CN112940131A - Rabbit monoclonal antibody aiming at TK1 in human serum and application - Google Patents
Rabbit monoclonal antibody aiming at TK1 in human serum and application Download PDFInfo
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Abstract
The invention discloses a rabbit monoclonal antibody aiming at TK1 in human serum, which is produced by immunizing a rabbit source by using an antigen; the antigen is formed by coupling peptide with an amino acid sequence of SEQ ID NO. 1 or peptide with an amino acid sequence of SEQ ID NO. 2 with a first carrier protein or a second carrier protein respectively; the rabbit monoclonal antibodies include rabbit monoclonal antibody a and rabbit monoclonal antibody B.
Description
Technical Field
The invention relates to a rabbit monoclonal antibody aiming at TK1 in human serum and application, belonging to the technical field of biology.
Background
Thymidine kinase 1(TK1) is a special kinase, catalyzes thymidine (TdR) to be 1-thymidylate phosphate (TMP), is a precursor necessary for the DNA synthesis of cancer cells, participates in the DNA salvage synthesis pathway, and is a special marker enzyme for DNA synthesis. TK1 is a cell cycle dependent enzyme, primarily found in the cytoplasm. The expression of TK1 is closely related to tumor cell proliferation, is a recognized cell proliferation specific marker and is clinically used for monitoring and dynamically evaluating abnormal proliferation rate of cells in vivo.
The healthy human cells are mostly in a static state and are non-proliferative cells, the TK1 enzyme content in serum is very low, when proliferative diseases occur, particularly tumors and tissue abnormal proliferative diseases, the cells are hyperproliferated, and the TK1 enzyme content in the serum is obviously increased and is 2 times or even more than 10 times higher than the average level of healthy people. Therefore, by detecting the change of the TK1 concentration level in serum, the cell proliferation abnormity can be sensitively found, the cell proliferation development trend can be dynamically evaluated, and important information is provided for early intervention and treatment of high-risk precancerous lesions and suppression of tumor progress. The difference from the common tumor markers is that the tumor markers CEA, AFP, PSA, etc. can only find the clinical tumors of partial organs, and the TK1 cell proliferation examination can more accurately examine the canceration risk of the lesion part, which cannot be replaced by the tumor markers.
At present, the tumor detection method adopting imaging hardly has the capacity of detecting early tumors, and the immunodetection method is the most ideal method for detecting the TK1 level in serum. Qualitative/semi-quantitative analysis based on immunohistochemistry, or qualitative/quantitative analysis based on ELISA and immunochemiluminescence, do not depart from high quality TK 1-specific antibodies. The TK1 specific antibody currently used in the industry includes polyclonal antibody and monoclonal antibody, wherein the former mainly is IgY from chicken, and the latter mainly is IgG from mouse; the emergence of rabbit monoclonal antibodies brings about a number of different advantages, which are more advantageous than the conventional murine monoclonal antibodies; first, rabbit antisera, which typically contain high affinity antibodies, can recognize a wider variety of epitopes than murine antisera; second, rabbit monoclonal antibodies are capable of recognizing many antigens that do not produce immunity in mice; thirdly, as the spleen of the rabbit is larger, more fusion experiments can be carried out, so that the high-throughput screening of the fusion cells becomes possible; from the aspect of immunogenicity, at present, the immunogenic polypeptide (epitope peptide) in the TK1 protein, including the carboxyl-terminal polypeptide and the amino-terminal polypeptide, is mainly used for immunizing chickens or mice to obtain antibodies, wherein part of the antibodies are obtained by using the full-length TK1 protein as an immunogen.
In conclusion, in the prior art, no report of preparing TK1 rabbit monoclonal antibody by adopting rabbit monoclonal antibody preparation technology exists, so that the key points are to search for a proper TK1 epitope peptide with immunogenicity, use the TK1 epitope peptide with immunogenicity, prepare a specific TK1 rabbit monoclonal antibody and develop a kit for quantitatively detecting TK 1.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a rabbit monoclonal antibody aiming at TK1 in human serum, the rabbit monoclonal antibody is applied to an enzyme-linked immunoassay method for establishing TK1 in human serum, and simultaneously the rabbit monoclonal antibody is used for preparing an in-vitro TK1 detection kit in human serum.
