dihydrogen, carbonate buffer, tetramethylbenzidine (TMB), hydrogen peroxide and urea (CO(NH 2 ) 2 ·H 2 O 2 ), Tween-20, Freund's complete adjuvant, Freund Incomplete adjuvant (Sigma-Aldrich); IMDM medium (Invitrogen); Dimethyl sulfoxide (DMSO) (Amresco); BupH PBS buffer (Thermo Fisher Scientific); Lysine (Shanghai source Ye Biotechnology Co., Ltd.); Anhydrous ether, acetic acid, isopropanol, methanol, hydrochloric acid, sulfuric acid, sodium hydroxide (Beijing Chemical Plant); MC-LR, MC-RR, MC-YR, MC-LA, MC- LF, MC-LW, MC-LY, MC-WR, MC-HtyR, MC-HilR, [D-Asp3]MC-LR, [D-Asp3]MC-RR (Enzo Life Sciences).
实施例1、阳性杂交瘤细胞株的获得、鉴定以及广谱型微囊藻毒素单克隆抗体的制备Example 1. Obtainment and identification of positive hybridoma cell lines and preparation of broad-spectrum microcystin monoclonal antibodies
1、免疫原KLH-MCs的合成1. Synthesis of immunogen KLH-MCs
上述12种微囊藻毒素小分子结构变异体，分别在其第七位氨基酸(Mdha)上通过2-巯基乙胺化学修饰引入一个氨基活性基团，经固相萃取纯化后采用戊二醛两步法分别将修饰后的微囊藻毒素分子与KLH偶联，再经凝胶过滤层析后得到12种完全抗原，等量混合后得到免疫原KLH-MCs。具体方法如下：The above-mentioned 12 microcystin small molecular structural variants, respectively, introduced an amino active group on the seventh amino acid (Mdha) through 2-mercaptoethylamine chemical modification, and purified by solid phase extraction using glutaraldehyde two The modified microcystin molecules were coupled with KLH by step method, and 12 kinds of complete antigens were obtained after gel filtration chromatography, and the immunogen KLH-MCs was obtained after equal mixing. The specific method is as follows:
步骤1氨基修饰的微囊藻毒素分子与KLH偶联物的制备 Step 1 Preparation of conjugate of amino-modified microcystin molecule and KLH
1.1、氨基修饰的MC-LR与KLH偶联物的制备1.1. Preparation of amino-modified MC-LR and KLH conjugate
将摩尔比3000:1的2-巯基乙胺和微囊藻毒素小分子MC-LR溶于碱性碳酸盐缓冲液中(pH＝8.0)，50℃水浴中反应1-3小时；降至室温后加入乙酸使反应停止得到中间产物；将中间产物采用固相萃取技术在SPE固相萃取C18小柱(500mg/6ml)(Agela Technologies)上进行纯化，得到氨基修饰的MC-LR，具体纯化步骤为：Dissolve 2-mercaptoethylamine and microcystin small molecule MC-LR with a molar ratio of 3000:1 in alkaline carbonate buffer (pH=8.0), and react in a water bath at 50°C for 1-3 hours; After room temperature, acetic acid was added to stop the reaction to obtain the intermediate product; the intermediate product was purified on a SPE solid-phase extraction C18 cartridge (500mg/6ml) (Agela Technologies) using solid-phase extraction technology to obtain amino-modified MC-LR, which was specifically purified The steps are:
活化：先用甲醇活化柱子，15分钟后将超纯水以一定的流速流过柱子；Activation: first activate the column with methanol, and flow ultrapure water through the column at a certain flow rate after 15 minutes;
上样：将样品以一定的流速流过柱子进行富集浓缩，重力过滤；Sample loading: the sample flows through the column at a certain flow rate for enrichment and concentration, and gravity filtration;
淋洗：上样完毕后，用5％体积比的甲醇水溶液淋洗以净化样品；Rinse: After loading the sample, rinse with 5% by volume methanol aqueous solution to purify the sample;
洗脱：用甲醇洗脱样品并收集，得到经氨基修饰的MC-LR。Elution: The sample was eluted with methanol and collected to obtain the amino-modified MC-LR.
20mg载体蛋白KLH，先用少量PBS充分溶解，再加入2mL 1.25％的戊二醛溶液，室温反应12h以上，活化产物经Sephadex G-25 PD-10 Desalting Column(GE Healthcare)进行层析，其中流动相为超纯水，得到活化后的KLH，并用PBS溶液稀释；0.5mg活化后的KLH与30μg氨基修饰的MC-LR充分混合，室温下反应24h，得到氨基修饰的MC-LR与KLH偶联物，以下简称为NH 2-MC-LR-KLH，将NH 2-MC-LR-KLH放置于-20℃冰箱中备用。 20mg carrier protein KLH, first fully dissolved in a small amount of PBS, then added 2mL 1.25% glutaraldehyde solution, reacted at room temperature for more than 12h, the activated product was chromatographed on Sephadex G-25 PD-10 Desalting Column (GE Healthcare), and the flow The phase is ultrapure water, the activated KLH is obtained, and diluted with PBS solution; 0.5mg of activated KLH is mixed with 30μg amino-modified MC-LR, and reacted at room temperature for 24h to obtain amino-modified MC-LR coupled with KLH The substance, hereinafter referred to as NH 2 -MC-LR-KLH, put the NH 2 -MC-LR-KLH in a refrigerator at -20°C for later use.
1.2、氨基修饰的MC-RR与KLH偶联物的制备1.2. Preparation of amino-modified MC-RR and KLH conjugate
将步骤1.1中的MC-LR替换为MC-RR，其它操作完全相同，得到氨基修饰的MC-RR与KLH偶联物，以下简称为NH 2-MC-RR-KLH，将NH 2-MC-RR-KLH放置于-20℃冰箱中备用。 Replace the MC-LR in step 1.1 with MC-RR, and the other operations are exactly the same to obtain the amino-modified MC-RR and KLH conjugate, hereinafter referred to as NH 2 -MC-RR-KLH, and NH 2 -MC- RR-KLH is placed in a refrigerator at -20℃ for later use.
1.3、氨基修饰的MC-YR与KLH偶联物的制备1.3. Preparation of amino-modified MC-YR and KLH conjugate
将步骤1.1中的MC-LR替换为MC-YR，其它操作完全相同，得到氨基修饰的MC-YR与KLH偶联物，以下简称为NH 2-MC-YR-KLH，将NH 2-MC-YR-KLH放置于-20℃冰箱中备用。 Replace MC-LR in step 1.1 with MC-YR, and other operations are exactly the same to obtain amino-modified MC-YR and KLH conjugate, hereinafter referred to as NH 2 -MC-YR-KLH, and NH 2 -MC- YR-KLH is placed in a refrigerator at -20℃ for later use.
1.4、氨基修饰的MC-LA与KLH偶联物的制备1.4. Preparation of amino-modified MC-LA and KLH conjugate
将步骤1.1中的MC-LR替换为MC-LA，其它操作完全相同，得到氨基修饰的MC-LA与KLH偶联物，以下简称为NH 2-MC-LA-KLH，将NH 2-MC-LA-KLH放置于-20℃冰箱中备用。 Replace the MC-LR in step 1.1 with MC-LA, and the other operations are exactly the same to obtain the amino-modified MC-LA and KLH conjugate, hereinafter referred to as NH 2 -MC-LA-KLH, and NH 2 -MC- LA-KLH is placed in a refrigerator at -20°C for later use.
1.5、氨基修饰的MC-LF与KLH偶联物的制备1.5. Preparation of amino-modified MC-LF and KLH conjugate
将步骤1.1中的MC-LR替换为MC-LF，其它操作完全相同，得到氨基修饰的MC-LF与KLH偶联物，以下简称为NH 2-MC-LF-KLH，将NH 2-MC-LF-KLH放置于-20℃冰箱中备用。 Replace MC-LR in step 1.1 with MC-LF, and other operations are exactly the same to obtain amino-modified MC-LF and KLH conjugate, hereinafter referred to as NH 2 -MC-LF-KLH, and NH 2 -MC- LF-KLH is placed in a refrigerator at -20℃ for later use.
1.6、氨基修饰的MC-LW与KLH偶联物的制备1.6. Preparation of amino-modified MC-LW and KLH conjugate
将步骤1.1中的MC-LR替换为MC-LW，其它操作完全相同，得到氨基修饰的MC-LW与KLH偶联物，以下简称为NH 2-MC-LW-KLH，将NH 2-MC-LW-KLH放置于-20℃冰箱中备用。 Replace MC-LR in step 1.1 with MC-LW, and other operations are exactly the same to obtain amino-modified MC-LW and KLH conjugate, hereinafter referred to as NH 2 -MC-LW-KLH, and NH 2 -MC- Place LW-KLH in a refrigerator at -20°C for later use.
1.7、氨基修饰的MC-LY与KLH偶联物的制备1.7. Preparation of amino-modified MC-LY and KLH conjugate
将步骤1.1中的MC-LR替换为MC-LY，其它操作完全相同，得到氨基修饰的MC-LY与KLH偶联物，以下简称为NH 2-MC-LY-KLH，将NH 2-MC-LY-KLH放置于-20℃冰箱中备用。 Replace MC-LR in step 1.1 with MC-LY, and the other operations are exactly the same to obtain amino-modified MC-LY and KLH conjugate, hereinafter referred to as NH 2 -MC-LY-KLH, and NH 2 -MC- LY-KLH is placed in a refrigerator at -20℃ for later use.
