Broad-spectrum microcystin monoclonal antibody and preparation method thereof
阅读说明：本技术 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 ) 是由 周小红 刘金钏 于 2020-03-27 设计创作，主要内容包括：本发明公开了一种广谱型微囊藻毒素单克隆抗体及其制备方法,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,得到广谱型的微囊藻毒素单克隆抗体MCAB1、MCAB2、MCAB3、MCAB4和MCAB5。本发明的5株单克隆抗体对12种微囊藻毒素均具有较高的亲和力,可用于实际样品中的微囊藻毒素检测。(The invention discloses a broad-spectrum microcystin monoclonal antibody and a preparation method thereof, in order to obtain an antibody with high specificity aiming at the whole cyclopeptide structure of microcystin, an amino active group is respectively introduced on the seventh amino acid (Mdha) of 12 kinds of microcystin through chemical modification by 2-mercaptoethylamine, then the amino active group is coupled with KLH to respectively obtain conjugates of 12 kinds of microcystin and KLH, and the broad-spectrum microcystin monoclonal antibodies MCAB1, MCAB2, MCAB3, MCAB4 and MCAB5 are obtained by adopting a mixed immunogen method for immunization. The 5 monoclonal antibodies of the invention have higher affinity to 12 kinds of microcystins and can be used for detecting the microcystins in actual samples.)
1. A monoclonal antibody, wherein the monoclonal antibody is a, b, c, d or e as follows:
a) the monoclonal antibody produced by hybridoma cell strain MCAB1 with the preservation number of CGMCCNo.19194;
b) the monoclonal antibody produced by hybridoma cell strain MCAB2 with the preservation number of CGMCC No. 19195;
c) the monoclonal antibody produced by hybridoma cell strain MCAB3 with the preservation number of CGMCCNo.19196;
d) the monoclonal antibody produced by hybridoma cell strain MCAB4 with the preservation number of CGMCC No. 19197;
e) the monoclonal antibody produced by hybridoma cell strain MCAB5 with the preservation number of CGMCCNo.19198.
2. The composition for preparing the broad-spectrum monoclonal antibody against microcystin of claim 1, wherein the composition is composed of the following 12 complete antigens: NH (NH)2-MC-LR-KLH、NH2-MC-RR-KLH、NH2-MC-YR-KLH、NH2-MC-LA-KLH,NH2-MC-LF-KLH、NH2-MC-LW-KLH,、NH2-MC-LY-KLH、NH2-MC-WR-KLH、NH2-MC-HtyR-KLH、NH2-MC-HilR-KLH、NH2-[D-Asp3]MC-LR-KLH and NH2-[D-Asp3]MC-RR-KLH; wherein said NH2-MC-LR-KLH is an amino-modified MC-LR and KLH conjugate; the NH2-MC-RR-KLH is an amino modified MC-RR and KLH conjugate; the NH2-MC-YR-KLH is an amino modified MC-YR and KLH conjugate; the NH2-MC-LA-KLH is an amino modified MC-LA and KLH conjugate; the NH2-MC-LF-KLH is an amino-modified MC-LF and KLH conjugate; the NH2-MC-LW-KLH is an amino-modified MC-LW and KLH conjugate; the NH2-MC-LY-KLH is a conjugate of amino-modified MC-LY and KLH; the NH2-MC-WR-KLH is an amino modified MC-WR-KLH conjugate; the NH2-MC-HtyR-KLH is a conjugate of amino-modified MC-HtyR and KLH; the NH2-MC-HilR-KLH is an amino modified MC-HilR-KLH conjugate; the NH2-[D-Asp3]MC-LR-KLH is amino modified [ D-Asp3]MC-LR conjugated with KLH; the NH2-[D-Asp3]MC-RR-KLH is amino modified [ D-Asp3]MC-RR and KLH conjugate;
the amino-modified MC-LR is a compound of formula 2 wherein R1 is leucine, R2 is arginine, and R3 is methyl; the amino modified MC-RR is a compound of formula 2, wherein R1 is arginine, R2 is arginine and R3 is methyl; the amino modified MC-YR is a compound in which R1 is tyrosine, R2 is arginine and R3 is methyl in formula 2; the amino modified MC-LA is a compound in which R1 is leucine, R2 is alanine, and R3 is methyl in formula 2; the amino modified MC-LF is a compound in which R1 is leucine, R2 is phenylalanine and R3 is methyl in a formula 2; the amino-modified MC-LW is a compound of formula 2 wherein R1 is leucine, R2 is tryptophan, and R3 is methyl; the amino-modified MC-LY is a compound of formula 2 wherein R1 is leucine, R2 is tyrosine, and R3 is methyl; the amino modified MC-WR is a compound in which R1 is tryptophan, R2 is arginine and R3 is methyl in formula 2; the amino modified MC-HtyrR is a compound in which R1 is homotyrosine, R2 is arginine and R3 is methyl in a formula 2; the amino modified MC-HilR is a compound in which R1 is isoisoleucine, R2 is arginine and R3 is methyl in formula 2; the amino modified [ D-Asp3] MC-LR is a compound in which R1 is leucine, R2 is arginine and R3 is hydrogen in formula 2; the amino modified [ D-Asp3] MC-RR is a compound of formula 2 wherein R1 is arginine, R2 is arginine and R3 is hydrogen.
