CN116462755B - Monoclonal antibody or antigen binding fragment of anti-microcystin LR - Google Patents
Monoclonal antibody or antigen binding fragment of anti-microcystin LR Download PDFInfo
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- CN116462755B CN116462755B CN202310714490.0A CN202310714490A CN116462755B CN 116462755 B CN116462755 B CN 116462755B CN 202310714490 A CN202310714490 A CN 202310714490A CN 116462755 B CN116462755 B CN 116462755B
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Classifications
-
- 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
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- 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
-
- 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
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention belongs to the field of biotechnology, and provides an anti-microcystin LR monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody or the antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region; the heavy chain variable region comprises immunoglobulin heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3; the light chain variable region comprises immunoglobulin light chain complementarity determining regions LCDR1, LCDR2, LCDR3. The anti-MC-LR monoclonal antibody is used for detecting microcystin LR, especially for detecting microcystin LR in food or water, for example, a common indirect competition ELISA detection method or a colloidal gold test strip can be established based on the monoclonal antibody, the detection accuracy is high, the stability is good, the detection result is accurate, the detection cost is low, the high-flux rapid detection requirement of the actual market can be met, and the monitoring and monitoring of polluted water quality and polluted food sources at any time can be well realized.
Description
Technical Field
The invention relates to a monoclonal antibody of anti-microcystin LR or antigen binding fragment thereof, belonging to the field of biotechnology.
Background
Microcystin LR (MC-LR) is one of the most widely distributed and most harmful freshwater cyanobacterials, so World Health Organization (WHO) prescribes a minimum limit of MC-LR of 1ng/mL in the standard guidelines of drinking water. The compound belongs to a cyclic heptapeptide compound with biological activity, is mainly produced by fresh water algae microcystis aeruginosa (Microcystis aeruginosa), can strongly inhibit the activity of protein phosphatase 1&2A, has strong liver cancer rate, can not effectively destroy the structure under the boiling and heating condition of 100 ℃, and can not be completely adsorbed by activated carbon. At present, the harm of MC-LR to the water environment and the health of people is one of the important environmental problems of global attention. In addition to entering the human body through drinking water, MC-LR can also accumulate in aquatic organisms, and it has been reported that fish, molluscs and plankton can accumulate a large amount of microcystin LR, which in turn enters the human body through the food chain. Laboratory work at home and abroad has been largely studied on detection of MC-LR, and specific detection methods include chemical analysis, immunoassay, protease inhibition, cytotoxicity, bioanalytical methods, and the like. The existing national standard detection method of MCs is mainly liquid chromatography, and has the problems of high cost, large sample sampling amount, complex pretreatment method and the like although the method has higher selectivity. In contrast, the immunoassay detection method has the technical advantages of rapid detection, high sensitivity, easy execution, low cost and the like. At present, monoclonal antibodies aiming at MC-LR with low detection cost, strong specificity and high sensitivity are urgently needed, and are combined with a conventional matched immune rapid detection technology to be applied to the fields of food safety, water pollution and the like in a larger range.
Antibodies to microcystin LR have been obtained by some screening methods in the prior art, but these antibodies have the following disadvantages: 1. antibodies are of non-uniform nature and have limited industrial application. 2. The antibody has low specificity, is easy to be interfered by microcystin MC-RR when being applied to immunodetection, and has low detection sensitivity. 3. The stability is poor, and the industrial cost is relatively high due to the limitation of the active condition during application. Therefore, at present, a monoclonal antibody aiming at MC-LR with low detection cost, strong specificity and high sensitivity is still urgently needed, so that the monoclonal antibody can be combined with a conventional matched immune rapid detection technology, and is widely applied to the fields of food safety, water pollution and the like.
Disclosure of Invention
The invention aims to solve the technical problems of providing the MC-LR monoclonal antibody of the microcystin, which has strong specificity, high sensitivity and stronger broad-spectrum recognition capability, can be used for rapidly detecting the microcystin, and lays a foundation for the accurate detection of MC-LR.