1.8、氨基修饰的MC-WR与KLH偶联物的制备1.8. Preparation of amino-modified MC-WR and KLH conjugate
将步骤1.1中的MC-LR替换为MC-WR，其它操作完全相同，得到氨基修饰的MC-WR与KLH偶联物，以下简称为NH 2-MC-WR-KLH，将NH 2-MC-WR-KLH放置于-20℃冰箱中备用。 Replace MC-LR in step 1.1 with MC-WR, and other operations are exactly the same to obtain amino-modified MC-WR and KLH conjugate, hereinafter referred to as NH 2 -MC-WR-KLH, and NH 2 -MC- WR-KLH is placed in a refrigerator at -20℃ for later use.
1.9、氨基修饰的MC-HtyR与KLH偶联物的制备1.9. Preparation of amino-modified MC-HtyR and KLH conjugate
将步骤1.1中的MC-LR替换为MC-HtyR，其它操作完全相同，得到氨基修饰的MC-HtyR与KLH偶联物，以下简称为NH 2-MC-HtyR-KLH，将NH 2-MC-HtyR-KLH放置于-20℃冰箱中备用。 Replace MC-LR in step 1.1 with MC-HtyR, and other operations are exactly the same to obtain amino-modified MC-HtyR and KLH conjugate, hereinafter referred to as NH 2 -MC-HtyR-KLH, and NH 2 -MC- HtyR-KLH is placed in a refrigerator at -20°C for later use.
1.10、氨基修饰的MC-HilR与KLH偶联物的制备1.10. Preparation of amino-modified MC-HilR and KLH conjugate
将步骤1.1中的MC-LR替换为MC-HilR，其它操作完全相同，得到氨基修饰的MC-HilR与KLH偶联物，以下简称为NH 2-MC-HilR-KLH，将NH 2-MC-HilR-KLH放置于-20℃冰箱中备用。 Replace MC-LR in step 1.1 with MC-HilR, and other operations are exactly the same to obtain amino-modified MC-HilR and KLH conjugate, hereinafter referred to as NH 2 -MC-HilR-KLH, and NH 2 -MC- HilR-KLH is placed in a refrigerator at -20℃ for later use.
1.11、氨基修饰的[D-Asp3]MC-LR与KLH偶联物的制备1.11 Preparation of amino-modified [D-Asp3] MC-LR and KLH conjugate
将步骤1.1中的MC-LR替换为[D-Asp3]MC-LR，其它操作完全相同，得到氨基修饰的[D-Asp3]MC-LR与KLH偶联物，以下简称为NH 2-[D-Asp3]MC-LR-KLH，将NH 2-[D-Asp3]MC-LR-KLH放置于-20℃冰箱中备用。 Replace MC-LR in step 1.1 with [D-Asp3]MC-LR, and other operations are exactly the same to obtain amino-modified [D-Asp3]MC-LR and KLH conjugate, hereinafter referred to as NH 2 -[D -Asp3]MC-LR-KLH, put NH 2 -[D-Asp3]MC-LR-KLH in a refrigerator at -20℃ for standby.
1.12、氨基修饰的[D-Asp3]MC-RR与KLH偶联物的制备1.12 Preparation of amino-modified [D-Asp3] MC-RR and KLH conjugate
将步骤1.1中的MC-LR替换为[D-Asp3]MC-RR，其它操作完全相同，得到氨基修饰的[D-Asp3]MC-RR与KLH偶联物，以下简称为NH 2-[D-Asp3]MC-RR-KLH，将NH 2-[D-Asp3]MC-RR-KLH放置于-20℃冰箱中备用。 Replace MC-LR in step 1.1 with [D-Asp3]MC-RR, and other operations are exactly the same to obtain amino-modified [D-Asp3]MC-RR and KLH conjugate, hereinafter referred to as NH 2 -[D -Asp3]MC-RR-KLH, put NH 2 -[D-Asp3]MC-RR-KLH in a refrigerator at -20℃ for use.
上述步骤中，12种微囊藻毒素小分子结构变异体的结构通式如式(1)所示，修饰后的12种微囊藻毒素小分子结构变异体的结构通式如式(2)所示，12种微囊藻毒素小分子结构变异体的R1、R2和R3的具体基团分别如表1所示。In the above steps, the general structural formulas of the 12 microcystin small molecular structural variants are as shown in formula (1), and the structural general formulas of the 12 modified microcystin small molecular structural variants are as shown in formula (2) As shown, the specific groups of R1, R2 and R3 of the 12 microcystin small molecular structure variants are shown in Table 1, respectively.
Figure PCTCN2021082941-appb-000003
Figure PCTCN2021082941-appb-000003
表1、12种微囊藻毒素小分子结构变异体在可变位点的特定基团Table 1. Specific groups at variable positions of 12 microcystins small molecular structure variants
Figure PCTCN2021082941-appb-000004
Figure PCTCN2021082941-appb-000004
Figure PCTCN2021082941-appb-000005
Figure PCTCN2021082941-appb-000005
2免疫原的制备2 Preparation of immunogen
将步骤1.1-1.12中制备的偶联物NH 2-MC-LR-KLH,NH 2-MC-RR-KLH,NH 2-MC-YR-KLH,NH 2-MC-LA-KLH,NH 2-MC-LF-KLH,NH 2-MC-LW-KLH,NH 2-MC-LY-KLH,NH 2-MC-WR-KLH,NH 2-MC-HtyR-KLH,NH 2-MC-HilR-KLH,NH 2-[D-Asp3]MC-LR-KLH和NH 2-[D-Asp3]MC-RR-KLH等质量混合后，得到免疫原KLH-MCs溶液。 Combine the conjugates prepared in steps 1.1-1.12 NH 2 -MC-LR-KLH, NH 2 -MC-RR-KLH, NH 2 -MC-YR-KLH, NH 2 -MC-LA-KLH, NH 2- MC-LF-KLH,NH 2 -MC-LW-KLH,NH 2 -MC-LY-KLH,NH 2 -MC-WR-KLH,NH 2 -MC-HtyR-KLH,NH 2 -MC-HilR-KLH NH 2 -[D-Asp3]MC-LR-KLH and NH 2 -[D-Asp3]MC-RR-KLH are mixed with equal mass to obtain the immunogen KLH-MCs solution.
(2)动物免疫与效价测定(2) Animal immunity and titer determination
将步骤2的免疫原KLH-MCs溶液与免疫佐剂等体积混合后免疫小鼠。The immunogen KLH-MCs solution of step 2 was mixed with an equal volume of the immune adjuvant to immunize mice.
选取适龄的SPF级Balb/c雌性小鼠，采用低剂量长程免疫法进行免疫，方法为：皮下多点注射，按照50μg KLH-MCs/只小鼠的剂量，对5只小鼠(编号为：1、2、3、4、5)进行初次免疫，每次每只小鼠注射0.1mL液体，其中，该0.1mL液体由0.05mL免疫原KLH-MCs溶液和0.05mL弗氏完全佐剂组成；之后按照30μg KLH-MCs/只小鼠的量对5只小鼠进行加强免疫，免疫间隔时间为2周，共免疫6次，每次每只小鼠注射0.06mL液体，其中，该0.06mL液体由0.03mL免疫原KLH-MCs溶液和0.03mL弗氏不完全佐剂组成。在第6次加强免疫1周后，对小鼠进行眼眶取血，用间接ELISA法测定效价，其中，包被抗原为12种BSA-MC的等量混合物，包被浓度为2μg/mL。对抗血清效价达到要求的小鼠，按照50μg免疫原/只小鼠的量进行一次腹腔注射冲击免疫，此次所用免疫原为12种BSA-MC的等量混合物(BSA-MCs)，3天后再次测定效价，选取血清效价最高的小鼠脾细胞进行细胞融合。Select age-appropriate SPF-grade Balb/c female mice and use the low-dose long-term immunization method for immunization. The method is: subcutaneous multi-point injection, according to the dose of 50μg KLH-MCs/mouse, to 5 mice (number: 1, 2, 3, 4, 5) for the first immunization, each mouse is injected with 0.1 mL of liquid each time, wherein the 0.1 mL of liquid is composed of 0.05 mL of immunogen KLH-MCs solution and 0.05 mL of Freund's complete adjuvant; After that, 5 mice were boosted with the amount of 30μg KLH-MCs/mouse. The interval of immunization was 2 weeks. A total of 6 immunizations were carried out. Each mouse was injected with 0.06mL liquid each time, of which the 0.06mL liquid It consists of 0.03mL immunogen KLH-MCs solution and 0.03mL Freund's incomplete adjuvant. One week after the sixth booster immunization, blood was taken from the orbit of the mice, and the titer was determined by indirect ELISA. The coating antigen was an equal mixture of 12 kinds of BSA-MC, and the coating concentration was 2 μg/mL. For mice with the required antiserum titer, an intraperitoneal injection of 50μg of immunogen/mouse was performed for impulse immunization. The immunogen used this time was an equal mixture of 12 kinds of BSA-MC (BSA-MCs), 3 days later The titer was determined again, and the mouse spleen cells with the highest serum titer were selected for cell fusion.