3. The composition of claim 2, wherein each of the 12 complete antigens is the same mass in the composition.
4. A reagent for detecting microcystin or a kit containing the same, wherein the reagent comprises at least one of the monoclonal antibodies described in a), b), c), d) and e) of claim 1.
5. The kit of claim 4, wherein the microcystin is at least one of 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.
6. A method for preparing a broad-spectrum monoclonal antibody of microcystin is characterized by comprising the following steps:
1) respectively introducing an amino group on the seventh amino acid residue N-methyl dehydroalanine of 12 microcystin molecular structure variants to obtain 12 amino-modified microcystin molecules; then coupling the 12 amino modified microcystin molecules with carrier protein to obtain 12 complete antigens;
2) mixing the 12 complete antigens obtained in the step 1) in equal mass to obtain a mixed immunogen, immunizing a mouse, taking spleen cells of the mouse with the highest serum titer to fuse with myeloma cells of the mouse, screening out positive hybridoma cell strains, injecting the positive hybridoma cell strains into the abdominal cavity of a homologous mouse to induce ascites, and obtaining a broad-spectrum monoclonal antibody which can be combined with 12 microcystin micromolecules in high affinity;
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. The method of claim 6, wherein: the carrier protein in the step 1) is bovine serum albumin, hemocyanin, human serum albumin, ovalbumin, mouse serum albumin, thyroglobulin or rabbit serum albumin.
8. Use of 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 to 7 for detecting and/or purifying microcystins.
9. Use according to claim 8, characterized in that: the use is for non-disease diagnostic purposes.
10. Use according to claim 9, characterized in that: the application is detecting microcystin in environmental samples or food; the microcystin is at least one of 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.
Technical Field
The invention relates to a broad-spectrum monoclonal antibody aiming at multiple structural variants of microcystin and a preparation method thereof.
Background
The frequent outbreak of the toxic cyanobacterial bloom brings serious influence on the ecological environment and the economy, and is now a global important environmental problem. Microcystis are the most common cyanobacteria in the poisonous cyanobacteria water bloom, Microcystins (MCs) generated by the Microcystins are small molecules with a common cyclic heptapeptide structure, and consist of 5 non-proteinogenic amino acids (such as dehydroalanine derivatives, special beta-amino acids Adda and the like) and 2 proteinogenic amino acids (variable amino acids at the 2 nd position and the 4 th position), and the chemical structural formula of the Microcystins is shown as a formula 1. More than 150 structural variants of microcystins with different degrees of toxicity have been reported, wherein microcystins-LR (MC-LR), MC-RR and MC-YR are the most common and most toxic microcystins in cyanobacterial bloom water, and acute toxicity caused by the structural variants can cause cell function and structure loss, and liver bleeding and even death. Therefore, the development of a reliable rapid screening technology for microcystins in water has important practical significance.
(formula 1)
The immunoassay method based on the antigen-antibody specific recognition and binding reaction has the advantages of high sensitivity, low cost, high throughput and the like. Aiming at the common structure of microcystins, the antibody with broad-spectrum specificity is prepared, all microcystins structural variants can be identified theoretically, and as long as the cross reactivity of each structural variant is not obviously different, all structural variant standards are not required to be provided, the total microcystins in the water can be directly and quantitatively detected, so that the method is very suitable for large-scale rapid screening and early warning monitoring of microcystins.