The invention adopts the following technical scheme:
a monoclonal antibody against microcystin LR, or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region;
the heavy chain variable region comprises immunoglobulin heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3; the amino acid sequence of the HCDR1 is RYNMS, the amino acid sequence of the HCDR2 is SINALGITQWPDSVKG, and the amino acid sequence of the HCDR3 is YSLPGSSYVSFDV;
the light chain variable region comprises immunoglobulin light chain complementarity determining regions LCDR1, LCDR2, LCDR3; the amino acid sequence of LCDR1 is GKMASVSADDYSYML, the amino acid sequence of LCDR2 is LAYNLEI, and the amino acid sequence of LCDR3 is QLHRSEPWT.
Alternatively, the heavy chain variable region has an amino acid sequence having at least 95% identity to SEQ ID No. 1;
the light chain variable region has an amino acid sequence having at least 95% identity to SEQ ID NO. 2.
Alternatively, the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO. 1;
the light chain variable region has an amino acid sequence shown as SEQ ID NO. 2;
alternatively, the monoclonal antibody or antigen binding fragment thereof against microcystin LR comprises a heavy chain IgG1 constant region and a light chain kappa constant region.
In a preferred embodiment, the 3 CDRs contained in the heavy chain variable region and/or the 3 CDRs contained in the light chain variable region are defined by the Chothia numbering system; in a preferred embodiment, the 3 CDRs contained in the heavy chain variable region and/or the 3 CDRs contained in the light chain variable region are defined by the ABM numbering system; in a preferred embodiment, the 3 CDRs contained in the heavy chain variable region and/or the 3 CDRs contained in the light chain variable region are defined by the Kabat numbering system; in a preferred embodiment, the 3 CDRs contained in the heavy chain variable region and/or the 3 CDRs contained in the light chain variable region are defined by the IMGT numbering system. Further, 3 CDRs contained in the heavy chain variable region and/or 3 CDRs contained in the light chain variable region are defined by the Kabat numbering system.
Alternatively, the monoclonal antibody is a murine antibody, chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody.
Alternatively, the antigen binding fragment is selected from Fab, fab ', (Fab') 2, fv, disulfide-linked Fv, scFv, diabody, or single domain antibody (sdAb).
Optionally, the monoclonal antibody further comprises: a heavy chain constant region (CH) of a mammalian immunoglobulin or variant 1 thereof, variant 1 having a substitution, deletion or addition of one or more amino acids compared to the sequence from which it is derived; and, a light chain constant region (CL) of a mammalian immunoglobulin or variant 2 thereof, variant 2 having up to 20 amino acid conservative substitutions compared to the sequence from which it is derived.
In the present invention, a monoclonal antibody may include variant 3, which differs from the antibody from which it is derived only by conservative substitutions of one or more amino acid residues; for example, conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids; has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the antibody from which it is derived, and substantially retains the above-described biological function of the antibody from which it is derived.
Alternatively, the monoclonal antibody is labeled.
The antibody labeling aims to link a label to an antibody, specifically react with an object to be detected to form a multi-component compound, and directly microscopic observation or automatic measurement of test results are carried out by means of a fluorescent microscope, a ray measuring instrument, an enzyme-labeled detector, an electron microscope, a light-emitting immunoassay instrument and other precise instruments. The label in the invention does not particularly refer to a certain labeling mode, and concretely can be an enzyme label, a biotinylation label, a fluorescent label, a colloidal gold label and the like; and are not limited herein.
The invention also provides a preparation method of the monoclonal antibody or the antigen binding fragment thereof for resisting microcystin LR, which comprises the following steps:
culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof against microcystin LR, and recovering the monoclonal antibody or antigen-binding fragment thereof against microcystin LR from the cell or culture of the cell.