上述实验中，包被抗原BSA-MC的合成方法具体为：包被抗原的合成方法同步骤1中所述的步骤，将步骤1的中的KLH替换为BSA，最后再加入20μL浓度为0.2M的赖氨酸溶液于室温下反应1～4h，以阻断未反应的醛基，得到的包被抗原放置于-20℃冰箱中备用。对12种BSA-MC分别用MALDI-TOF/MS进行质谱鉴定，测定的12种微囊藻毒素小分子与BSA的偶联比均为1:1。In the above experiment, the synthesis method of the coated antigen BSA-MC is specifically: the synthesis method of the coated antigen is the same as that described in step 1, replace KLH in step 1 with BSA, and finally add 20 μL to a concentration of 0.2M The lysine solution is reacted at room temperature for 1 to 4 hours to block unreacted aldehyde groups, and the obtained coated antigen is placed in a refrigerator at -20°C for later use. The 12 kinds of BSA-MC were identified by MALDI-TOF/MS by mass spectrometry, and the coupling ratios of the 12 kinds of microcystin small molecules and BSA were all 1:1.
(2)细胞融合(2) Cell fusion
1)将状态良好的小鼠骨髓瘤细胞SP2/0细胞轻轻地从培养瓶壁上吹打下来，转移至50mL离心管中；小鼠摘眼球取血后拉颈处死，放入75％的酒精中浸泡5min；1) The mouse myeloma cell SP2/0 cells in good condition are gently pipetted off the wall of the culture flask and transferred to a 50mL centrifuge tube; the mouse is put to death by pulling the neck after removing the eyeballs and blood, and putting in 75% alcohol Soak in medium for 5min;
2)在平皿中倒入少量不含血清的IMDM培养基，将细胞筛及注射器内芯放入平皿中；2) Pour a small amount of serum-free IMDM medium into the dish, and put the cell sieve and the inner core of the syringe into the dish;
3)在无菌状态下取出小鼠脾脏，置于细胞筛上，用注射器内芯轻轻地将脾脏充分碾碎，再将碾好的细胞吸入到装有SP2/0细胞的离心管中，室温1500r/min下离心5min；3) Take out the mouse spleen in a sterile state, place it on the cell sieve, gently crush the spleen with a syringe core, and then suck the crushed cells into a centrifuge tube containing SP2/0 cells. Centrifuge at 1500r/min for 5min at room temperature;
4)取小鼠的胸腺并碾碎，转移至15mL离心管中，再加入1mL HAT培养液，于孵箱中(37℃，5％CO 2)进行培养。 4) Take the mouse thymus and crush it, transfer it to a 15mL centrifuge tube, add 1mL HAT culture solution, and culture in an incubator (37°C, 5% CO 2 ).
5)将离心好的细胞弃上清液，用不含血清的IMDM培养基将细胞小心轻柔地吹匀，再次于室温1500r/min下离心5min。5) Discard the supernatant of the centrifuged cells, use the serum-free IMDM medium to blow the cells gently and evenly, and centrifuge again at 1500r/min for 5min at room temperature.
6)将离心好的细胞弃上清液，轻拍离心管底使细胞充分悬浮，再将离心管置于37℃温水浴中，并在1min内缓慢加入1mL聚乙二醇(PEG)，静置1min后，在2min内缓慢加入2mL不含血清的IMDM培养基，接着在2min内缓慢加入8mL不含血清的IMDM培养基，室温1000r/min下离心5min。6) Discard the supernatant of the centrifuged cells, tap the bottom of the centrifuge tube to fully suspend the cells, then place the centrifuge tube in a 37°C warm water bath, and slowly add 1 mL polyethylene glycol (PEG) within 1 min. After 1 min, slowly add 2 mL serum-free IMDM medium within 2 min, and then slowly add 8 mL serum-free IMDM medium within 2 min, and centrifuge for 5 min at 1000 r/min at room temperature.
7)弃上清，加入10mL血清，并小心将细胞吹匀，加入步骤4)备好的胸腺细胞，再加入25mL无菌的半固体培养基，充分混匀后均匀倒入30个细胞培养皿中并置于湿盒中，于孵箱中(37℃，5％CO 2)培养。 7) Discard the supernatant, add 10 mL of serum, and carefully blow the cells evenly, add the thymocytes prepared in step 4), and then add 25 mL of sterile semi-solid medium, mix well and pour into 30 cell culture dishes evenly And placed in a humid box, cultured in an incubator (37°C, 5% CO 2 ).
8)在第11天左右，开始挑克隆，此时细胞团大小肉眼可见，在显微镜下能够很好的分辩(注意防止培养基颜色变黄，导致营养不够，细胞团出现死亡；若细胞融合团较多，可提前挑克隆)。挑10板×93个细胞单克隆于96孔细胞培养板中培养(事先用含小鼠胸腺细胞的20％IMDM+2％HT培养液铺板，120μL/孔)，第3天换液一次，第5天细胞已铺满96孔板，此时采用间接ELISA检测培养基上清液中的抗体。8) On the 11th day or so, start to pick clones. At this time, the size of the cell cluster is visible to the naked eye and can be distinguished well under the microscope. More, you can pick clones in advance). Pick 10 plates × 93 single cells and culture them in a 96-well cell culture plate (previously plate with 20% IMDM+2% HT medium containing mouse thymocytes, 120μL/well), and change the medium once on the third day. After 5 days, the cells have spread to the 96-well plate. At this time, indirect ELISA was used to detect the antibodies in the medium supernatant.
(5)阳性克隆株筛选(5) Screening of positive clones
1)用包被液(50mM碳酸盐缓冲液，pH 9.6)稀释“BSA-MC”使其终浓度为2μg/mL，100μL/孔加入到96孔酶联板中进行包被，于4℃包被过夜后用PBS-T(0.05％吐温-20-PBS)洗液洗涤3次。1) Dilute "BSA-MC" with a coating solution (50mM carbonate buffer, pH 9.6) to a final concentration of 2μg/mL, and add 100μL/well to a 96-well ELISA plate for coating, at 4°C After coating overnight, it was washed 3 times with PBS-T (0.05% Tween-20-PBS) washing solution.
2)用封闭液(2％脱脂牛奶-PBS)封闭，200μL/孔，37℃孵育2h后用洗 液洗涤3次。2) Block with blocking solution (2% skimmed milk-PBS), 200 µL/well, incubate at 37°C for 2 hours, and wash with washing solution 3 times.
3)分别于相应孔中加入一抗(96孔板中的细胞培养上清液)、阴性对照(SP2/0培养上清液)、空白对照(PBS溶液)、阳性对照(PBS稀释1000倍阳性血清)，均为100μL/孔，37℃孵育1h后用洗液洗涤3次。3) Add primary antibody (cell culture supernatant in 96-well plate), negative control (SP2/0 culture supernatant), blank control (PBS solution), and positive control (PBS diluted 1000 times positive) to the corresponding wells. Serum), all 100μL/well, incubated at 37°C for 1h and washed 3 times with lotion.
4)加入PBS稀释20000倍的酶标二抗(山羊抗小鼠IgG/HRP)，100μL/孔，37℃孵育1h后用洗液洗涤3次。4) Add enzyme-labeled secondary antibody (goat anti-mouse IgG/HRP) diluted 20000 times in PBS, 100μL/well, incubate at 37°C for 1h, and wash with lotion 3 times.
5)加显色液(1％A液+10％B液+超纯水(A液：1％TMB-DMSO；B液：0.1％H 2O 2-柠檬酸缓冲液))，100μL/孔，显色时间为5min左右。 5) Add color developing solution (1% A solution + 10% B solution + ultrapure water (A solution: 1% TMB-DMSO; B solution: 0.1% H 2 O 2 -citrate buffer)), 100 μL/well , The color development time is about 5min.
6)加终止液(2M浓硫酸)，50μL/孔，终止反应。6) Add stop solution (2M concentrated sulfuric acid), 50μL/well to stop the reaction.
7)酶标仪测450nm处吸光值，记录保存数据。7) Measure the absorbance at 450nm with a microplate reader, record and save the data.
A 450值为阴性对照2.1倍的为阳性杂交瘤细胞株。对于一筛呈阳性的31株克隆株，进一步培养后，按照同样方法进行第二次筛选，得到23株阳性杂交瘤细胞株。 A 450 value of 2.1 times the negative control is a positive hybridoma cell line. For the 31 clones that were positive in one screen, they were further cultured and then screened for the second time according to the same method to obtain 23 positive hybridoma cell lines.
(6)阳性克隆株亚类鉴定(6) Identification of subtypes of positive clones
1)用100mM PBS(pH7.4)溶液稀释包被抗体(各亚类抗体混合物)至0.5μg/mL,0.1mL/孔，4℃孵育过夜。1) Dilute the coated antibody (antibody mixture of each subclass) with 100mM PBS (pH7.4) solution to 0.5μg/mL, 0.1mL/well, and incubate overnight at 4°C.