The key to establishing the microcystin total amount immunoassay is to obtain high-quality broad-spectrum antibodies. The microcystins belong to hapten small molecules, and are required to be coupled with carrier proteins to be modified into complete antigens (immunogens) and then to be immunized into animals to obtain antibodies. In order to produce high titers of broad spectrum specific antibodies, hapten molecular design is the first key point for successful establishment of immunoassay methods, while immunization protocols are the second key point.
At present, few research reports about the preparation of broad-spectrum antibodies of microcystins exist, the broad spectrum of the obtained antibodies cannot meet the ideal requirements, the measured cross-reactivity data is relatively limited, and the cross-reactivity difference among structural variants is obvious. In 2014, an Ingunn A.Samdal subject group adopts a mixture of 5 microcystin molecules with differences in variable amino acid sites to be coupled with Bovine Serum Albumin (BSA) to prepare immunogen, and the broad spectrum and the cross reactivity of the obtained sheep polyclonal antibody are improved greatly. However, polyclonal antibodies have large inter-immunoassay batch errors. Aiming at the problems, the invention provides a preparation method of a broad-spectrum microcystin monoclonal antibody.
Disclosure of Invention
The invention aims to invent a broad-spectrum monoclonal antibody suitable for immunoassay of total amount of microcystin in water and establish a preparation method thereof.
The invention claims a monoclonal antibody, which is a, b, c, d or e as follows:
a) monoclonal antibody produced by hybridoma cell strain MCAB1 with preservation number of CGMCC No. 19194;
b) monoclonal antibody produced by hybridoma cell strain MCAB2 with preservation number of CGMCC No. 19195;
c) monoclonal antibody produced by hybridoma cell strain MCAB3 with preservation number of CGMCC No. 19196;
d) monoclonal antibody produced by hybridoma cell strain MCAB4 with preservation number of CGMCC No. 19197;
e) the monoclonal antibody produced by hybridoma cell strain MCAB5 with the preservation number of CGMCC No. 19198.
The invention also claims a composition for preparing broad-spectrum monoclonal antibodies against microcystins, which comprises 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, which are separately packaged and used separately.
The invention also claims a composition for preparing the broad-spectrum monoclonal antibody of the microcystin, which consists of the following 12 complete antigens: NH (NH)2-MC-LR-KLH、NH2-MC-RR-KLH、 NH2-MC-YR-KLH、NH2-MC-LA-KLH,NH2-MC-LF-KLH、NH2-MC-LW-KLH,、 NH2-MC-LY-KLH、NH2-MC-WR-KLH、NH2-MC-HtyR-KLH、NH2-MC-HilR-KLH、 NH2-[D-Asp3]MC-LR-KLH and NH2-[D-Asp3]MC-RR-KLH; wherein said NH2-MC-LR-KLH is an amino-modified MC-LR and KLH conjugate; the NH2-MC-RR-KLH is an amino modified MC-RR and KLH conjugate; the NH2-MC-YR-KLH is a conjugate of amino-modified MC-YR and KLH; the NH2-MC-LA-KLH is an amino modified MC-LA and KLH conjugate; the NH2-MC-LF-KLH is an amino-modified MC-LF and KLH conjugate; the NH2-MC-LW-KLH is an amino-modified MC-LW and KLH conjugate; the NH2-MC-LY-KLH is a conjugate of amino-modified MC-LY and KLH; the NH2-MC-WR-KLH is an amino modified MC-WR-KLH conjugate; the NH2-MC-HtyR-KLH is a conjugate of amino-modified MC-HtyR and KLH; the NH2-MC-HilR-KLH is an amino modified MC-HilR-KLH conjugate; the NH2-[D-Asp3]MC-LR-KLH is amino modified [ D-Asp3]MC-LR conjugated with KLH; the NH2-[D-Asp3]MC-RR-KLH is amino modified [ D-Asp3]MC-RR and KLH conjugate; wherein KLH is hemocyanin.
The amino-modified MC-LR is a compound obtained by introducing an amino group to the seventh amino acid residue N-methyl dehydroalanine of the MC-LR, namely a compound in which R1 is leucine, R2 is arginine and R3 is methyl in the
The structural general formula of the microcystins is shown as a formula (1), and the structural general formula of the amino-modified microcystins is shown as a formula (2).
