CN104862283A - Pair of high-specificity high-affinity monoclonal antibodies capable of binding to human myoglobin and application thereof - Google Patents
Pair of high-specificity high-affinity monoclonal antibodies capable of binding to human myoglobin and application thereof Download PDFInfo
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Abstract
The invention discloses a pair of hybridoma cell strains 3M4 and 5M7 (both preserved in China Center for Type Culture Collection on March 25, 2015, with accession numbers of CCTCC C201529 and CCTCC C201530, respectively) secreting high-specificity high-affinity monoclonal antibodies capable of binding to human myoglobin, and the secreted monoclonal antibodies 3M4 and 5M7. The pair of monoclonal antibodies have high specificity and high affinity, can specifically bind to different epitopes of human myoglobin to form a double-antibody sandwich mode so as to realize quantitative determination of myoglobin in human serum, and show good detection specificity and sensitivity. The detection sensitivity of the monoclonal antibodies is close to detection sensitivity of an imported kit; and a double-antibody sandwich Elisa method established in the invention has a greater linear scope, can be clinically used for detection of myoglobin in human serum, is of great guiding significance to early diagnosis of myocardial infarction and has a commercial application value.
Description
Technical field
The invention belongs to biological technical field.More specifically, a pair high specific high-affinity is related in conjunction with the monoclonal antibody of human muscle hemoglobin and application thereof.
Background technology
All the time, cardiovascular disorder, particularly Acute Myocardial Infarction (AMI) are the arch-criminals having a strong impact on human health, once myocardial infarction outbreak, if can not get clear and definite diagnosis and effective treatment at short notice, Autopsy Cases will cause irreversible necrosis because of hypoxic-ischemic.Therefore, the diagnosis that Acute Myocardial Infarction is correct in time in early days seems particularly important concerning reduction mortality ratio.The diagnosis being found to be Acute Myocardial Infarction of serum cardiac damage markers provides important foundation.
The diagnosis utilizing myocardial injury markers to carry out Acute Myocardial Infarction has had the history of more than 50 year, roughly experienced by three important developmental stage.In 50 to the sixties of eighties of last century, the amino saccharase (AST) of Aspartic Acid, serum lactic dehydrogenase (LDH) and creatine kinase (CK) are the leading indicators that Acute Myocardial Infarction detects, the isoenzymes of creatine kinase (CK-MB) that heartspecific is higher has been found subsequently in 70 to the eighties, to the initial stage nineties, the discovery and application of myohaemoglobin, Troponin I (TnI), TnT (TnT) then becomes more special, the more responsive Testing index of Diagnosis of AMI.Following advantages should be possessed: (1) high density in myocardial cell, and the material not existing in other histocytes or exist on a small quantity as a kind of desirable myocardial injury markers; (2) just can be released into blood at once when myocardial cell has slight damage, and can detect by current detection means; (3) early stage myocardial damage can be detected, and window phase is longer; (4) infarction size size judging prognosis can be estimated, and can thrombolytic effect etc. be assessed.Myohaemoglobin is owing to being present in myocardial cell's tenuigenin, and molecular weight is little, is the myocardial injury markers be discharged into the earliest after Acute Myocardial Infarction occurs in blood, makes the timely diagnosis of disease become possibility.In addition due to its in blood the transformation period very short, so be infer myocardial infarct size and judge the important indicator of efficiency of thrombolysis.If 8h after pectoralgia, myohaemoglobin level is still in normal value, so can get rid of the possibility of AMI, and negative elimination factor is 100%.Although because it also exists in a large number in skeletal muscle, the parafunctional disease of renal excretion also may cause the rising of myoglobin content, its specificity is not high especially, but then can significantly improve its diagnostic value in conjunction with the index that other specificitys such as troponin, creatine kinase are higher.Therefore, consider, myohaemoglobin is the diagnosis marker of an extraordinary Acute Myocardial Infarction.
At present, mainly adopt the immunological detection method based on monoclonal antibody to detect myohaemoglobin clinically, this wherein double-antibody sandwich Elisa because it is highly sensitive, high specificity, be applicable to detect multiple sample simultaneously, and simple to operate, do not need the advantages such as special plant and instrument and be used widely.Successfully have developed the double-antibody sandwich Elisa test kit that can be used for detecting human muscle hemoglobin abroad at present, but develop the domestic reagent box that can match in excellence or beauty with import reagent box, the monopolization of breaking external product, remain the striving direction of domestic reagent box research and development.For the antibody of double-antibody sandwich Elisa except possessing high specific, high-affinity, also want the different epi-positions that can identify same antigen.Other two strain antibodies, except responding with immunogen, are also wanted to react with the natural antigen in sample.Therefore, the quality of the monoclonal antibody of preparation successfully researches and develops the key of double-antibody sandwich Elisa test kit.