2)PBS-T洗2次，加入封闭液，200μL/孔，37℃孵育2h。2) Wash twice with PBS-T, add blocking solution, 200μL/well, and incubate at 37°C for 2h.
3)PBS-T洗3次，加入阳性杂交瘤上清液，100μL/孔，37℃孵育1h。3) Wash with PBS-T 3 times, add the positive hybridoma supernatant, 100 μL/well, and incubate at 37°C for 1 hour.
4)PBS-T洗3次，相应孔中分别加入封闭液(2％BSA+3％蔗糖于PBS中)1：1000(轻链：κ，λ)或1：2000(重链：M,G1,G2a,G2b,G3,A)稀释的各亚类酶标二抗，0.1mL/孔，37℃孵育1h。4) Wash 3 times with PBS-T, add blocking solution (2% BSA+3% sucrose in PBS) 1:1000 (light chain: κ, λ) or 1:2000 (heavy chain: M, G1) to the corresponding wells. , G2a, G2b, G3, A) Diluted enzyme-labeled secondary antibodies of each subclass, 0.1 mL/well, incubate at 37°C for 1 h.
5)PBS-T洗3次，加入显色溶液，100μL/孔，5min后加终止液，50μL/孔，10-20min内用酶标仪测450nm处吸光值，记录保存数据。5) Wash 3 times with PBS-T, add coloring solution, 100μL/well, add stop solution after 5min, 50μL/well, measure the absorbance at 450nm with a microplate reader within 10-20min, record and save the data.
23株阳性细胞株进行亚类鉴定后，确认其中5株为IgG1型阳性杂交瘤细胞株。分别用12种BSA-MC单体和BSA再次包板，采用间接ELISA方法做筛选，确认5株IgG1型阳性杂交瘤细胞株对12种微囊藻毒素均具有特异性响应。5株阳性杂交瘤细胞株，名称分别为MCAB1，MCAB2，MCAB3，MCAB4，MCAB5，于2019年12月10日保藏于中国普通微生物菌种保藏管理中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，杂交瘤细胞株MCAB1的保藏编号分别为CGMCC No.19194，杂交瘤细胞株MCAB2的保藏编号分 别为CGMCC No.19195，杂交瘤细胞株MCAB3的保藏编号分别为CGMCC No.19196，杂交瘤细胞株MCAB4的保藏编号分别为CGMCC No.19197，杂交瘤细胞株MCAB5的保藏编号分别为CGMCC No.19198。After subclass identification of 23 positive cell lines, 5 of them were confirmed to be IgG1 positive hybridoma cell lines. Re-coated with 12 kinds of BSA-MC monomer and BSA respectively, and screened by indirect ELISA method, confirming that 5 strains of IgG1-positive hybridoma cell lines have specific responses to 12 kinds of microcystins. Five positive hybridoma cell lines, named MCAB1, MCAB2, MCAB3, MCAB4, MCAB5, were deposited at the China Common Microbial Culture Collection and Management Center (abbreviated as CGMCC, address: Beichen West, Chaoyang District, Beijing) on December 10, 2019 No. 1, No. 3, Institute of Microbiology, Chinese Academy of Sciences), the deposit number of the hybridoma cell line MCAB1 is CGMCC No.19194, the deposit number of the hybridoma cell line MCAB2 is CGMCC No.19195, the hybridoma cell line MCAB3 The deposit number is CGMCC No. 19196, the deposit number of the hybridoma cell line MCAB4 is CGMCC No. 19197, and the deposit number of the hybridoma cell line MCAB5 is CGMCC No. 19198.
分别取上述5种杂交瘤细胞株按照步骤(7)和步骤(8)的方法制备单克隆抗体，并将杂交瘤细胞株MCAB1分泌的单克隆抗体命名为抗体MCAB1；杂交瘤细胞株MCAB2分泌的单克隆抗体命名为抗体MCAB2；杂交瘤细胞株MCAB3分泌的单克隆抗体命名为抗体MCAB3；杂交瘤细胞株MCAB4分泌的单克隆抗体命名为抗体MCAB4；杂交瘤细胞株MCAB5分泌的单克隆抗体命名为抗体MCAB5。The above five hybridoma cell lines were taken to prepare monoclonal antibodies according to the method of step (7) and step (8), and the monoclonal antibody secreted by the hybridoma cell line MCAB1 was named antibody MCAB1; the monoclonal antibody secreted by the hybridoma cell line MCAB2 The monoclonal antibody is named antibody MCAB2; the monoclonal antibody secreted by the hybridoma cell line MCAB3 is named antibody MCAB3; the monoclonal antibody secreted by the hybridoma cell line MCAB4 is named antibody MCAB4; the monoclonal antibody secreted by the hybridoma cell line MCAB5 is named Antibody MCAB5.
(7)腹水制备(7) Ascites preparation
1)将恒温水浴箱温度调节至37℃-40℃。1) Adjust the temperature of the constant temperature water bath to 37°C-40°C.
2)从液氮罐中取出冻存的细胞株，立即放入37℃-40℃温水中迅速晃动，直至完全溶解。2) Take out the frozen cell line from the liquid nitrogen tank and immediately put it in 37°C-40°C warm water and shake it quickly until it is completely dissolved.
3)将细胞冻存悬浮液转移到离心管中，加入5mL完全培养基(IMDM+15％新生牛血清)，轻轻吹打混匀。3) Transfer the frozen cell suspension to a centrifuge tube, add 5 mL of complete medium (IMDM + 15% newborn calf serum), and gently pipette to mix.
4)将细胞悬浮液于室温1000r/min下离心5min，弃上清液。4) Centrifuge the cell suspension at 1000 r/min at room temperature for 5 min, and discard the supernatant.
5)向细胞沉淀内加入完全培养基，轻轻吹打混匀后，转移到细胞培养皿内，置于37℃细胞培养箱中培养。5) Add complete medium to the cell pellet, gently pipette to mix, transfer to a cell culture dish, and place it in a 37°C cell incubator for culture.
6)将细胞培养到对数生长期，PBS溶液洗涤并对细胞计数。6) Culture the cells to the logarithmic growth phase, wash with PBS solution and count the cells.
7)小鼠腹腔在接种一周前采用腹腔注射方式打石蜡油，500μL/Balb/c小鼠。7) The mouse's abdominal cavity was injected with paraffin oil, 500 μL/Balb/c mice, by intraperitoneal injection one week before inoculation.
8)采用腹腔注射方式接种细胞，每只小鼠注射1mL(细胞量5×10 5-9×10 5个/mL)。 8) The cells were inoculated by intraperitoneal injection, and each mouse was injected with 1 mL (cell volume 5×10 5 -9×10 5 cells/mL).
9)一周后取腹水，5000r/min下离心10min，取上清液，于-20℃保存。9) Take the ascites one week later, centrifuge at 5000r/min for 10min, take the supernatant, and store it at -20°C.
(8)抗体纯化(8) Antibody purification
1)腹水样本预处理：用偶联缓冲液(20mM磷酸钠溶液，pH 7.0)将解冻后的腹水上清液稀释3倍，12000r/min，4℃下离心10min，再用0.22μm滤膜过滤，以除去脂肪、细胞残渣及小颗粒物质。1) Pretreatment of ascites sample: Dilute the thawed ascites fluid 3 times with coupling buffer (20mM sodium phosphate solution, pH 7.0), 12000r/min, centrifuge at 4℃ for 10min, and filter with 0.22μm filter membrane , To remove fat, cell residues and small particulate matter.
2)平衡：用10倍抗体亲和预装柱(HiTrap rProtein A FF)体积的偶联缓冲液平衡柱子，保持流速为1mL/min。2) Equilibrium: Equilibrate the column with a coupling buffer of 10 times the volume of the antibody affinity prepacked column (HiTrap rProtein A FF), and keep the flow rate at 1 mL/min.
3)上样：柱中加入样品，收集流出液，保持流速为1mL/min。3) Sample loading: Add sample to the column, collect the effluent, and keep the flow rate at 1 mL/min.
4)洗杂：用5倍柱体积的偶联缓冲液过柱，保持流速为1mL/min。4) Wash impurities: Pass the column with 5 times the column volume of coupling buffer and keep the flow rate at 1 mL/min.
5)洗脱：用5倍柱体积的洗脱缓冲液(0.1M柠檬酸钠溶液，pH 3.5)洗脱抗体，收集于离心管中，保持流速为1mL/min，之后立即用1M pH 9.0的Tris-HCl缓冲液将收集液的pH调整至7.0。5) Elution: Use 5 column volumes of elution buffer (0.1M sodium citrate solution, pH 3.5) to elute the antibody, collect it in a centrifuge tube, keep the flow rate at 1 mL/min, and immediately use 1M pH 9.0 Adjust the pH of the collection solution to 7.0 with Tris-HCl buffer.
6)透析：使用0.01M PBS缓冲液将抗体透析过夜，换液3次。6) Dialysis: Use 0.01M PBS buffer to dialyze the antibody overnight and change the fluid 3 times.