The preparation method of the invention cultures host cells under conditions that allow for expression of the monoclonal antibodies; monoclonal antibodies are recovered from the cultured host cell culture. The monoclonal antibodies provided herein can be prepared by various methods known in the art, such as by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then the host cell is transfected. The transfected host cells are then cultured under specific conditions and express the antibodies of the invention.
The invention also provides a nucleic acid encoding the monoclonal antibody against microcystin LR or antigen-binding fragment thereof.
Alternatively, the nucleic acid sequence of the heavy chain variable region gene of the antibody is shown as SEQ ID NO.9, and the nucleic acid sequence of the light chain variable region gene of the antibody is shown as SEQ ID NO. 10.
The invention also provides a host cell, which comprises the nucleic acid.
The present invention also provides a composition comprising the above monoclonal antibody against microcystin LR or an antigen-binding fragment thereof.
The invention also provides a kit comprising the monoclonal antibody or antigen binding fragment thereof against microcystin LR.
The kit can adopt a double-antibody sandwich ELISA kit and a preparation method thereof, and comprises the monoclonal antibody mc-3LR9. Specifically, the ELISA kit adopts a double-antibody sandwich method, and consists of an ELISA plate coated with a capture antibody, an enzyme-labeled detection antibody, a substrate color development liquid, a stop solution and a concentrated washing liquid.
The invention also provides application of the monoclonal antibody or antigen binding fragment, nucleic acid, host cell, composition and kit for resisting microcystin LR in detection of microcystin LR. Further, the method comprises the application of detecting microcystin LR in food or water.
Compared with the prior art, the invention has the following technical effects:
the present invention provides a monoclonal antibody or antigen-binding fragment thereof against microcystin LR, comprising a light chain variable region and a heavy chain variable region; the heavy chain variable region comprises immunoglobulin heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3; the amino acid sequence of the HCDR1 is RYNMS, the amino acid sequence of the HCDR2 is SINALGITQWPDSVKG, and the amino acid sequence of the HCDR3 is YSLPGSSYVSFDV; the light chain variable region comprises immunoglobulin light chain complementarity determining regions LCDR1, LCDR2, LCDR3; the amino acid sequence of the LCDR1 is GKMASVSADDYSYML, the amino acid sequence of the LCDR2 is LAYNLEI, and the amino acid sequence of the LCDR3 is QLHRSEPWT; the anti-MC-LR monoclonal antibody has higher cross reaction rate to microcystin, which shows that the monoclonal antibody has better broad-spectrum recognition capability, and has higher specificity and sensitivity to MC-LR.
Therefore, the anti-MC-LR monoclonal antibody can be used for detecting microcystin LR, especially microcystin LR in food or water, for example, a common indirect competition ELISA detection method or a colloidal gold test strip can be established based on the monoclonal antibody, the detection accuracy is high, the stability is good, the detection result is accurate, the detection cost is low, the high-flux rapid detection requirement of the actual market can be met, and the monitoring and monitoring of polluted water quality and polluted food sources at any time can be well realized.
Drawings
FIG. 1 shows the SDS-PAGE identification of recombinantly produced antibody mc-3LR9.
FIG. 2a shows the result of SDS-PAGE gel electrophoresis silver staining for identification of antibody purification effect, lanes: protein labeling; lanes 1-2 are the antibody-mc-3 LR9 eluted with the elution buffer, the desalted antibody mc-3LR9, and the purified antibody mc-3LR9 was mostly in the elution buffer.
FIG. 2b shows the results of Western blotting experiments, lanes: protein markers, for the identification of the purification effect of antibodies; lanes 2-4 are antibody mc-3LR9 in different buffers of binding buffer, washing buffer and elution buffer, and most of purified antibody mc-3LR9 is already in elution buffer and is consistent with silver staining results.
FIG. 3 shows the results of purification of antibody mc-3LR9 by HPLC.
FIG. 4 shows the determination of the affinity constant (in units of μg/mL on the abscissa) of monoclonal antibody mc-3LR9.