The technical scheme of the invention is as follows:
the invention discloses a rabbit monoclonal antibody aiming at TK1 in human serum, which is produced by immunizing a rabbit source by using an antigen; the antigen is formed by coupling peptide with an amino acid sequence of SEQ ID NO. 1 or peptide with an amino acid sequence of SEQ ID NO. 2 with a first carrier protein or a second carrier protein respectively; the rabbit monoclonal antibodies include rabbit monoclonal antibody a and rabbit monoclonal antibody B.
The invention discloses application of the rabbit monoclonal antibody aiming at TK1 in human serum in establishing an enzyme-linked immunoassay method for TK1 in human serum.
Further, the enzyme-linked immunoassay method adopts a double-antibody sandwich method for detection; the rabbit monoclonal antibody A is a capture antibody; the rabbit monoclonal antibody B is a binding antibody.
The invention also discloses application of the rabbit monoclonal antibody aiming at TK1 in human serum in preparation of a kit for detecting TK1 in human serum.
The invention also discloses an in-vitro detection kit for TK1 in human serum, which comprises an ELISA plate coated with the rabbit monoclonal antibody A, a binding antibody, an enzyme marker, a color developing agent, a stop solution, a TK1 standard substance, a washing buffer solution and a dilution buffer solution.
Compared with the prior art, the invention has the beneficial effects that:
1. the antigen formed by coupling the peptide with the amino acid sequence of SEQ ID NO. 1 or the peptide with the amino acid sequence of SEQ ID NO. 2 and the first carrier protein or the second carrier protein has good antigenicity, so that rabbit-derived immunity generates a rabbit monoclonal antibody aiming at TK1 in human serum, and the rabbit monoclonal antibody can be combined with TK1 in a serum sample in a high specificity manner.
2. The rabbit monoclonal antibody aiming at TK1 in human serum is applied to establishing a TK1 enzyme-linked immunoassay method in human serum, and is also applied to preparing a kit for detecting TK1 in human serum, so that the TK1 level in a serum sample can be effectively detected, and the TK1 can be used for early screening of tumors.
Detailed Description
The present invention will be further described with reference to specific examples, which are provided to assist understanding of the present invention and are not intended to be limiting.
The invention discloses a rabbit cloned antibody aiming at TK1 in human serum and application thereof, which are specifically described in the following embodiments;
example 1 preparation of Rabbit monoclonal antibody
Rabbit monoclonal antibody a or rabbit monoclonal antibody B was obtained by:
1. preparation of antigen:
having the amino acid sequence of SEQ ID NO 1 by BDB methodThe peptide or the peptide with the amino acid sequence of SEQ ID NO. 2 is coupled with a first carrier protein or a second carrier protein respectively to prepare an antigen A or an antigen B, and the first carrier protein and the second carrier protein are the same or different keyhole limpet hemocyanin; taking the amino acid sequence as peptide 1 or peptide 210.0 mg, dissolving with 1mL of 0.1M PBS buffer solution (pH is 7.4); keyhole Limpet Hemocyanin (KLH)10mg dissolved in 20mL of 0.2M borate buffer (pH 8.6); mixing the two solutions, cooling to 0 deg.C, and collecting BDBCl2Reacting at room temperature for 1.5h, dialyzing overnight, packaging, and storing at-20 deg.C to obtain antigen A or antigen B;
wherein, SEQ ID NO: 1: YAKDTRYSSSFCTHDRNTMEA
SEQ ID NO:2：AALDGTFQRKPFGA
2. Animal immunization:
first immunization: taking the prepared antigen A or antigen B (l.0mg antigen/antigen), dissolving the antigen A or antigen B in PBS buffer solution in equal volume, adding homogeneous Freund's complete adjuvant, violently shaking to emulsify the antigen A or antigen B, and extracting emulsion with 1mL syringe to remove bubbles; taking out the rabbit from the cage, fixing the rabbit by using a fixer, performing subcutaneous injection at 5 different parts, and selecting 4-6 points on two sides of a spine (two points of the neck, the chest and the lumbar) of the rabbit for subcutaneous injection; removing hair on the back of the rabbit, sterilizing with iodophor, inserting needle at 15 deg.C relative to skin, wherein the insertion depth is l-2cm, and injecting antigen solution without penetrating into muscle; after each injection, the needle was placed at the injection site for 10 seconds, then gently pulled out, sterilized at the injection site, and the above operation was repeated at 5 sites;