得到的抗体采用SDS-PAGE(12％分离胶浓度)检测纯度，采用紫外分光光度计检测浓度。确认得到的5株抗体纯度都在90％以上，满足下游分析需求。The obtained antibody was tested for purity by SDS-PAGE (12% separation gel concentration) and concentration by UV spectrophotometer. It was confirmed that the purity of the five antibodies obtained was above 90%, which met the needs of downstream analysis.
实施例2、分别检测5株杂交瘤细胞株分泌的单克隆抗体与12种微囊藻毒素的亲和力Example 2: Detect the affinity of monoclonal antibodies secreted by 5 hybridoma cell lines to 12 microcystins
采用间接ELISA方法检测，具体操作步骤如下：Using indirect ELISA method to detect, the specific steps are as follows:
1)用包被液分别稀释12种“BSA-MC”，使其终浓度分别为4，2，1，0.5，0.25μg/mL，共5个梯度，100μL/孔加入到96孔酶联板中进行包被，于4℃包被过夜后用PBS-T洗液洗涤3次。1) Dilute 12 kinds of "BSA-MC" with the coating solution to make the final concentration of 4, 2, 1, 0.5, 0.25μg/mL respectively, 5 gradients in total, 100μL/well added to the 96-well enzyme-linked plate After coating overnight at 4°C, it was washed 3 times with PBS-T washing solution.
2)用封闭液(2％脱脂牛奶-PBS)封闭，200μL/孔，37℃孵育2～4h后用洗液洗涤3次。2) Block with blocking solution (2% skimmed milk-PBS), 200 μL/well, incubate at 37°C for 2 to 4 hours, and wash with washing solution 3 times.
3)将纯化后的5株抗体(抗体MCAB1、抗体MCAB2、抗体MCAB3、抗体MCAB4和抗体MCAB5)，用PBS分别稀释成1：250，再作一系列2倍梯度稀释，分别为1：500，1：1000，1：2000，1：4000，1：8000，1：16000，1：32000，1：64000，1：128000，1：256000，1：512000，共12个梯度；分别于相应孔中加入一抗(上述浓度梯度抗体)、空白对照(PBS溶液)，均为100μL/孔，37℃孵育1～4h后用洗液洗涤3次。3) Dilute the 5 purified antibodies (antibody MCAB1, antibody MCAB2, antibody MCAB3, antibody MCAB4, and antibody MCAB5) with PBS to 1:250, and then make a series of 2-fold dilutions to 1:500. 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, a total of 12 gradients; respectively in the corresponding holes Add the primary antibody (the above-mentioned concentration gradient antibody) and the blank control (PBS solution), both of which are 100 μL/well. After incubating at 37°C for 1 to 4 hours, they are washed 3 times with lotion.
4)加入PBS稀释20000倍的酶标二抗(山羊抗小鼠IgG/HRP)，100μL/孔，37℃孵育1～4h后用洗液洗涤3次。4) Add enzyme-labeled secondary antibody (goat anti-mouse IgG/HRP) diluted 20000 times in PBS, 100 μL/well, incubate at 37°C for 1 to 4 hours, and wash 3 times with lotion.
5)加显色液，100μL/孔，显色时间为5～10min左右。5) Add color developing solution, 100μL/well, and the color development time is about 5-10min.
6)加终止液(2M浓硫酸)，50μL/孔，终止反应。6) Add stop solution (2M concentrated sulfuric acid), 50μL/well to stop the reaction.
7)酶标仪测450nm处吸光值，记录保存数据。7) Measure the absorbance at 450nm with a microplate reader, record and save the data.
以阳性杂交瘤克隆株MCAB2分泌的抗体MCAB2与MC-LR的反应为例，取包被抗原浓度分别为2，1，0.5，0.25μg/mL测定抗原抗体反应曲线，结果如图1所示。以包被抗原浓度为2μg/mL的抗原抗体反应曲线为例，计算方法如下：Taking the reaction between the antibody MCAB2 secreted by the positive hybridoma clone MCAB2 and MC-LR as an example, the concentration of the coating antigen was 2, 1, 0.5, and 0.25 μg/mL to determine the antigen-antibody reaction curve. The results are shown in Figure 1. Taking the antigen-antibody reaction curve with a coating antigen concentration of 2μg/mL as an example, the calculation method is as follows:
1)通过对反应曲线进行Logistic模型拟合，得到2μg/mL包被抗原浓度对应最大吸光度一半(即1/2A max)时，抗体的稀释倍数。 1) By fitting the logistic model of the reaction curve, the dilution factor of the antibody when the concentration of 2μg/mL coating antigen corresponds to half of the maximum absorbance (ie 1/2A max) is obtained.
2)根据经过纯化的抗体MCAB2的浓度，可计算出在上述稀释倍数下对应的抗体总浓度Ab t，其中抗体分子量按150,000(IgG分子量)计算。 2) According to the concentration of the purified antibody MCAB2, the total antibody concentration Ab t corresponding to the above dilution factor can be calculated, where the antibody molecular weight is calculated as 150,000 (IgG molecular weight).
3)按照同样方法计算出包被抗原浓度为1μg/mL条件下，对应最大吸光度的一半时抗体的稀释倍数，由此计算出抗体总浓度Ab t’。 3) According to the same method, calculate the antibody dilution factor when the coating antigen concentration is 1μg/mL, which corresponds to half of the maximum absorbance, and thus calculate the total antibody concentration Ab t '.
4)根据以下抗体亲和力常数计算公式(n为两个包被抗原的浓度倍数关系，即n＝2)，即可计算出一个亲和常数K 1。 4) According to the following antibody affinity constant calculation formula (n is the multiple relationship of the concentration of the two coated antigens, that is, n=2), an affinity constant K 1 can be calculated.
Figure PCTCN2021082941-appb-000006
Figure PCTCN2021082941-appb-000006
5)以此类推，可得出三个亲和常数K 1，K 2及K 3，计算平均值即得抗体MCAB2与MC-LR的亲和常数。相同方法计算抗体MCAB2与其他11种微囊藻毒素的亲和力常数，12个亲和常数取平均值得到抗体MCAB2与12种微囊藻毒素的平均亲和力常数，具体结果见表2。 5) By analogy, three affinity constants K 1 , K 2 and K 3 can be obtained. Calculate the average value to obtain the affinity constants of the antibody MCAB2 and MC-LR. The same method is used to calculate the affinity constants of antibody MCAB2 and other 11 types of microcystins. The 12 affinity constants are averaged to obtain the average affinity constants of antibody MCAB2 and 12 types of microcystins. The specific results are shown in Table 2.
按照上述相同的方法将抗体MCAB2替换为抗体MCAB1，得到抗体MCAB1与12种微囊藻毒素的亲和力常数及其平均亲和力常数，结果如表2所示。The antibody MCAB2 was replaced with the antibody MCAB1 according to the same method as above, and the affinity constants of the antibody MCAB1 and 12 kinds of microcystins and their average affinity constants were obtained. The results are shown in Table 2.
按照上述相同的方法将抗体MCAB2替换为抗体MCAB3，得到抗体MCAB3与12种微囊藻毒素的亲和力常数及其平均亲和力常数，结果如表2所示。The antibody MCAB2 was replaced with the antibody MCAB3 according to the same method as above, and the affinity constants of the antibody MCAB3 and 12 kinds of microcystins and their average affinity constants were obtained. The results are shown in Table 2.
按照上述相同的方法将抗体MCAB2替换为抗体MCAB4，得到抗体MCAB4与12种微囊藻毒素的亲和力常数及其平均亲和力常数，结果如表2所示。The antibody MCAB2 was replaced with the antibody MCAB4 according to the same method as above, and the affinity constants of the antibody MCAB4 and 12 kinds of microcystins and their average affinity constants were obtained. The results are shown in Table 2.
按照上述相同的方法将抗体MCAB2替换为抗体MCAB5，得到抗体MCAB5与12种微囊藻毒素的亲和力常数及其平均亲和力常数，结果如表2所示。The antibody MCAB2 was replaced with the antibody MCAB5 according to the same method described above, and the affinity constants of the antibody MCAB5 and 12 kinds of microcystins and their average affinity constants were obtained. The results are shown in Table 2.
表2、5株单克隆抗体分别与12种微囊藻毒素的亲和力常数及其平均亲和力常数Table 2. Affinity constants and average affinity constants of 5 monoclonal antibodies to 12 kinds of microcystins
Figure PCTCN2021082941-appb-000007
Figure PCTCN2021082941-appb-000007
Figure PCTCN2021082941-appb-000008
Figure PCTCN2021082941-appb-000008
从表2中可以看出，抗体MCAB1平均亲和力常数为4.10×10 8L/mol，抗体MCAB2与12种微囊藻毒素的平均亲和力常数为1.12×10 9L/mol，抗体MCAB3平均亲和力常数为5.35×10 8L/mol，抗体MCAB4平均亲和力常数为6.64×10 8L/mol，抗体MCAB5平均亲和力常数为3.92×10 8L/mol。上述结果表明5株单克隆抗体对12种微囊藻毒素均具有较高的亲和力，兼备广谱特异性和高亲和力的优势，通过合理设计免疫传感分析方法，以期可以直接定量检测水体中总微囊藻毒素，从而实现微囊藻毒素的大规模样品快速筛查和预警监测。 It can be seen from Table 2 that the average affinity constant of antibody MCAB1 is 4.10×10 8 L/mol, the average affinity constant of antibody MCAB2 and 12 kinds of microcystins is 1.12×10 9 L/mol, and the average affinity constant of antibody MCAB3 is 5.35×10 8 L/mol, the average affinity constant of antibody MCAB4 is 6.64×10 8 L/mol, and the average affinity constant of antibody MCAB5 is 3.92×10 8 L/mol. The above results indicate that the 5 monoclonal antibodies have high affinity for 12 kinds of microcystins, and have the advantages of broad-spectrum specificity and high affinity. By rationally designing immunosensing analysis methods, it is possible to directly quantitatively detect the total amount of water in the water body. Microcystin, so as to realize the rapid screening and early warning monitoring of large-scale samples of microcystin.