In the composition, each antigen of the 12 complete antigens has the same mass.
The invention also claims a reagent for detecting microcystin or a kit containing the reagent, wherein the reagent comprises at least one of the monoclonal antibodies in a), b), c), d) and e).
Wherein the molecular structure variant of the microcystin is at least one of 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 invention also claims a method for preparing the broad-spectrum monoclonal antibody of the microcystin, which comprises the steps of immunizing a mouse with the complete antigen composition to obtain an immunized mouse, fusing splenocytes of the mouse with the highest serum titer in the immunized mouse with myeloma cells of the mouse, screening out positive hybridoma cell strains, injecting the positive hybridoma cell strains into the abdominal cavity of a homologous mouse to induce ascites, and obtaining the broad-spectrum monoclonal antibody which can be combined with 12 microcystin micromolecules with high affinity;
the method specifically comprises the following steps:
1) respectively introducing an amino group on the seventh amino acid residue N-methyl dehydroalanine of 12 microcystin molecular structure variants to obtain 12 amino-modified microcystin molecules; then coupling the 12 amino modified microcystin molecules with carrier protein to obtain 12 complete antigens;
2) mixing the 12 complete antigens obtained in the step 1) in equal mass to obtain a mixed immunogen, taking splenocytes of a mouse after immunizing the mouse, fusing the splenocytes with myeloma cells of the mouse, and screening out positive hybridoma cell strains to obtain a broad-spectrum monoclonal antibody containing microcystins;
the 12 molecular structural variants of the 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, mouse serum albumin, thyroglobulin or rabbit serum albumin.
The immunogen in the step 2) is an equal mass mixture of 12 microcystin molecular structure variants coupled with KLH or BSA respectively.
The immune mouse in the step 2) is a Balb/c mouse; the immunization method comprises the following steps: the dosage of the immunogen for each immunization is 30-50 mug/mouse, and the interval time of the two immunizations is 10-30 days; the immunization mode is subcutaneous multi-point injection; the mouse myeloma cell is a mouse
The method for screening the positive hybridoma cell strain in the step 2) comprises the following steps: firstly, screening by using a coating antigen, and then preparing an immunogen and screening by using a protein monomer for the coating antigen; the coating antigen is obtained by coupling the amino-modified microcystin molecules obtained in the step 1) with Bovine Serum Albumin (BSA); the coating antigen is not the same as the carrier protein in the immunogen.
The application of the monoclonal antibody, the composition, the immunogen, the kit or the method in detecting and/or purifying microcystins is also within the protection scope of the invention.
The application is detecting microcystin in environmental samples or food; the molecular structure variant of the microcystin in the application is at least one of 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 use is for non-disease diagnostic purposes.
In order to obtain antibodies with high specificity to the whole cyclic peptide structure of the microcystins, 12 kinds of microcystins (MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW, MC-LY, MC-WR, MC-HtyR, MC-HilR, [ D-Asp3] are respectively used]MC-LR,[D-Asp3]Introducing an amino active group on a seventh amino acid (Mdha) of MC-RR) through chemical modification of 2-mercaptoethylamine, then coupling with Keyhole Limpet Hemocyanin (KLH) to respectively obtain 12 conjugates of microcystins and KLH, and immunizing by adopting a 'mixed immunogen method', namely mixing the 12 conjugates of microcystins and KLH (12 complete antigens) in equal mass and then immunizing a mouse by using an immunogen; respectively coupling the 12 small molecules with or Bovine Serum Albumin (BSA), mixing the coupled small molecules in equal amount to serve as coating antigens, and measuring the serum titer by adopting an indirect ELISA method; selecting spleen cells of immunized mice with serum titer meeting requirementsFusing cell with SP2/0 myeloma cell, screening out positive hybridoma cell strain, culturing positive hybridoma cell strain, identifying its subtype, injecting the IgG1 subtype positive hybridoma cell strain into the abdominal cavity of homologous mouse to induce ascites to obtain broad-spectrum microcystin monoclonal antibodies MCAB1, MCAB2, MCAB3, MCAB4 and MCAB5, and the average affinity constant of MCAB1 and 12 microcystins is 4.10 × 108L/mol, the average affinity constant of antibody MCAB2 with 12 microcystins was 1.12 × 109L/mol, the average affinity constant of antibody MCAB3 with 12 microcystins was 5.35 × 108L/mol, the average affinity constant of antibody MCAB4 with 12 microcystins was 6.64 × 108L/mol, the average affinity constant of antibody MCAB5 with 12 microcystins was 3.92 × 108L/mol, the results show that the 5 monoclonal antibodies have higher affinity to 12 kinds of microcystins and can be used for detecting the microcystins in actual samples (such as environmental samples or food).