second immunization (0.5mg antigen/mouse): after 21 days (every 3 weeks), the same antigen with half of Freund's incomplete adjuvant is injected by the same method and injection operation as the first time, but the site selection is different from the first time; collecting blood on the 28 th day after injection, and collecting 4mL for 2-tube anticoagulation flow detection and 4mL for 1-tube anticoagulation ELISA detection; the operation steps are basically similar to the ear margin intravenous injection;
third immunization (0.5mg antigen/mouse): on day 35 (after 2 weeks), the injection method and dose were the same as the second, and blood was collected on day 42 after injection;
fourth immunization (0.5mg antigen/mouse): on day 49 (after 2 weeks), the injection method and dose were the same as the second, and blood was collected 56 days after injection;
3. cell fusion
Measuring the serum titer by an ELISA method after four times of immunization, taking a rabbit with high serum titer, and taking the spleen aseptically after collecting whole blood from the carotid artery; the spleen separation method comprises the following steps: fat and other tissue surrounding the spleen was first removed in a petri dish. After flushing with 1640 culture solution, placing the spleen on a stainless steel screen, extruding and screening, suspending with the 1640 culture solution, centrifuging at the rotating speed of 1400rpm for 5min, discarding the supernatant, repeatedly washing once, re-suspending the cells with the 1640 culture solution, taking a small amount of cell suspension, diluting, and dyeing with trypan blue to calculate the number of viable cells; based on cell count, 2X 10 of the total8A suspension of individual splenic lymphocytes is ready for use.
Subculturing 240E cells about one week before cell fusion experiment, collecting the vigorous 240E cells in logarithmic growth phase on the day of fusion to fuse with spleen cells of the immunized rabbit, wherein the fusion agent is 50% PEG, and taking culture supernatant of the 240E cells as negative supernatant control before fusion; respectively taking 2 × 108Spleen lymphocytes and 1X 108Mixing 240E cells uniformly, adding 1640 culture solution, separating, removing supernatant, mixing the two cells uniformly to form paste, pre-heating in a water bath at 37 ℃, slowly adding 50% PEG solution pre-heated at 37 ℃, adding 1640 culture solution pre-heated at 37 ℃, diluting PEG to lose effect, supplementing 1640 culture solution, centrifuging, removing supernatant, and suspending cell precipitate in HAT culture solution;
4. screening of hybridoma cells
The fused cells were split into 40 feeder cells-containing 96-well cell culture plates using HAT as a selective medium, and placed at 37 ℃ in 6% CO2Culturing in an incubator; performing initial detection after three weeks, taking cell culture hole supernatant, screening out positive clones by using an indirect ELISA method, and transferring hybridoma cells positive in the initial detection into a 24-hole cell culture plate; after one week, hybridoma cells with strong affinity of secreted antibody and good cell growth state are screened out by adopting indirect ELISA method and indirect competition ELISA method, and the hybridoma cells are usedCloning by a limiting dilution method; selecting five polyclonal clones for cloning, and subpackaging each polyclonal clone in 3-5 96-hole cell culture plates; after three weeks, positive clones were screened by indirect ELISA and each polyclonal clone was selected>3 (about 10) positive wells were transferred to 24-well cell culture plates; after one week, screening hybridoma cells with strong affinity and good cell growth state of the secreted antibody by adopting an indirect ELISA method and an indirect competitive ELISA method, and culturing to obtain the cloned antibody in the list;
5. antibody affinity assay
Adopting an indirect competition ELISA method to carry out antibody affinity determination, simultaneously adding TK1 solution with series concentration and rabbit monoclonal antibody A or rabbit monoclonal antibody B (after being diluted by 2500 times) into a 96-hole enzyme label plate coated with coating antigen, drawing an affinity curve by taking the inhibition rate as a vertical coordinate and the TK1 concentration logarithm as a horizontal coordinate, determining the mass concentration value (IC50 value) of TK1 when the inhibition rate reaches 50%, and determining the affinity of the rabbit monoclonal antibody to the TK 1;
the formula for calculating the inhibition rate is as follows:
inhibition rate is 100% - (OD)450sam/OD450max)×100％
Wherein, OD450samIndicates the OD of the competitive well, OD450maxRepresents the OD of the positive well.