实施例3、采用杂交瘤细胞株CGMCC No.19198分泌的单克隆抗体MCAB5分别检测水样中的MC-LR、MC-RR和MC-YRExample 3. The monoclonal antibody MCAB5 secreted by the hybridoma cell line CGMCC No. 19198 was used to detect MC-LR, MC-RR and MC-YR in water samples.
采用间接ELISA方法检测，具体操作步骤如下：Using indirect ELISA method to detect, the specific steps are as follows:
1)将MC-LR、MC-RR和MC-YR标准品采用超纯水分别作一系列3倍梯度稀释，浓度分别为：1000、333、111、37、12.34、4.12、1.37、0.46、0.15、0.05μg/L，作为水样。1) The MC-LR, MC-RR and MC-YR standards were diluted with ultrapure water in a series of 3-fold gradients respectively, the concentrations were: 1000, 333, 111, 37, 12.34, 4.12, 1.37, 0.46, 0.15 , 0.05μg/L, as a water sample.
2)采用NH 2-MC-LR-BSA作为包被抗原(所述NH 2-MC-LR-BSA为将实施例1中步骤1.1中的KLH替换为BSA，得到的氨基修饰的MC-LR与BSA偶联物)，用包被液将其稀释为2μg/mL，100μL/孔加入到96孔酶联板中进行包被，于4℃包被过夜后用PBS-T洗液洗涤3次。 2) Using NH 2 -MC-LR-BSA as the coating antigen (the NH 2 -MC-LR-BSA is the replacement of KLH in step 1.1 in Example 1 with BSA, the resulting amino-modified MC-LR and BSA conjugate), diluted to 2μg/mL with coating solution, 100μL/well was added to 96-well enzyme-linked plate for coating, coated overnight at 4°C and washed with PBS-T washing solution 3 times.
3)用封闭液(2％脱脂牛奶-PBS)封闭，200μL/孔，37℃孵育2～4h后用 洗液洗涤3次，将步骤1)的标准水样溶液、空白对照(标准液用PBS溶液代替)，分别加入酶标板相应孔中，均为100μL/孔。3) Block with blocking solution (2% skimmed milk-PBS), 200μL/well, incubate at 37°C for 2 to 4 hours, wash with washing solution 3 times, and replace the standard water sample solution and blank control (standard solution with PBS) from step 1) Solution instead), respectively add 100μL/well to the corresponding wells of the ELISA plate.
4)加入纯化后的抗体MCAB5(用PBS稀释成1：1000)，均为100μL/孔，37℃孵育1～4h后用洗液洗涤3次。4) Add the purified antibody MCAB5 (diluted with PBS to 1:1000), all of which are 100 μL/well, incubate at 37° C. for 1 to 4 hours, and then wash with lotion 3 times.
5)加入PBS稀释20000倍的酶标二抗(山羊抗小鼠IgG/HRP)，100μL/孔，37℃孵育1～4h后用洗液洗涤3次。5) Add enzyme-labeled secondary antibody (goat anti-mouse IgG/HRP) diluted 20000 times in PBS, 100 μL/well, incubate at 37°C for 1 to 4 hours, and wash 3 times with lotion.
6)加显色液，100μL/孔，显色时间为5～10min左右。6) Add color developing solution, 100μL/well, and the color development time is about 5-10min.
7)加终止液(2M浓硫酸)，50μL/孔，终止反应。7) Add stop solution (2M concentrated sulfuric acid), 50μL/well to stop the reaction.
8)酶标仪测450nm处吸光值，记录保存数据。8) Measure the absorbance at 450nm with a microplate reader, record and save the data.
结果如图2所示。表明3条标准曲线均具有完整的反S形状，具有上平台和下平台，误差线为n＝3次平行实验的标准偏差，相对标准偏差(变异系数)均在20％以内，表明检测精密度良好。The result is shown in Figure 2. It shows that the three standard curves all have a complete reverse S shape, with an upper platform and a lower platform. The error bar is n = standard deviation of 3 parallel experiments, and the relative standard deviation (coefficient of variation) is within 20%, indicating the detection precision good.
采用4参数Logistic模型作为间接竞争ELISA检测的数据分析和评价基础，模型如下：The 4-parameter Logistic model is used as the basis for data analysis and evaluation of indirect competitive ELISA detection. The model is as follows:
Figure PCTCN2021082941-appb-000009
Figure PCTCN2021082941-appb-000009
其中：in:
x：目标物浓度，自变量；x: target concentration, independent variable;
A：x对应的吸光度(Absorbance)，因变量；A: Absorbance corresponding to x, dependent variable;
A 1：上端渐近线值(x＝0)，即最大吸光度； A 1 : The upper asymptote value (x=0), that is, the maximum absorbance;
A 2：下端渐近线值(x→∞)，即最小吸光度； A 2 : The asymptotic value of the lower end (x→∞), that is, the minimum absorbance;
p：线性区间斜率；p: slope of linear interval;
x 0：曲线的中点，即拐点。 x 0 : The midpoint of the curve, that is, the inflection point.
在竞争ELISA中，半抑制浓度IC 50＝x 0；依据上述Logistic模型，结合率Y计算公式如下，标准曲线的最低检测限为Y＝90％时对应的目标物质的浓度，定量检测区间为Y＝80％～20％对应的目标物浓度区间。 In the competitive ELISA, the half-inhibitory concentration IC 50 = x 0 ; According to the above Logistic model, the binding rate Y is calculated as follows, the minimum detection limit of the standard curve is the concentration of the target substance corresponding to Y = 90%, and the quantitative detection interval is Y =80%～20% corresponding target concentration range.
Figure PCTCN2021082941-appb-000010
Figure PCTCN2021082941-appb-000010
分别对图2中的3条标准曲线采用四参数Logistic模型拟合，分析结果表明单克隆抗体MCAB5对MC-LR的半抑制浓度为IC 50＝2.44μg/L，最低检测限为0.25μg/L，定量区间为0.58μg/L-10.33μg/L；对MC-RR的半抑制浓度为 IC 50＝6.66μg/L，最低检测限为0.36μg/L，定量区间为1.06μg/L-41.67μg/L；对MC-YR的半抑制浓度为IC 50＝5.47μg/L，最低检测限为0.40μg/L，定量区间为1.05μg/L-28.62μg/L。 The three standard curves in Figure 2 were fitted with a four-parameter Logistic model. The analysis results showed that the half-inhibitory concentration of the monoclonal antibody MCAB5 on MC-LR was IC 50 =2.44μg/L, and the minimum detection limit was 0.25μg/L The quantification interval is 0.58μg/L-10.33μg/L; the half-inhibitory concentration of MC-RR is IC 50 =6.66μg/L, the minimum detection limit is 0.36μg/L, and the quantification interval is 1.06μg/L-41.67μg /L; The half-inhibitory concentration of MC-YR is IC 50 =5.47μg/L, the minimum detection limit is 0.40μg/L, and the quantitative interval is 1.05μg/L-28.62μg/L.
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention has given specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principles of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes that deviate from the scope disclosed in this application and use conventional techniques known in the art. According to the scope of the appended claims below, some basic features can be applied.
