CN116162165A - Antihuman thrombin-antithrombin complex antibody, preparation method, detection reagent and application - Google Patents
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Abstract
The invention discloses an antihuman thrombin-antithrombin complex antibody, a preparation method, a detection reagent and application, wherein a pTRIOZ-mIgG1e2 expression plasmid containing thrombin A chain and B chain coding sequences is firstly constructed, HEK293F cells are transfected, cell culture supernatant is collected, and thrombin recombinant proteins are obtained through purification of Ni columns and Strep-Tactin columns; then constructing pCDNA3.4 expression plasmid containing antithrombin coding sequence, transfecting HEK293F cells, collecting cell culture supernatant, purifying by Ni column to obtain antithrombin recombinant protein; preparing thrombin-antithrombin complex by in vitro reaction of thrombin recombinant protein and antithrombin recombinant protein; the prepared thrombin-antithrombin complex is used as an immunogen to immunize a Balb/c mouse, spleen cells of the mouse are fused with myeloma cells SP2/0 to obtain hybridoma cells, and thrombin, antithrombin and thrombin-antithrombin complex are used for screening clone strains.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis, in particular to a preparation method of an antithrombin-antithrombin compound specific antibody and application of the antibody in a thrombin-antithrombin compound detection kit.
Background
Thrombin-antithrombin complex (TAT) is a complex which is produced by rapid binding to antithrombin 1:1 after in vivo prothrombin has been activated to thrombin. Thrombin consists of two polypeptide chains, one containing 36 amino acid residues (a-chain) and the other containing 259 residues (B-chain), which are covalently linked by disulfide bonds. It is difficult to directly measure thrombin level, and measuring the concentration of TAT in plasma can reflect thrombin level combined with antithrombin, and the TAT content in plasma has diagnostic effect on thrombosis such as disseminated intravascular coagulation. The content of free antithrombin in blood is about hundred thousand times that of TAT, and accurate determination of TAT is difficult.
Currently, the mainstream TAT quantification method is a chemiluminescent immunoassay (CLEIA) kit of hsomekang in japan, and the detection principle is that thrombin monoclonal antibody and antithrombin monoclonal antibody are used to realize quantitative detection of TAT through double antibody sandwich, and the detection method is a solid-liquid phase separation assay method, and requires complicated cleaning operation and automatic detection equipment.
The detection of TAT is gradually focused, and the development of high-quality TAT specific antibodies and domestic high-quality and low-cost detection kits has great significance.
Disclosure of Invention
The invention aims to provide an antihuman thrombin-antithrombin complex antibody, a preparation method, a detection reagent and application thereof, and the application of the prepared antibody in a thrombin-antithrombin complex detection kit is adopted, so that the thrombin-antithrombin complex content in a sample can be rapidly and accurately detected by the kit, and the detection result is not influenced by antithrombin.
In order to achieve the above purpose, the present invention provides the following technical solutions: the preparation method of the antithrombin-antithrombin complex specific antibody comprises the following steps:
first step, preparation of thrombin-antithrombin complex
Firstly constructing pTRIOZ-mIgG1e2 expression plasmid containing thrombin A chain and B chain coding sequences, transfecting HEK293F cells, collecting cell culture supernatant, and purifying by a Ni column and a Strep-Tactin column to obtain thrombin recombinant protein; then constructing pCDNA3.4 expression plasmid containing an antithrombin chain coding sequence, transfecting HEK293F cells, collecting cell culture supernatant, and purifying by a Ni column to obtain antithrombin recombinant protein; mixing thrombin recombinant protein and antithrombin recombinant protein according to the ratio of 0.5:1, reacting to prepare thrombin-antithrombin complex, and separating and purifying by molecular sieve to obtain high-purity thrombin-antithrombin complex;
second step, preparation of antithrombin-antithrombin complex specific antibodies
Immunization of Balb/c mice with thrombin-antithrombin complexes prepared in the first step as immunogens, ELISA indirect method for detecting mice serum titers exceeding 10 5 Then, fusing the mouse spleen cells with myeloma cells SP2/0 to obtain hybridoma cells, and screening clone strains by thrombin, antithrombin and thrombin-antithrombin complexes to obtain cell strains capable of secreting antithrombin-antithrombin complex specific antibodies; the method comprisesInjecting hybridoma cells into the abdominal cavity of a mouse, collecting ascites, and purifying to obtain a specific monoclonal antibody of the antithrombin-antithrombin complex;
the antithrombin-antithrombin complex specific antibody prepared by the invention is a new conformational epitope which is identified in the thrombin-antithrombin complex forming process and is not combined with thrombin and antithrombin;
the invention relates to an application of an antithrombin-antithrombin compound specific antibody in a thrombin-antithrombin compound detection kit, which can accurately detect the content of thrombin-antithrombin compound in a sample by a double-antibody sandwich method by adopting the antibody and thrombin antibody combination, and the detection method is not influenced by antithrombin, thus being a method for specifically detecting human thrombin-antithrombin compound.