The results show that: the concentration of TK1 is between 200 pg/mL-600 pg/mL when the rabbit monoclonal antibody A reaches 50% inhibition rate, and the concentration of TK1 is between 100 pg/mL-300 pg/mL when the rabbit monoclonal antibody B reaches 50% inhibition rate, which indicates that the prepared rabbit monoclonal antibody has high affinity to TK 1;
6. specificity identification of Rabbit monoclonal antibodies
Detecting the specificity of the rabbit monoclonal antibody by ELISA, respectively taking human TK1 protein, TK2 protein, S-100B protein and neuron specific enolase NSE as detection antigen coated ELISA plates, respectively detecting the specific reactions of the rabbit monoclonal antibody A and the rabbit monoclonal antibody B prepared by the invention and the human TK1 protein by an ELISA method, taking rabbit serum as a negative control, and taking PBS liquid as a blank control; the result shows that the rabbit monoclonal antibody A and the rabbit monoclonal antibody B prepared by the invention only react with TK1 positively, and react with S-100B protein, neuron-specific enolase NSE and TK2 protein negatively, which indicates that the rabbit monoclonal antibody A and the rabbit monoclonal antibody B have specificity.
Example 2 establishment of an enzyme-linked immunoassay for Rabbit monoclonal antibody to TK1 in human serum
The enzyme-linked immunoassay method adopts a double-antibody sandwich method, the rabbit monoclonal antibody A aiming at TK1 in human serum provided by the invention is a capture antibody, and the rabbit monoclonal antibody B is labeled by HRP and then is used as a binding antibody;
the enzyme-linked immunoassay method comprises the following specific steps:
(1) coating the capture antibody with carbonate buffer (pH 9.6, concentration 0.05M), incubating at 4 deg.C overnight, and washing the plate with the washing buffer;
(2) blocking with 1 (w/v)% BSA-0.05M;
(3) washing and diluting a standard sample and a sample to be detected by using a sample buffer solution, adding the standard sample and the sample to be detected into an enzyme label plate, incubating for 30-60min under the condition of normal temperature, and washing the plate by using a washing buffer solution;
(4) adding a rabbit monoclonal antibody B marked by HRP diluted by phosphate buffer solution containing 1% of bovine serum albumin and 0.05% of Tween-20, incubating for 30-60min at normal temperature, and washing the plate by using a washing solution;
(5) adding color developing agent, developing at room temperature for 15min, adding stop solution to stop developing, and measuring absorbance, OD at 450nm and 630nm respectively450Subtract OD630Is the corrected absorbance value.
Example 3 establishment of an enzyme-linked immunoassay for Rabbit monoclonal antibody to TK1 in human serum
The enzyme-linked immunoassay method adopts a double-antibody sandwich method, and utilizes the existing mouse monoclonal antibody aiming at TK1 in human serum as a capture antibody; the rabbit monoclonal antibody A or B provided by the invention is used as a binding antibody.
The enzyme-linked immunoassay method comprises the following specific steps:
(1) coating the capture antibody with carbonate buffer (pH 9.6, concentration 0.05M), incubating at 4 deg.C overnight, and washing the plate with the washing buffer;
(2) blocking with 1 (w/v)% BSA-0.05M;
(3) washing and diluting a standard sample and a sample to be detected by using a sample buffer solution, adding the standard sample and the sample to be detected into an enzyme label plate, incubating for 30-60min under a normal temperature condition, and washing the plate by using a washing buffer solution;
(4) adding rabbit monoclonal antibody A or B diluted by phosphate buffer solution containing 1% bovine serum albumin and 0.05% Tween-20, incubating at normal temperature for 30-60min, and washing the plate with washing solution;
(5) adding goat anti-rabbit IgG labeled with HRP, incubating for 30-60min at normal temperature, and washing the plate with washing liquid;
(6) adding color developing agent, developing at room temperature for 15min, adding stop solution to stop developing, and measuring absorbance, OD at 450nm and 630nm respectively450Subtract OD630Is the corrected absorbance value.