FIG. 5 shows a plotted monoclonal antibody mc-3LR9 inhibition curve (abscissa unit ng/mL).
Detailed Description
The following examples are provided only for the purpose of illustrating the invention in more detail. Thus, for the purposes of this disclosure, it will be apparent to those skilled in the art that the examples should not be construed as limiting the scope of this disclosure.
Example 1
1. Preparation of anti-MC-LR monoclonal antibodies
Preparing MC-LR samples, namely, salvaging blue algae mud for foot experiments from a Taihu blue algae treatment experiment base, sieving to remove large mechanical impurities, centrifuging to remove water, freezing at-80 ℃ in a low-temperature refrigerator, freeze-drying to obtain algae powder by using a freeze dryer after freezing, leaching the algae powder in glacial acetic acid, adding according to the volume-weight ratio of 1g/100mL, extracting for 2h, collecting supernatant, purifying by a Spe-pak C18 column, performing rotary evaporation on methanol collection liquid, and further performing refined MC-LR by using HPLC (high performance liquid chromatography), and collecting solids for later use.
Synthesis of MC-LR artificial complete antigen
By utilizing the characteristic of stronger nucleophilic addition reaction of ethylamine thiolate colored radicals, 1 amino group is introduced at the seventh amino acid unsaturated double bond Mdha position of microcystin and is coupled with carboxyl of carrier protein through the amino group, and the characteristics of Bovine Serum Albumin (BSA), chicken Ovalbumin (OVA) and keyhole limpet hemocyanin (keyhole limpet hemocyanin, KLH) which are known at present are considered, MC-LR is analyzed at about 1kD, the OVA cost is low, but the coupled complex is unstable, the KLH molecular weight is large and the structure is complex, the application technology of BSA is mature, the complex has stable physicochemical properties and morphological structure, the coupled state is stable, the solubility of the complex is good, and the cost is low, so that the complex is selected as the carrier protein. And (3) a coupling step: based on the more common coupling technique, MC-LR is coupled to carrier protein BSA by glutaraldehyde two-step method. To the solution was slowly added dropwise 400. Mu.L of 10 mmol/L CdCl 2 The solution is evenly mixed, the pH value is regulated to 7.0 by using 1mol/L KOH solution, then the solution is slowly oscillated to react in a shaking table at the constant temperature of 25 ℃ for 6 h, the solution is taken out and dialyzed in deionized water for 2 d, the deionized water is changed once in the morning, the evening and the morning, and the MC-LR complete antigen MC-LR-BSA is obtained after the dialysis is completed.
3. Hybridoma cell screening and antibody detection
Female BALB/c mice of 6 weeks old were immunized 4 times for 3 total immunizations. Each mouse was immunized with 80. Mu.L of artificial complete antigen MC-LR-BSA, and the specific immunization procedure is shown in the following Table. After the 2 nd immunization, 10 th and d th veins were collected, and the antibody titer was measured by an ELISA indirect method to determine whether or not antibodies against the antigen were produced. And if the potency does not reach the standard, continuing the subcutaneous immunization of Freund's incomplete adjuvant for the 3 rd time. The final direct injection of artificial complete antigen MC-LR-BSA, taking mouse spleen after 3d, selecting spleen cells, and preparing for subsequent fusion.