Compared with the prior art, the invention has the beneficial effects that:
the invention has the advantages that the preparation method is simple, the prepared antibody has strong specificity, the detection mode is not influenced by antithrombin when the antibody is applied to a kit for detecting the content of thrombin-antithrombin complex, and compared with a main flow detection kit (Japanese Hizimeric TAT chemiluminescent detection kit), the kit has lower cost and stronger specificity, and can provide reliable reference for clinical diagnosis and treatment of doctors.
Drawings
FIG. 1 shows the results of thrombin-antithrombin complex non-denaturing polyacrylamide gel electrophoresis;
FIG. 2 is a Western blot identification of antithrombin-antithrombin murine monoclonal antibodies;
FIG. 3 is a test linearity of the kit of the present invention;
FIG. 4 is a correlation of the kit of the invention with a Hizimeric kit;
FIG. 5 is a sequence diagram of SEQ ID NO.1 according to the present invention;
FIG. 6 is a sequence diagram of SEQ ID NO.2 according to the present invention;
FIG. 7 is a sequence diagram of SEQ ID NO.3 according to the present invention;
FIG. 8 is a sequence diagram of SEQ ID NO.4 according to the present invention;
FIG. 9 is a sequence diagram of SEQ ID NO.5 according to the present invention;
FIG. 10 is a sequence diagram of SEQ ID NO.6 according to the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Referring to fig. 1-10, the present invention provides a technical solution: an antihuman thrombin-antithrombin complex antibody, a preparation method, a detection reagent and application,
preparation of thrombin-antithrombin complexes
1. Thrombin A chain, B chain and antithrombin coding gene synthesis
From NCBI (National Center for Biotechnology Information), the mRNA sequence of Prothrombin (NM-000506.5) was queried, the thrombin A chain amino acid sequence of which was (SEQ ID NO. 1) and the thrombin B chain amino acid sequence of which was (SEQ ID NO. 2);
the mRNA sequence of antithrombin (AH 002614.2) was queried from NCBI (National Center for Biotechnology Information), which has the amino acid sequence of (SEQ ID NO. 3);
under the precondition of not changing amino acid sequence, carrying out codon optimization on the gene sequences for encoding the recombinant proteins so as to improve the expression quantity in HEK293F cells, wherein the optimized specific sequence is shown as a sequence table SEQ ID No: 4. SEQ ID No: 5. SEQ ID No:6, entrusting the Nanjing Jinsri biotechnology Co.
2. Construction of recombinant thrombin A-and B-chain expression vectors and recombinant antithrombin expression vectors
The synthetic thrombin A chain gene is used as a template, primers respectively comprising SgrAI and PshAI restriction enzyme cutting sites are designed, an external secretion signal peptide sequence is introduced into an upstream primer, and a Strep tag sequence is introduced into a downstream primer, so that external secretion expression and later purification are facilitated. The target gene is amplified by using an amplification primer, and a fusion fragment with the structure of SgrAI enzyme cutting site-signal peptide sequence-thrombin A chain sequence-Strep label-PshAI enzyme cutting site is amplified. The target gene fragment and pTRIOZ-mIgG1e2 vector were digested with SgrAI and PshAI restriction enzymes, and after digestion, the gel was recovered using 1% agarose gel. The digested and recovered thrombin A chain fragment was then ligated to the cut pTRIOZ-mIgG1e2 vector using T4 DNA ligase.
The synthetic thrombin B chain gene is taken as a template, primers respectively comprising AgeI and AvrII restriction enzyme cutting sites are designed, an external secretion signal peptide sequence is introduced into an upstream primer, and a His tag sequence is introduced into a downstream primer, so that external secretion expression and later purification are facilitated. Amplifying the target gene by using an amplification primer to obtain a fusion fragment with the structure of AgeI enzyme cutting site-signal peptide sequence-thrombin B chain sequence-His tag-AvrII enzyme cutting site. The target gene fragment and the recombinant thrombin A chain vector constructed above were subjected to double digestion with AgeI and AvrII restriction enzymes, and after digestion, gel recovery was performed using 1% agarose gel. The digested recovered thrombin B-chain fragment is then ligated with T4 DNA ligase onto the cleaved recombinant thrombin A-chain vector.