Example 4TK1 in vitro test kit
In this example, the kit for in vitro detection of TK1 comprises an elisa plate coated with a murine monoclonal antibody, a rabbit monoclonal antibody prepared according to example 1, an enzyme label, a chromogenic agent, a stop solution, a TK1 standard, a wash buffer, and a dilution buffer;
the preparation and operation of TK1 in vitro detection kit are as follows:
1. preparation of various buffers and reagents:
A. coating buffer solution: carbonate buffer with concentration of 0.050M, pH of 9.4-9.6
Na2CO3：16.0g、NaHCO3: 29.0g of distilled water is dissolved in 1000ml of distilled water;
B. sample/wash buffer: 10 XPBS-Tween 20 at pH 7.2
Na2HPO4·12H2O：58g、KH2PO4: 4g, NaCl: 100g, KCl: 4g, dissolving in distilled water to 1000ml, adding Tween 20: 20ml of the solution;
C. enzyme marker diluent
10 XPBS-Tween 20: 10ml, FCS (calf serum): 20ml, distilled water to 1000ml, enzyme stabilizer: 1g, biological preservative: 1 ml;
D. color-developing agent A
Citric acid: 35.5g, carbamide peroxide: 10g, distilled water to 1000ml, Tween 20: 10 ml;
E. color-developing agent B
Citric acid: 120g, EDTA-2 Na: 1g, TMB.2HCl: 2g, dissolving in distilled water to 1000 ml;
F. stopping liquid: h at a concentration of 2M2SO4
Concentrated sulfuric acid (95-98%): 22.2ml, distilled water: 177.3ml, slowly dripping concentrated sulfuric acid into distilled water during timing, and shaking up;
2. preparation of a coated plate:
dissolving a mouse monoclonal antibody aiming at TK1 in human serum in 0.05M carbonate buffer solution with pH of 9.6 to prepare pre-coating solution, adding 100 mu L of the pre-coating solution into each hole of an enzyme label plate according to 0.1 mu g/hole, standing at 4 ℃ for 18-24h, taking out, throwing off the coating solution, washing with sample/washing buffer solution, sealing by 1 (w/v)% BSA-0.05M ethanolamine for 16h, drying overnight, filling into an aluminum platinum bag, vacuumizing, sealing, and storing at 4 ℃.
3. The dilution ratio of the binding antibody and the enzyme conjugate (horseradish peroxidase-labeled goat anti-rabbit IgG antibody) is determined by a matrix titration experiment, and the horseradish peroxidase-labeled goat anti-rabbit IgG antibody is diluted by using an enzyme-labeled diluent.
4. The kit comprises the following components:
coating a plate: 48/96 hole
TK1 calibrator: 6, the number of the cells is as follows: 6X 1.0ml (concentration of 20pM, 10pM, 5pM, 2pM, 1pM, 0pM, respectively)
Binding of antibody: 1X 10ml (diluted 1: 5000)
Enzyme conjugate: 1X 10ml (diluted 1: 5000)
Concentrated wash (25 × PBS-Tween 20): 1X 20ml
Color-developing agent A: 1X 6.0ml
And a color developing agent B: 1X 6.0ml
Stopping liquid: 1X 6.0ml
5. The kit comprises the following steps:
adding 100 mu L/hole of the serum to be detected and the calibrator into each hole of the coated plate, wherein the serum to be detected and the calibrator are both double-hole, incubating for 60 minutes at 37 ℃, washing for 5 times by using 1 multiplied by washing buffer solution, and patting dry; adding 100 mu L/well of TK1 binding antibody into each well, incubating for 30 minutes at 37 ℃, washing for 5 times by using 1 × washing buffer solution, and patting dry; adding 100 mu L of enzyme conjugate into each hole, incubating for 30min at 37 ℃, washing for 5 times by using 1 Xwashing buffer solution, and patting to dry; adding 50 μ L of color development agent A, B solution into each well, mixing, and incubating at 37 deg.C for 15 min; the reaction was stopped by adding 50. mu.L of stop solution to each well, and absorbance was measured by using a dual wavelength (450nm, 620nm) using an enzyme-linked detector (model RT-6000, available from Redu Co.).
6. And (4) judging a result:
table 1: calibrator concentration and corresponding average absorbance (OD) value
| Concentration pM | 0 | 1 | 2 | 5 | 10 | 20 |
| Average OD value | 0.038 | 0.162 | 0.268 | 0.499 | 0.871 | 1.559 |
Drawing a standard curve by using the concentration of the calibrator and the logarithmic value of the corresponding absorbance, and obtaining the R of the standard curve20.9982; the TK1 concentration results in the samples tested were calculated from the standard curve.
Example 5 in vitro assay kit Performance test
The kit of example 3 was used to perform a minimum detection limit and specificity comparison study with a commercially available TK1 detection kit.