Table 1: complete antigen MC-LR-BSA immunization program
Antibody detection is carried out on the supernatant of all cell culture wells, and the specific method is as follows: (1) Coating, namely diluting MC-LR with a coating solution with pH of 9.6, adding 50-uL to each hole of an ELISA plate, wherein the antigen amount of each hole is 20ng, and coating at 40 ℃ overnight; wash 3 times with wash solution and pat dry. (2) Blocking, namely diluting the mouse serum to about 12% by using a diluent, and dripping the diluent into an ELISA plate, wherein each hole is 50uL. (3) Hybridoma culture supernatant, 50-100 uL/well, cell primary well supplemented with equal amount of RPMI-1640 medium, positive control was serum of 200X immunized mice diluted, negative control was Sp2/0 cell supernatant, and after incubation for 2h, 3 beats were washed. (4) Horseradish peroxidase-labeled goat anti-mouse secondary antibody at 1:10000 dilution was added dropwise, and incubation was repeated at 50 per well uL. (5) And (3) dropwise adding an o-phenylenediamine (OPD) substrate solution 100 uL into each hole, placing the ELISA plate at a dark place, dropwise adding a proper amount of stop solution into each hole when the color of the substrate is moderately developed, and measuring by using an automatic ELISA analyzer. The test is provided with 8 concentration gradients, namely 1:500,1:1000,1:2000, 1:4000, 1:8000,1:16000 and 1:32000,1:64000, wherein each concentration is provided with three parallel control holes, and finally the titer is determined according to the ratio of the average value of the three holes to the negative control hole, so that the cost effectiveness of serum is found to be close to 1:32000, and the test mice for cell fusion have good immune effect and can be used for cell fusion.
Spleen cells were selected for fusion with PEG4000 and SP2/0 myeloma cells. The ELISA plate is coated with 20 ng/mL, and the ELISA plate is screened by an indirect ELISA method, and cloned by a limiting dilution method. Through detection screening of three positive cell holes, 41 hole detection values are determined to be relatively close to positive control hole values, and then the holes with relatively good repeatability are subjected to clone screening.
4. Purification and subtype identification of monoclonal antibodies
Through the preliminary subcloning screening, 2 hybridoma cell strains capable of stably producing the anti-MC-LR monoclonal antibody are finally obtained, one of the hybridoma cell strains is named MC-3LR9, and the cell strains are subjected to expansion culture to prepare ascites. The 2 hybridoma cell strains are respectively injected into mice, and the generated ascites is collected. Specifically, 10 Balb/c mice produced by the warp and each mouse is injected with 500 uL Freund's incomplete adjuvant in the abdominal cavity to perform ' pre-stimulation ' on the mice, so that the normal immune function of the mice is reduced, and the generation of ascites is promoted. 7-10 days after injection of Freund's incomplete adjuvant, each mouse was intraperitoneally injected with l X10 6 Mice were observed daily for enlargement of the peritoneal cavity after injection of hybridoma cells. After 11 days of hybridoma cell injection, the abdominal cavity of the mouse is obviously enlarged, the abdominal cavity is punctured by an 18-gauge needle, and ascites is drained by gravity so as to obtain the ascites. Centrifuging the collected ascites at 3000 rpm for 10 min, collecting supernatant, and preserving at-20deg.C.
The collected ascites was filtered through a 0.5 μm filter membrane and purified by a Protein G column. Purity was determined by SDS-PAGE gel electrophoresis. After thermal cleavage of the antibody, the antibody heavy chains (molecular weight about 50 kD) and light chains (molecular weight about 25 kD) were obtained. The eluate of 12 tubes of antibody mc-3LR9 was collected, and the final antibody was pooled and concentrated to give a concentration of 7.73mg/mL of antibody mc-3LR9.
5. Recombinant production of antibody mc-3LR9
After separating single hybridoma cells, according to the need, extracting corresponding RNA from hybridoma cell strain of antibody mc-3LR9 by using RNA extraction kit instruction, taking hybridoma cells with the best growth state, centrifuging, washing, and counting living cells every 10 5 Individual cells were packed into an EP tube, and after adding 1 mL TRIzon Reagent, the mixture was thoroughly mixed, and allowed to stand for 5 minutes, specifically according to the Taka.9108 Trizol RNA extraction protocol. The mouse cDNA sequence was amplified using the extracted RNA as template according to the reverse transcription kit instructions. Amplification of antibodies using the obtained cDNA as a template and Universal primers for mouse antibody variable region genesLight and heavy chain genes. After PCR product purification, a cDNA library was constructed and examined by Agilent 2100 Bioanalyzer with Agilent high-sensitivity DNA chip
The size distribution of cDNA fragments was obtained. In order to obtain a good library, the resulting product should have little or no short fragments (< 500 bp) and peak at a fragment size of 1.5-2 kb. And selecting samples with better quality, and carrying out library construction work. And finally, detecting the quality of the cDNA library, wherein the ideal average size of the fragmented DNA library is 300-800bp broad peak. Library sequencing was done by Beijing Boolodian Biolimited using the illuminea sequencing platform 150bp paired-end double ended sequencing approach.