Biological material preservation instructions
1. Taxonomic nomenclature of biological materials: a positive mouse hybridoma cell strain secreting a broad-spectrum antibody of microcystin.
Numbering of the biological material: MCAB 1.
The preservation unit is called as follows: china general microbiological culture Collection center.
The preservation unit is abbreviated as: CGMCC.
Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The preservation date is as follows: 12 and 10 months in 2019.
The preservation number is: CGMCC No. 19194.
2. Taxonomic nomenclature of biological materials: a positive mouse hybridoma cell strain secreting a broad-spectrum antibody of microcystin.
Numbering of the biological material:
The preservation unit is called as follows: china general microbiological culture Collection center.
The preservation unit is abbreviated as: CGMCC.
Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The preservation date is as follows: 12 and 10 months in 2019.
The preservation number is: CGMCC No. 19195.
3. Taxonomic nomenclature of biological materials: a positive mouse hybridoma cell strain secreting a broad-spectrum antibody of microcystin.
Numbering of the biological material: MCAB 3.
The preservation unit is called as follows: china general microbiological culture Collection center.
The preservation unit is abbreviated as: CGMCC.
Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The preservation date is as follows: 12 and 10 months in 2019.
The preservation number is: CGMCC No. 19196.
4. Taxonomic nomenclature of biological materials: a positive mouse hybridoma cell strain secreting a broad-spectrum antibody of microcystin.
Numbering of the biological material: MCAB 4.
The preservation unit is called as follows: china general microbiological culture Collection center.
The preservation unit is abbreviated as: CGMCC.
Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The preservation date is as follows: 12 and 10 months in 2019.
The preservation number is: CGMCC No. 19197.
5. Taxonomic nomenclature of biological materials: a positive mouse hybridoma cell strain secreting a broad-spectrum antibody of microcystin.
Numbering of the biological material: MCAB 5.
The preservation unit is called as follows: china general microbiological culture Collection center.
The preservation unit is abbreviated as: CGMCC.
Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The preservation date is as follows: 12 and 10 months in 2019.
The preservation number is: CGMCC No. 19198.
Drawings
FIG. 1 is the binding curve of monoclonal antibody secreted by the positive hybridoma cell strain MCAB2 screened by the invention and the coating antigen BSA-MC-LR with different concentrations.
FIG. 2 is a standard curve for detecting MC-LR, MC-RR and MC-YR in a water sample respectively by using the monoclonal antibody secreted by the screened positive hybridoma cell strain MCAB 5.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Myeloma cell SP2/0 seed (Beijing large protein research center); SPF grade Balb/c pure female mice (sbeft Beijing Biotechnology, Inc.); newborn bovine serum (Nanjing Victort Biotechnology Co., Ltd.); enzyme-labeled secondary antibody (goat anti-mouse IgG/HRP) (Beijing Zhonghua Jinqiao Biotechnology Co., Ltd.); PEG 1500(Roche corporation); HAT stock solution, HT stock solution, paraffin oil, BSA, hemocyanin (KLH), Tris-HCl, 2.2% methylcellulose, 2-mercaptoethylamine, glutaraldehyde, citric acid, sodium citrate, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, carbonate buffer, Tetramethylbenzidine (TMB), urea hydrogen peroxide (CO (NH)2)2·H2O2) Tween-20, Freund's complete adjuvant, Freund's incomplete adjuvant (Sigma-Aldrich Co.); IMDM medium (Invitrogen); dimethylsulfoxide (DMSO) (Amresco corporation); BupH PBS buffer (Thermo Fisher Scientific Co.); lysine (Shanghai-derived leaf Biotech Co., Ltd.); anhydrous ether, acetic acid, isopropanol, and methylAlcohol, 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 Co.).
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