工业应用Industrial application
本发明中为了获得针对微囊藻毒素整个环肽结构具有高度特异性的抗体，分别在12种微囊藻毒素(MC-LR,MC-RR,MC-YR,MC-LA,MC-LF,MC-LW,MC-LY,MC-WR,MC-HtyR,MC-HilR,[D-Asp3]MC-LR,[D-Asp3]MC-RR)的第七位氨基酸(Mdha)上通过2-巯基乙胺化学修饰引入一个氨基活性基团，然后与钥孔血蓝蛋白(KLH)偶联，分别得到12种微囊藻毒素与KLH的偶联物，采用“混合免疫原法”进行免疫，即分别将上述12种微囊藻毒素与KLH的偶联物(12种完全抗原)等质量混合后作为免疫原来免疫小鼠；再将上述12种小分子分别与或牛血清白蛋白(BSA)偶联并等量混合后作为包被抗原，采用间接ELISA法测定血清效价；选取血清效价满足要求的免疫小鼠的脾细胞，与SP2/0骨髓瘤细胞融合，筛选出阳性杂交瘤细胞株；培养阳性杂交瘤细胞株并进行亚型鉴定；将获得的IgG1亚型阳性杂交瘤细胞株注入同系小鼠腹腔诱生腹水，得到广谱型的微囊藻毒素单克隆抗体MCAB1、MCAB2、MCAB3、MCAB4和MCAB5。抗体MCAB1与12种微囊藻毒素的平均亲和力常数为4.10×10 8L/mol，抗体MCAB2与12种微囊藻毒素的平均亲和力常数为1.12×10 9L/mol，抗体MCAB3与12种微囊藻毒素的平均亲和力常数为5.35×10 8L/mol，抗体MCAB4与12种微囊藻毒素的平均亲和力常数为6.64×10 8L/mol，抗体MCAB5与12种微囊藻毒素的平均亲和力常数为3.92×10 8L/mol，上述结果表明5株单克隆抗体对12种微囊藻毒素均具有较高的亲和力，可用于实际样品(如环境样品或者食品)中的微囊藻毒素检测。 In the present invention, in order to obtain highly specific antibodies against the entire cyclic peptide structure of microcystin, 12 kinds of microcystin (MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, MC-LY, MC-WR, MC-HtyR, MC-HilR, [D-Asp3]MC-LR, [D-Asp3]MC-RR) on the seventh amino acid (Mdha) through 2- The chemical modification of mercaptoethylamine introduces an amino active group, and then couples with keyhole limpet hemocyanin (KLH) to obtain 12 kinds of conjugates of microcystin and KLH respectively. The "mixed immunogen method" is used for immunization. That is, the above-mentioned 12 kinds of microcystins and KLH conjugates (12 kinds of complete antigens) are mixed and used as immunogens to immunize mice; then the above-mentioned 12 kinds of small molecules are respectively combined with or bovine serum albumin (BSA) After being coupled and mixed in equal amounts, the serum titer is determined by indirect ELISA method; the spleen cells of immunized mice whose serum titer meets the requirements are selected and fused with SP2/0 myeloma cells to screen out positive hybridoma cells Strains; culture positive hybridoma cell lines and perform subtype identification; inject the obtained IgG1 subtype positive hybridoma cell lines into the abdominal cavity of the same strain of mice to induce ascites, and obtain broad-spectrum microcystin monoclonal antibodies MCAB1, MCAB2 MCAB3, MCAB4 and MCAB5. The average affinity constant of antibody MCAB1 and 12 kinds of microcystins is 4.10×10 8 L/mol, the average affinity constant of antibody MCAB2 and 12 kinds of microcystins is 1.12×10 9 L/mol, and the antibody MCAB3 has 12 kinds of microcystins. The average affinity constant of cystins is 5.35×10 8 L/mol, the average affinity constant of antibody MCAB4 and 12 kinds of microcystins is 6.64×10 8 L/mol, the average affinity of antibody MCAB5 and 12 kinds of microcystins The constant is 3.92×10 8 L/mol. The above results indicate that the 5 monoclonal antibodies have high affinity for 12 kinds of microcystins, and can be used for the detection of microcystins in actual samples (such as environmental samples or food) .

Claims (14)

  1. 一种单克隆抗体，其特征在于，所述单克隆抗体为如下a、b、c、d或e：A monoclonal antibody, characterized in that the monoclonal antibody is the following a, b, c, d or e:
    a)由保藏号为CGMCC No.19194的杂交瘤细胞株MCAB1产生的单克隆抗体；a) Monoclonal antibodies produced by the hybridoma cell line MCAB1 with the deposit number CGMCC No. 19194;
    b)由保藏号为CGMCC No.19195的杂交瘤细胞株MCAB2产生的单克隆抗体；b) Monoclonal antibodies produced by the hybridoma cell line MCAB2 with the deposit number CGMCC No. 19195;
    c)由保藏号为CGMCC No.19196的杂交瘤细胞株MCAB3产生的单克隆抗体；c) Monoclonal antibodies produced by the hybridoma cell line MCAB3 with the deposit number CGMCC No. 19196;
    d)由保藏号为CGMCC No.19197的杂交瘤细胞株MCAB4产生的单克隆抗体；d) Monoclonal antibodies produced by the hybridoma cell line MCAB4 with the deposit number CGMCC No. 19197;
    e)由保藏号为CGMCC No.19198的杂交瘤细胞株MCAB5产生的单克隆抗体。e) Monoclonal antibodies produced by the hybridoma cell line MCAB5 with the deposit number CGMCC No. 19198.
  2. 用于制备权利要求1所述微囊藻毒素广谱型单克隆抗体的组合物，其特征在于，所述组合物由下述12种完全抗原组成：NH 2-MC-LR-KLH、NH 2-MC-RR-KLH、NH 2-MC-YR-KLH、NH 2-MC-LA-KLH,NH 2-MC-LF-KLH、NH 2-MC-LW-KLH,、NH 2-MC-LY-KLH、NH 2-MC-WR-KLH、NH 2-MC-HtyR-KLH、NH 2-MC-HilR-KLH、NH 2-[D-Asp3]MC-LR-KLH和NH 2-[D-Asp3]MC-RR-KLH；其中所述NH 2-MC-LR-KLH为氨基修饰的MC-LR与KLH偶联物；所述NH 2-MC-RR-KLH为氨基修饰的MC-RR与KLH偶联物；所述NH 2-MC-YR-KLH为氨基修饰的MC-YR与KLH偶联物；所述NH 2-MC-LA-KLH为氨基修饰的MC-LA与KLH偶联物；所述NH 2-MC-LF-KLH为氨基修饰的MC-LF与KLH偶联物；所述NH 2-MC-LW-KLH为氨基修饰的MC-LW与KLH偶联物；所述NH 2-MC-LY-KLH为氨基修饰的MC-LY与KLH偶联物；所述NH 2-MC-WR-KLH为氨基修饰的MC-WR与KLH偶联物；所述NH 2-MC-HtyR-KLH为氨基修饰的MC-HtyR与KLH偶联物；所述NH 2-MC-HilR-KLH为氨基修饰的MC-HilR与KLH偶联物；所述NH 2-[D-Asp3]MC-LR-KLH为氨基修饰的[D-Asp3]MC-LR与KLH偶联物；所述NH 2-[D-Asp3]MC-RR-KLH为氨基修饰的[D-Asp3]MC-RR与KLH偶联物； The composition for preparing the microcystin broad-spectrum monoclonal antibody of claim 1, wherein the composition is composed of the following 12 complete antigens: NH 2 -MC-LR-KLH, NH 2 -MC-RR-KLH, NH 2 -MC-YR-KLH, NH 2 -MC-LA-KLH, NH 2 -MC-LF-KLH, NH 2 -MC-LW-KLH,, NH 2 -MC-LY -KLH, NH 2 -MC-WR-KLH, NH 2 -MC-HtyR-KLH, NH 2 -MC-HilR-KLH, NH 2 -[D-Asp3]MC-LR-KLH and NH 2 -[D- Asp3] MC-RR-KLH; wherein the NH 2 -MC-LR-KLH is an amino-modified MC-LR and KLH conjugate; the NH 2 -MC-RR-KLH is an amino-modified MC-RR and KLH conjugate; the NH 2 -MC-YR-KLH is an amino-modified MC-YR and KLH conjugate; the NH 2 -MC-LA-KLH is an amino-modified MC-LA and KLH conjugate The NH 2 -MC-LF-KLH is an amino-modified MC-LF and KLH conjugate; the NH 2 -MC-LW-KLH is an amino-modified MC-LW and KLH conjugate; the NH 2 -MC-LY-KLH is an amino-modified MC-LY and KLH conjugate; the NH 2 -MC-WR-KLH is an amino-modified MC-WR and KLH conjugate; the NH 2 -MC- HtyR-KLH is a conjugate of amino-modified MC-HtyR and KLH; the NH 2 -MC-HilR-KLH is a conjugate of amino-modified MC-HilR and KLH; the NH 2 -[D-Asp3]MC -LR-KLH is a conjugate of amino-modified [D-Asp3]MC-LR and KLH; the NH 2 -[D-Asp3]MC-RR-KLH is amino-modified [D-Asp3]MC-RR and KLH conjugate;
    所述氨基修饰的MC-LR为式2中R1为亮氨酸、R2为精氨酸和R3为甲基的化合物；所述氨基修饰的MC-RR为式2中R1为精氨酸、R2为精氨酸和R3为甲基的化合物；所述氨基修饰的MC-YR为式2中R1为酪氨酸、R2为精氨酸和R3为甲基的化合物；所述氨基修饰的MC-LA为式2中R1为亮氨酸、R2为丙氨酸和R3为甲基的化合物；所述氨基修饰的MC-LF为式2中R1为亮氨酸、R2为苯丙氨酸和R3为甲基的化合物；所述氨基修饰的MC-LW为式2中R1为亮氨酸、R2为色氨酸和R3为甲基的化合物；所述氨基修饰的MC-LY为式2中R1为亮氨酸、R2为酪氨酸和R3为甲基的化合物；所述氨基修饰的MC-WR为式2中R1为色氨 酸、R2为精氨酸和R3为甲基的化合物；所述氨基修饰的MC-HtyR为式2中R1为高酪氨酸、R2为精氨酸和R3为甲基的化合物；所述氨基修饰的MC-HilR为式2中R1为同异亮氨酸、R2为精氨酸和R3为甲基的化合物；所述氨基修饰的[D-Asp3]MC-LR为式2中R1为亮氨酸、R2为精氨酸和R3为氢的化合物；所述氨基修饰的[D-Asp3]MC-RR为式2中R1为精氨酸、R2为精氨酸和R3为氢的化合物。The amino-modified MC-LR is a compound in which R1 is leucine, R2 is arginine and R3 is methyl in formula 2; the amino-modified MC-RR is a compound in formula 2 where R1 is arginine and R2 Is a compound in which arginine and R3 are methyl; the amino-modified MC-YR is a compound in formula 2 where R1 is tyrosine, R2 is arginine and R3 is a methyl group; the amino-modified MC- LA is a compound in formula 2 where R1 is leucine, R2 is alanine and R3 is methyl; the amino-modified MC-LF is formula 2 where R1 is leucine, R2 is phenylalanine and R3 Is a methyl compound; the amino-modified MC-LW is a compound in formula 2 where R1 is leucine, R2 is tryptophan, and R3 is a methyl; the amino-modified MC-LY is a compound in formula 2 where R1 Is a compound in which R2 is tyrosine and R3 is methyl; the amino-modified MC-WR is a compound in formula 2 where R1 is tryptophan, R2 is arginine and R3 is methyl; The amino-modified MC-HtyR is a compound in formula 2 where R1 is homotyrosine, R2 is arginine and R3 is methyl; the amino-modified MC-HilR is a compound in formula 2 where R1 is homo-isoleucine , R2 is a compound in which arginine and R3 is a methyl group; the amino-modified [D-Asp3]MC-LR is a compound in formula 2 where R1 is leucine, R2 is arginine and R3 is hydrogen; The amino-modified [D-Asp3] MC-RR is a compound in formula 2 where R1 is arginine, R2 is arginine, and R3 is hydrogen.