Transcript de novo assembly was performed on scRNA-seq data using the rnaSPades in this experiment, with reference to the BOLGER method (BOLGER A M, LOHSE M, USADEL B. Trimmomatic: a flexible trimmer for Illumina sequence data [ J ]. Bioinformatics, 2014, 30 (15): 2114-2120.). Compared with transcriptome de novo splicing software such as Bridger and Trinity, the rnaSPades optimizes single-cell data, reduces the influence of noise of the single-cell data on splicing, and greatly reduces false positive transcripts of candidate transcripts. Igfinder was then used to find transcripts that might be antibody sequences. Finally, regions of the antibody sequence were annotated using IMGT v-quest and IMGT blast. The gene sequencing and the synthesis submitting Zhejiang Jinsri biotechnology Co., ltd, through the IMGT database mouse source antibody sequence comparison result and common mouse source antibody degenerate primer comparison, we further design the upstream and downstream primers for amplifying the heavy chain and light chain variable region of the antibody respectively, which are used for amplifying the light and heavy chain variable region, and the restriction enzyme sites Sgf I and Mlu I are introduced at both sides respectively.
TABLE 2 design primer PCR
Recombinant expression vectors were constructed using a maintainable Blue-White T Vector (Blue-White Vector T, available from Shanghai ze Biotechnology Co., ltd.) by cloning synthetic antibody heavy and light chain genes into the expression Vector Blue-White T Vector, and constructing recombinant expression Vector mc-3LR9-H and light chain expression Vector mc-3LR9-K, respectively. Wherein the nucleic acid sequence of the antibody heavy chain variable region gene is shown as SEQ ID NO.9, and the nucleic acid sequence of the antibody light chain variable region gene is shown as SEQ ID NO. 10. The two vectors were co-transfected together into HEK293 cells and after 4 days the cell supernatants containing the recombinant antibodies were collected. The supernatant of transiently transfected HEK293 cells and ascites from mice were purified using Protein G magnetic beads to obtain recombinantly expressed antibodies as well as antibodies secreted by hybridoma cells. Subsequently, the purified antibodies were identified by SDS-PAGE, and the results are shown in FIG. 1. Two distinct bands were seen from SDS-PAGE, with molecular weights of approximately 50kDa and 25kDa, respectively. The identification result is consistent with the clone screening result, and simultaneously accords with the molecular weight characteristics of the murine antibody, and no obvious interference protein exists. It can be seen that the recombinant vector was constructed successfully and it was expressed successfully as an intact antibody molecule in HEK293 cells.
The amino acid sequences of the antibodies are shown in the following table.
Table 3: anti-microcystin MC-LR monoclonal antibody MC-3LR9
In the next experiment, a stable solubilization system was developed for antibody mc-3LR9 protein, and the result of silver staining by SDS-PAGE gel electrophoresis after purification is shown in fig. 2a, and the result of Western blotting (Western blotting) after purification is shown in fig. 2 b. It can be seen that the western blot experimental results are consistent with the silver staining results, demonstrating that purified antibody mc-3LR9 was obtained.