The synthetic antithrombin gene is used as a template, primers respectively containing XbaI and AgeI restriction enzyme sites are designed, an exocrine signal peptide sequence is introduced into an upstream primer, and a His tag sequence is introduced into a downstream primer, so that exocrine expression and later purification are facilitated. The target gene is amplified by using an amplification primer, and a fusion fragment with a structure of XbaI enzyme cutting site-signal peptide sequence-antithrombin sequence-His tag-AgeI enzyme cutting site is amplified. The desired gene fragment and the pcdna3.4 vector were double digested with XbaI and agoi restriction enzymes, and after digestion, gel recovery was performed using 1% agarose gel. The digested and recovered antithrombin fragment was then ligated to the cut pcdna3.4 vector using T4 DNA ligase.
3. Preparation of recombinant thrombin and antithrombin proteins
HEK293F cells were cultured using 293 chemistry-defined high-density serum-free cell culture broth (Pearl sea Biotechnology Co., ltd.) and the HEK293F cell density was adjusted to 2X 10 on the day of transfection 6 cell/mL, the activity rate is not lower than 95%, 100 mug plasmid and 500 mug transfection reagent are added for each 100mL cell suspension transfected, and the mixture is slowly dropped into a shake flask, 5% CO 2 Shaking table culture was carried out at 37℃and 120rpm for 7 days, and the culture supernatant was collected by centrifugation at 9000 rpm.
Recombinant thrombin protein purification. After equilibration of the Ni column with 10mmol PBS buffer, the cell supernatant was slowly loaded. After eluting the hybrid protein with 10mmol PBS buffer containing 50mmol/L imidazole, the recombinant protein was eluted with 10mmol PBS buffer containing 500mmol/L imidazole. Dialyzing the eluted recombinant protein into a binding buffer solution of 100mM Tris-HCl,150mM NaCl,1mM EDTA,pH 8.0, loading the eluted recombinant protein onto a Strep Tactin chromatographic column, and eluting the eluted recombinant protein by using a binding buffer solution containing 2.5mM dehydrobiotin to obtain the recombinant thrombin protein.
Purifying recombinant antithrombin protein. After equilibration of the Ni column with 10mmol PBS buffer, the cell supernatant was slowly loaded. Eluting the hybrid protein by using 10mmol PBS buffer solution containing 50mmol/L imidazole, and eluting by using 10mmol PBS buffer solution containing 500mmol/L imidazole to obtain the recombinant antithrombin protein.
4. Formation and purification of thrombin-antithrombin complexes
The recombinant thrombin and antithrombin prepared above were adjusted to 1.0mg/mL, mixed in a volume ratio of 0.5:1, incubated at 37℃for 30 minutes, and equilibrated with PBS at 1.0mL/min over a dextran gel filtration medium (Boglan Bestdex G-75), and thrombin-antithrombin complex was collected.
Example 2
Referring to fig. 1-4, the present invention provides a technical solution: a preparation method of an antithrombin-antithrombin complex specific antibody and application of the antibody in an antithrombin-antithrombin complex detection kit,
preparation of antibodies specific for antithrombin-antithrombin complexes
1. Immunization of mice
The thrombin-antithrombin complex prepared above was taken and thoroughly mixed with an equal volume of Freund's complete adjuvant (purchased from sigma biological company) to immunize Balb/c mice at a priming dose of 100 ug/mouse, and the second and third immunizations were performed at 21 and 42 days intervals after the first immunization, respectively, with an immunization dose of 50 ug/mouse. Collecting blood from orbit 7 days after the third immunization, separating serum, detecting serum titer by using thrombin-antithrombin complex by indirect method, and collecting serum titer greater than 10 5 The mice of (2) were subjected to intra-spleen booster immunization at a dose of 100 ug/mouse.
2. Hybridoma cell preparation
Spleen cells and sp2/0 myeloma cells were fused at a ratio of 10:1 at 3 days after the boost, and the fused cells were cultured on HAT-containing DMEM medium. Detecting the content of specific antibody in the cell culture supernatant by a 96-well plate indirect method coated with thrombin-antithrombin complex about 6-7 days after fusion, selecting positive holes with OD value not lower than 0.5, and performing 3 rounds of subcloning by a limiting dilution method to finally obtain the hybridoma cell strain capable of stably secreting the antithrombin-antithrombin complex.