  3. 根据权利要求2所述的组合物，其特征在于，所述组合物中，所述12种完全抗原中的每种抗原质量相同。The composition according to claim 2, wherein, in the composition, each of the 12 complete antigens has the same quality.
  4. 用于检测微囊藻毒素的试剂或含有所述试剂的试剂盒，其特征在于，所述试剂包括权利要求1中a)、b)、c)、d)和e)中所述的单克隆抗体中的至少一种。A reagent for detecting microcystin or a kit containing the reagent, characterized in that the reagent comprises the monoclonal described in claim 1 a), b), c), d) and e) At least one of the antibodies.
  5. 根据权利要求4所述的试剂盒，其特征在于，所述微囊藻毒素为MC-LR,MC-RR,MC-YR,MC-LA,MC-LF,MC-LW,MC-LY,MC-WR,MC-HtyR,MC-HilR,[D-Asp3]MC-LR,[D-Asp3]MC-RR中的至少一种。The kit according to claim 4, wherein the microcystin is MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, MC-LY, MC -At least one of WR, MC-HtyR, MC-HilR, [D-Asp3] MC-LR, [D-Asp3] MC-RR.
  6. 一种制备微囊藻毒素广谱型单克隆抗体的方法，其特征在于，包括以下步骤：A method for preparing a broad-spectrum monoclonal antibody against microcystin, which is characterized in that it comprises the following steps:
    1)分别在12种微囊藻毒素分子结构变异体的第七位氨基酸残基N-甲基脱氢丙氨酸上引入一个氨基，得到12种经氨基修饰的微囊藻毒分子；再将该12种经氨基修饰的微囊藻毒素分子分别与载体蛋白偶联得到12种完全抗原；1) Introduce an amino group into the seventh amino acid residue N-methyldehydroalanine of 12 kinds of microcystin molecular structure variants to obtain 12 kinds of amino-modified microcystin molecules; The 12 amino-modified microcystin molecules are respectively coupled with carrier proteins to obtain 12 complete antigens;
    2)将步骤1)的12种完全抗原等质量混合，得到混合免疫原，对小鼠进行免疫后取血清效价最高的小鼠的脾细胞与小鼠骨髓瘤细胞进行融合，筛选出阳性杂交瘤细胞株，将其注入同系小鼠腹腔诱生腹水，得到可与12种微囊藻毒素小分子高亲和力结合的广谱型单克隆抗体；2) Mix the 12 complete antigens of step 1) with equal quality to obtain a mixed immunogen. After immunizing the mice, the spleen cells of the mouse with the highest serum titer are fused with the mouse myeloma cells, and the positive hybridization is selected. Tumor cell line, inject it into the abdominal cavity of the same strain of mice to induce ascites, and obtain a broad-spectrum monoclonal antibody that can bind to 12 kinds of microcystin small molecules with high affinity;
    所述12种微囊藻毒素为MC-LR,MC-RR,MC-YR,MC-LA,MC-LF,MC-LW,MC-LY,MC-WR,MC-HtyR,MC-HilR,[D-Asp3]MC-LR和[D-Asp3]MC-RR。The 12 kinds of microcystins are MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, MC-LY, MC-WR, MC-HtyR, MC-HilR, [ D-Asp3] MC-LR and [D-Asp3] MC-RR.
  7. 根据权利要求6所述的方法，其特征在于：所述步骤1)中的所述载体蛋白为牛血清白蛋白、血蓝蛋白、人血清白蛋白、卵清蛋白、鼠血清白蛋白、甲状腺球蛋白或兔血清白蛋白。The method according to claim 6, wherein the carrier protein in the step 1) is bovine serum albumin, hemocyanin, human serum albumin, ovalbumin, murine serum albumin, and thyroglobulin. Protein or rabbit serum albumin.
  8. 权利要求1所述的单克隆抗体、权利要求2或3所述的组合物、权利要求4或5所述的试剂盒或者权利要求6-7中任一所述的方法在检测微囊藻毒素 和/或纯化微囊藻毒素中的应用。The monoclonal antibody of claim 1, the composition of claim 2 or 3, the kit of claim 4 or 5, or the method of any one of claims 6-7 in the detection of microcystin And/or purification of microcystins.
  9. 根据权利要求8所述的应用，其特征在于：所述应用为非疾病诊断目的的应用。The application according to claim 8, wherein the application is an application for non-disease diagnosis purposes.
  10. 根据权利要求9所述的应用，其特征在于：所述应用为检测环境样品或者食品中的微囊藻毒素；所述微囊藻毒素为MC-LR,MC-RR,MC-YR,MC-LA,MC-LF,MC-LW,MC-LY,MC-WR,MC-HtyR,MC-HilR,[D-Asp3]MC-LR,[D-Asp3]MC-RR中的至少一种。The application according to claim 9, characterized in that: the application is the detection of microcystin in environmental samples or food; the microcystin is MC-LR, MC-RR, MC-YR, MC- At least one of LA, MC-LF, MC-LW, MC-LY, MC-WR, MC-HtyR, MC-HilR, [D-Asp3]MC-LR, [D-Asp3]MC-RR.
  11. 一种检测微囊藻毒素的方法，其特征在于，使用权利要求1所述的单克隆抗体、权利要求2或3所述的组合物、权利要求4或5所述的试剂盒或者权利要求6-7中任一所述的方法进行待测样品中微囊藻毒素的检测。A method for detecting microcystin, which is characterized by using the monoclonal antibody of claim 1, the composition of claim 2 or 3, the kit of claim 4 or 5, or claim 6. The method described in any one of -7 detects the microcystin in the sample to be tested.
  12. 根据权利要求11所述的检测微囊藻毒素的方法，其特征在于，所述微囊藻毒素为MC-LR,MC-RR,MC-YR,MC-LA,MC-LF,MC-LW,MC-LY,MC-WR,MC-HtyR,MC-HilR,[D-Asp3]MC-LR,[D-Asp3]MC-RR中的至少一种。The method for detecting microcystin according to claim 11, wherein the microcystin is MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, At least one of MC-LY, MC-WR, MC-HtyR, MC-HilR, [D-Asp3]MC-LR, [D-Asp3]MC-RR.
  13. 一种纯化微囊藻毒素的方法，其特征在于，使用权利要求1所述的单克隆抗体、权利要求2或3所述的组合物、权利要求4或5所述的试剂盒或者权利要求6-7中任一所述的方法纯化待测样品中的微囊藻毒素。A method for purifying microcystin, characterized by using the monoclonal antibody according to claim 1, the composition according to claim 2 or 3, the kit according to claim 4 or 5, or claim 6. The method described in any one of -7 purifies the microcystin in the sample to be tested.
  14. 根据权利要求13所述的纯化微囊藻毒素的方法，其特征在于，所述微囊藻毒素为MC-LR,MC-RR,MC-YR,MC-LA,MC-LF,MC-LW,MC-LY,MC-WR,MC-HtyR,MC-HilR,[D-Asp3]MC-LR,[D-Asp3]MC-RR中的至少一种。The method for purifying microcystin according to claim 13, wherein the microcystin is MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, At least one of MC-LY, MC-WR, MC-HtyR, MC-HilR, [D-Asp3]MC-LR, [D-Asp3]MC-RR.
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