1. Minimum detection Limit comparison study
Taking internal control reference substance with concentration of 0pM, respectively detecting with two kits for 20 times, and averaging 20 times of detection results (OD. values)
And standard deviation SD, average plus two standard deviations
The corresponding concentration values are the lowest detection limits as shown in table 2:
TABLE 2 calculation of minimum detection Limit results
The detection sensitivity of the TK1 in-vitro detection kit prepared by the embodiment of the invention is obviously superior to that of the kit products sold in the market.
2. Comparative study of kit specificity
Taking possible cross-reaction products, using two kits to detect, calculating the average value of the two parallel measured values (OD. values) of each cross-reaction product sample, calculating the concentration value of each sample from the corresponding kit calibration curve, comparing with the self concentration of the cross-reaction product, and calculating the cross-reaction rate as shown in the following table 3:
TABLE 3 calculation of Cross-reaction results
The result shows that the product detection specificity of the embodiment of the invention is obviously superior to the commercial kit product.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Sequence listing
<110> Fujian Yitong Biotech Ltd
<120> rabbit monoclonal antibody aiming at TK1 in human serum and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Tyr Ala Lys Asp Thr Arg Tyr Ser Ser Ser Phe Cys Thr His Asp Arg
1 5 10 15
Asn Thr Met Glu Ala
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<210> 2
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Ala Ala Leu Asp Gly Thr Phe Gln Arg Lys Pro Phe Gly Ala
1 5 10
Claims (5)
1. A rabbit monoclonal antibody to TK1 in human serum, characterized by: immunizing a rabbit source with an antigen to produce a rabbit monoclonal antibody; the antigen is formed by coupling peptide with an amino acid sequence of SEQ ID NO. 1 or peptide with an amino acid sequence of SEQ ID NO. 2 with a first carrier protein or a second carrier protein respectively; the rabbit monoclonal antibodies include rabbit monoclonal antibody a and rabbit monoclonal antibody B.
2. Use of a rabbit monoclonal antibody to TK1 in human serum according to claim 1 in the establishment of an enzyme linked immunoassay for TK1 in human serum.
3. Use according to claim 2, characterized in that: the enzyme-linked immunoassay adopts a double-antibody sandwich method for detection; the rabbit monoclonal antibody A is a capture antibody; the rabbit monoclonal antibody B is a binding antibody.
4. Use of a rabbit monoclonal antibody against TK1 in human serum according to claim 1 in the preparation of a kit for the detection of TK1 in human serum.
5. An in vitro detection kit for TK1 in human serum, which is characterized in that: the kit comprises an ELISA plate coated with the rabbit monoclonal antibody of claim 1, a binding antibody, an enzyme marker, a color developing agent, a stop solution, a TK1 standard, a washing buffer and a dilution buffer.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102432683A (en) * | 2011-10-28 | 2012-05-02 | 周际 | Preparation of multi-epitope TK1 antibody and application of multi-epitope TK1 antibody to evaluation on recurrence risk and prognosis of tumor patient at early stage |
| WO2015094106A1 (en) * | 2013-12-19 | 2015-06-25 | Arocell Ab | Monoclonal anti-tk1 antibodies |
| CN106554950A (en) * | 2015-09-24 | 2017-04-05 | 深圳市安群生物工程有限公司 | People's TK1 epitope peptides, antigen, antibody, application and test kit |
| CN109416358A (en) * | 2016-08-10 | 2019-03-01 | 阿尔贝蒂兽医诊断公司 | The determination of non-human mammal TK1 protein level |
-
2021
- 2021-02-04 CN CN202110156602.6A patent/CN112940131A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102432683A (en) * | 2011-10-28 | 2012-05-02 | 周际 | Preparation of multi-epitope TK1 antibody and application of multi-epitope TK1 antibody to evaluation on recurrence risk and prognosis of tumor patient at early stage |
| WO2015094106A1 (en) * | 2013-12-19 | 2015-06-25 | Arocell Ab | Monoclonal anti-tk1 antibodies |
| CN105980407A (en) * | 2013-12-19 | 2016-09-28 | 阿洛赛尔公司 | Monoclonal anti-TK1 antibodies |
| CN106554950A (en) * | 2015-09-24 | 2017-04-05 | 深圳市安群生物工程有限公司 | People's TK1 epitope peptides, antigen, antibody, application and test kit |
| CN109416358A (en) * | 2016-08-10 | 2019-03-01 | 阿尔贝蒂兽医诊断公司 | The determination of non-human mammal TK1 protein level |
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