3. Screening of hybridoma cell lines specifically recognizing thrombin-antithrombin complex
A hybridoma cell line in which the cell supernatant reacted with thrombin-antithrombin complex but not with thrombin and antithrombin was selected and identified as a cell line capable of secreting an antibody specific for thrombin-antithrombin complex, and the 1 cell line selected was designated TAT-D3.
4. Screening for thrombin-recognizing hybridoma cell lines
A hybridoma cell line that reacted with both thrombin and thrombin-antithrombin complex but did not react with antithrombin was selected and identified as a cell line capable of secreting thrombin-specific antibodies, and the 1-cell line selected was designated as T-D5.
Table 1 hybridoma cell screening assays.
5. Purification of thrombin and antithrombin-antithrombin complex murine monoclonal antibodies
Injecting the TAT-D3 and T-D5 hybridoma cells into the abdominal cavity of a mouse, collecting ascites, and purifying by SPA to obtain the anti-human thrombin-antithrombin complex with the purity of more than 95% and the anti-human thrombin mouse monoclonal antibody.
6. Identification of the type of the anti-thrombin-antithrombin Complex murine monoclonal antibody site
The thrombin-antithrombin complex prepared in example 1 was diluted to 0.1mg/ml with 10mmol PBS, 20. Mu.L of the diluted protein was subjected to 10% SDS-PAGE electrophoresis, and after the electrophoresis was completed, the gel was equilibrated in a transfer buffer for 15 minutes, and 300mA electrotransfer was performed for 30 minutes. The membrane was placed in a blocking solution, blocked overnight at room temperature, the blocking solution was discarded, and purified TAT-D3 monoclonal antibody was added at a concentration of 10. Mu.g/ml and incubated for 2 hours at 4 ℃. The membrane was washed 3 times with TBST for 3min each. A1/4000 dilution of horseradish peroxidase-labeled goat anti-mouse IgG was added, incubated at 37℃for 2 hours, and the membrane was washed with TBST 4 times for 3 minutes each. Adding the solution A and the solution B of the DAB kit which are uniformly mixed in equal proportion for developing. As can be seen from FIG. 2, the TAT-D3 antibody is not seen in the 66.4-97.2kD band, demonstrating that TAT-D3 is a conformational epitope that recognizes the thrombin-antithrombin complex.
Example 3
Referring to fig. 1-4, the present invention provides a technical solution: an antihuman thrombin-antithrombin complex antibody, a preparation method, a detection reagent and application,
method for detecting thrombin-antithrombin complex by double-antibody sandwich CLEIA
1. Biotin-conjugated TAT-D3 monoclonal antibodies
TAT-D3 monoclonal antibody was dissolved in a pH9.6 carbonate buffer to adjust the concentration to 10mg/mL. Biotin-N-hydroxysuccinimide ester (Biotin-NHS) was dissolved in DMF at a concentration of 10mg/mL. Biotin-NHS was slowly added dropwise in a proportion of 150. Mu.g per 1mg of antibody, and the reaction was carried out overnight at 4℃after gentle shaking. Separating and purifying by using a molecular sieve tube with a 30kD cut-off amount, and adding equal volume of glycerol to store at 4 ℃ for later use.
2. Enzyme-labeled streptavidin (T-D5-AP)
1mg of AP is weighed and dissolved in 0.5mL of distilled water, and 0.5mL of newly prepared 0.06M NaIO is added 4 The solution was allowed to stand at 4℃for 30min in the dark, and then 0.5mL of 0.16M ethylene glycol was added to terminate the reaction. 1mg of T-D5 monoclonal antibody is added, the mixture is placed in a dialysis bag after standing at room temperature for 30min, and the dialysis is carried out at 4 ℃ in 0.05M carbonate buffer solution for overnight. 0.2mL of freshly prepared 5mg/mL NaBH was added 4 Mixing, vibrating at room temperature for 2h, adding saturated ammonium sulfate solution with equal volume, standing at 4deg.C for 30min, centrifuging at low temperature (4deg.C, 12000rpm,30 min), and separating. The precipitate was dissolved in 0.2mL of 0.05M phosphate buffer, and then stored at-20℃with an equivalent amount of glycerol.
3. Reagent minimum detection limit and linear range determination
Diluting 20 μg streptavidin magnetic beads (purchased from Beijing Boer biotechnology Co., ltd.) to 20 μg/mL with 0.1MMES buffer (pH 5.0), adding a chemiluminescent special enzyme-labeled blackboard (purchased from Thermo Fisher Scientific) into 100 μl of each well, washing, re-suspending to 40 μg/mL, adding 5000-fold diluted biotin-coupled TAT-D3 monoclonal antibody into 50 μl of the well, shaking and reacting for 30min on a shaker, washing and spin-drying, adding 50 μl of standard or sample to be tested and 50 μl of 5000-fold diluted T-D5-AP on a shaker, shaking and reacting for 30min, washing and spin-drying, adding 100 μl of chromogenic substrate (Beijing Bo immunological technology Co., ltd.), shaking and reacting for 5min on a shaker, and reading PLU value in a chemiluminescent instrument.