From the aspect of immunity, the requirement on the purity of the antibody in detection is considered to be high, so that the high-performance liquid chromatography (HPLC) is adopted for precision purification, a chromatographic column TSK DEAE-5PW (TOYO Soda company), mobile phase A liquid (Tris-acetic acid 0.01mol/L, pH) and the flow rate is 1.0mL/min; mobile phase B solution (Tris-acetic acid 0.015mol/L, pH8.5, containing 1mol/L sodium acetate) with a flow rate of 1.0mL/min; the ultraviolet detection wavelength is 278nm, the sensitivity is 0.2AUFS, and the relation between the elution binary gradient time and the concentration of the mobile phase B is 0min-0% -33 min-60% -45 min-0%. Finally, we collect and obtain the anti-MC-LR monoclonal antibody with purity of more than 99.8%, the specific result is shown in figure 3, the collected peak is single peak, the purity of the sample is extremely high, and the specific requirement of the antibody for large-scale industrial application is met.
Typing kit (IsoStrip Roche) for the anti-MC-LR monoclonal antibody subtype with murine monoclonal antibody TM ) Identification and determination of OD 450 The subtype corresponding to the positive well with the highest value is the subtype to which the antibody mc-3LR9 belongs, and the results are shown in Table 4 below.
TABLE 4 identification of anti-MC-LR monoclonal antibody MC-3LR9 subtype
The results show that the anti-MC-LR monoclonal antibody MC-3LR9 is of the IgG1 type.
Example 2
1. Affinity assay of monoclonal antibody MC-3LR9 for MC-LR
The affinity constant Ka of the antibody indicates the affinity between the antibody and the antigen, and the high-affinity antibody can reduce the use amount of the antigen and the antibody in the actual use process, and simultaneously provides a lot of advantages for establishing an indirect competition ELISA detection method and preparing a colloidal gold immunochromatography test strip. Antibody affinity constant value Ka at 10 7 -10 12 High affinity is considered at mol/L, and Ka value is lower than 10 7 The lower affinity of the antibody can limit the further development and application of the antibody, so that the screening of the antibody with high affinity is important. The invention adopts an indirect non-competitive ELISA detection method (specific reference microcystin LR monoclonal antibody preparation and application research [ D ]]The affinity of the monoclonal antibodies was determined by the Anhui academy of science and technology, 2018.DOI:10.27869/d.cnki. Gakjx.2018.000017. The coated antigen (which was purified MC-LR prepared in example 1) was diluted to the appropriate 3 concentrations (1. Mu.g/mL, 0.33. Mu.g/mL, 0 in order) in a 3-fold gradient11. Mu.g/mL), the antibodies (the purified anti-MC-LR monoclonal antibodies prepared in example 1) were diluted by a 2-fold ratio to 8 concentrations, the logarithmic value of the antibody concentration was taken as the abscissa, the OD value at the corresponding concentration was taken as the ordinate, nonlinear fitting was performed by using Origin 8.5 software to obtain a standard curve, the antibody concentration value corresponding to the s0% absorbance was calculated from the fitted curve, each two antibody concentrations were set as a group, the affinity constant Ka of each group of antibodies was calculated, and finally the average value was taken. The formula is as follows:
Ka=(n-1)/2([Ab']t-[Ab]t)
n is the ratio of the concentration of the coating antigen in each group.
[Ab]t and [ Ab ]']t OD 50% at different coating antigen concentrations for each group max Corresponding antibody concentration (mol/L). As can be seen from the experimental results of FIG. 4, when the coating antigen concentration was 1. Mu.g/mL, 0.33. Mu.g/mL, 0.11. Mu.g/mL, 50% OD at each coating concentration max The corresponding antibody concentrations were 0.5. Mu.g/mL, 0.25. Mu.g/mL and 0.125. Mu.g/mL, respectively, and Ka of the monoclonal antibody mc-3LR9 was 5.37X10, calculated according to the formula of the antibody affinity calculation 8 The mol/L is an antibody with higher affinity.