4. Performance assessment of the kit of the invention
(1) Minimum detection limit and linear range of reagent
Drawing a standard curve with standard substance concentration and RLU value, wherein R of the standard curve 2 ＝0.9998。
And measuring for 20 times by using a zero standard, calculating the average RLU value and standard deviation of the standard, substituting the RLU value obtained by adding 2 times of standard deviation to the RLU average value into a standard curve equation, and calculating the concentration as the lowest detection amount of the standard curve equation.
The minimum detection limit of the reagent is 0.066ng/mL, and the effective linear range is 0.066-150 ng/mL.
TABLE 2 Standard concentration and corresponding RLU values
TABLE 3 RLU value for zero value standard detection
(2) Clinical sample detection
50 clinical samples were collected and simultaneously the thrombin-antithrombin complex was detected using the chemiluminescent immunoassay (CLEIA) kit of hispida and the CLEIA assay of the invention, the overall correlation R of the kit of the invention and the hispida kit 2 ＝0.9802。
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. An antihuman thrombin-antithrombin complex antibody, a preparation method, a detection reagent and application thereof are characterized in that: the method comprises the following steps:
first, preparation of thrombin-antithrombin complex:
firstly, constructing pTRIOZ-mIgG1e2 expression plasmids containing thrombin A chain and B chain coding sequences and pCDNA3.4 expression plasmids containing antithrombin coding sequences, respectively transfecting HEK293F cells, collecting cell culture supernatant, purifying thrombin and antithrombin recombinant proteins by affinity chromatography, mixing the thrombin and the antithrombin recombinant proteins according to the ratio of 0.5:1, reacting to prepare thrombin-antithrombin complex, and separating and purifying by molecular sieve to obtain the thrombin-antithrombin complex with high purity;
second, preparation of antithrombin-antithrombin complex specific antibodies:
immunization of Balb/c mice with thrombin-antithrombin complexes prepared in the first step as immunogens, ELISA indirect method for detecting mice serum titers exceeding 10 5 Then, fusing the mouse spleen cells with myeloma cells SP2/0 to obtain hybridoma cells, and screening clone strains by thrombin, antithrombin and thrombin-antithrombin complexes to obtain cell strains capable of secreting antithrombin-antithrombin complex specific antibodies; injecting the hybridoma cells into the abdominal cavity of a mouse, collecting ascites, and purifying to obtain the specific monoclonal antibody of the antithrombin-antithrombin complex.
2. The anti-human thrombin-antithrombin complex antibody, the preparation method, the detection reagent and the application according to claim 1, wherein: the prepared antithrombin-antithrombin complex specific antibody is a new conformational epitope antibody which is used for recognizing thrombin-antithrombin complex forming process and is not combined with thrombin and antithrombin.
3. The anti-human thrombin-antithrombin complex antibody, the preparation method, the detection reagent and the application according to claim 1, wherein: the prepared thrombin-antithrombin complex specific antibody is applied to a thrombin-antithrombin complex detection kit, the thrombin-antithrombin complex specific antibody is combined with thrombin antibody, the content of thrombin-antithrombin complex in a sample can be accurately detected by a double-antibody sandwich method, and the detection method is not influenced by antithrombin, so that the method is a method for specifically detecting human thrombin-antithrombin complex.
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| JPS62138187A (en) * | 1985-12-13 | 1987-06-20 | Yatoron:Kk | Anti-antithrombin-thrombin complex monoclonal antibody |
| GB9021370D0 (en) * | 1990-10-02 | 1990-11-14 | Ciba Geigy Ag | Monoclonal antibodies directed against complexes formed by thrombin and thrombin inhibitors |
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| JPS62138187A (en) * | 1985-12-13 | 1987-06-20 | Yatoron:Kk | Anti-antithrombin-thrombin complex monoclonal antibody |
| GB9021370D0 (en) * | 1990-10-02 | 1990-11-14 | Ciba Geigy Ag | Monoclonal antibodies directed against complexes formed by thrombin and thrombin inhibitors |
| CN115327121A (en) * | 2015-03-31 | 2022-11-11 | 美迪恩斯生命科技株式会社 | Reagent and method for measuring thrombin-antithrombin complex |
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