Example 3
Identification of sensitivity of monoclonal antibody MC-3LR9 to MC-LR
Determination of monoclonal antibody sensitivity, i.e., IC, according to an indirect competition ELISA detection procedure 50 The value, under the condition of the best working concentration of the antibody, the inhibition rate of the multiple ratio series concentration standard substance is measured, the standard curve is established by fitting with Origin 8.5 software, and the IC of the antibody is obtained by calculation 50 Values. Based on the results of optimizing the conditions of the indirect competition ELISA assay, PSB buffer solution with concentration of 0.01mol/L, pH of 8.0 was selected as a standard diluent, and the sensitivity of monoclonal antibody mc-3LR9 was further measured, and a standard curve established by means of Origin 8.5 software is shown in FIG. 5.
Standard curve R for antibody mc-3LR9 fitted according to Origin 8.5 software 2 Is 0.993, and the monoclonal antibody mc-3LR9 antibody IC is calculated 50 At 1.41 ng/mL, the limit of detection (LOD) was 0.07ng/mL, and the linear range (IC) 20 -IC 80 ) Is 0.11-3.63 ngThe antibody has higher sensitivity and can be widely applied to MC-LR detection at low cost.
Example 4
Determination of Cross-reactivity of monoclonal antibody mc-3LR9
Reference "preparation of microcystin LR monoclonal antibody and its use" studied the specific determination of the cross-reactivity in DOI:10.27869/d.cnki. Gakjx.2018.000017, we determined the recognition ability of monoclonal antibody MC-3LR9 to another common microcystin RR (MC-RR) similar to MC-LR in structure and function and toxicity.
Table 5: cross-reactivity of antibody mc-3LR9
Table 5 shows the results of determining MC-RR standard by indirect competition ELISA, IC of MC-LR against monoclonal antibody 50 The value of 1.41 ng/mL and the cross reaction rate with MC-RR is 109.3%, which proves that the monoclonal antibody MC-3LR9 prepared by the test has better broad-spectrum recognition capability and higher cross reaction rate with MC-RR.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (9)
1. A monoclonal antibody against microcystin LR or antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region;
the heavy chain variable region comprises immunoglobulin heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3; the amino acid sequence of the HCDR1 is RYNMS, the amino acid sequence of the HCDR2 is SINALGITQWPDSVKG, and the amino acid sequence of the HCDR3 is YSLPGSSYVSFDV;
the light chain variable region comprises immunoglobulin light chain complementarity determining regions LCDR1, LCDR2, LCDR3; the amino acid sequence of the LCDR1 is GKMASVSADDYSYML, the amino acid sequence of the LCDR2 is LAYNLEI, and the amino acid sequence of the LCDR3 is QLHRSEPWT;
the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 1;
the light chain variable region has an amino acid sequence shown as SEQ ID NO. 2;
the monoclonal antibody or antigen binding fragment thereof against microcystin LR comprises a heavy chain IgG1 constant region and a light chain kappa constant region.
2. The monoclonal antibody or antigen-binding fragment thereof against microcystin LR according to claim 1 wherein said monoclonal antibody against microcystin LR is a murine or humanized antibody.
3. The method for preparing the monoclonal antibody against microcystin LR or antigen-binding fragment thereof according to claim 1 or 2, comprising the steps of:
culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof against microcystin LR, and recovering the monoclonal antibody or antigen-binding fragment thereof against microcystin LR from the cell or culture of the cell.
4. A nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof against microcystin LR according to claim 1 or 2.
5. A host cell comprising the nucleic acid of claim 4.
6. A composition comprising the monoclonal antibody or antigen-binding fragment thereof against microcystin LR according to claim 1 or 2.
7. A kit comprising the monoclonal antibody or antigen-binding fragment thereof of anti-microcystin LR of claim 1 or 2.
8. Use of the monoclonal antibody or antigen binding fragment thereof against microcystin LR of claim 1 or 2, the nucleic acid of claim 4, the host cell of claim 5, the composition of claim 6, the kit of claim 7 for detecting microcystin LR.
9. The use according to claim 8, comprising the use of microcystin LR in the detection of food or water.
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