Summary of the invention
The technical problem to be solved in the present invention overcomes the defect and deficiency that in prior art, myohaemoglobin detection technique exists, the mouse monoclonal antibody of a pair different epi-position of targeted human myohaemoglobin (Human Myoglobin) is provided, the different epi-positions in conjunction with human muscle hemoglobin of this antagonism physical efficiency high-affinity, high specific, and form double-antibody sandwich pattern, thus detection by quantitative can be carried out to myohaemoglobin.
The object of this invention is to provide a pair high specific high-affinity monoclonal antibody in conjunction with human muscle hemoglobin.
Another object of the present invention is the application of described high specific high-affinity in conjunction with the monoclonal antibody of human muscle hemoglobin.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
Secrete the hybridoma cell strain 3M4 of high specific high-affinity in conjunction with the monoclonal antibody of human muscle hemoglobin, be preserved in China on March 25th, 2015. Wuhan. the China typical culture collection center of Wuhan University, deposit number is CCTCC C201529.
Secrete the hybridoma cell strain 5M7 of high specific high-affinity in conjunction with the monoclonal antibody of human muscle hemoglobin, be preserved in China on March 25th, 2015. Wuhan. the China typical culture collection center of Wuhan University, deposit number is CCTCC C201530.
A pair high specific high-affinity, in conjunction with the monoclonal antibody 3M4 of human muscle hemoglobin and 5M7, is secreted by above-mentioned hybridoma cell strain 3M4 and hybridoma cell strain 5M7 respectively and is obtained.
Wherein, the variable region gene of described monoclonal antibody 3M4 and 5M7 includes heavy chain variable region gene and chain variable region gene; The gene order of the variable region of heavy chain of monoclonal antibody 3M4 is as shown in SEQ ID NO.1, and the gene order of variable region of light chain is as shown in SEQ ID NO.2; The gene order of the variable region of heavy chain of monoclonal antibody 5M7 is as shown in SEQ ID NO.3, and the gene order of variable region of light chain is as shown in SEQ ID NO.4.
The aminoacid sequence of the variable region of heavy chain of described monoclonal antibody 3M4 is as shown in SEQ ID NO.5, and the aminoacid sequence of variable region of light chain is as shown in SEQ ID NO.6; The aminoacid sequence of the variable region of heavy chain of described monoclonal antibody 5M7 is as shown in SEQ ID NO.7, and the aminoacid sequence of variable region of light chain is as shown in SEQ ID NO.8.
Said monoclonal antibody 3M4 and 5M7 is detecting the application in human serum in myohaemoglobin, and preparing the application in the reagent or test kit detecting myohaemoglobin in human serum, all within protection scope of the present invention.
Detect a double-antibodies sandwich ELISA for myohaemoglobin in human serum, be using said monoclonal antibody 3M4 as detection antibody, set up using monoclonal antibody 5M7 as capture antibody.
Preferably, the step of described double-antibodies sandwich ELISA is as follows:
S1. extremely wrapping by concentration with the carbonate buffer solution dilution capture antibody 5M7 of pH9.6 is 2.5 μ g/ml, and 4 DEG C of bags are spent the night;
S2., after washing 3 times with PBST, adopt 5% skim-milk or 2% BSA to close, sealing condition is 37 DEG C, 1h;
S3. the serum sample to be detected of 80 μ l Sample dilution twice dilutions is added, add the low cross reaction diluted of detection antibody 3M4 of the detection antibody 3M4(HRP mark that 120 μ l HRP mark subsequently to working concentration 0.5 μ g/ml), 37 DEG C of reaction 1h;
S4. wash five times with PBST, each 3min, adds nitrite ion after patting dry, and room temperature lucifuge stops after hatching 10min, and microplate reader reads OD450 value.
Wherein, Sample dilution used in the embodiment of the present invention and low cross reaction diluent are purchased from German Candor Bioscience company.
Meanwhile, in the detection human serum set up based on above-mentioned double-antibodies sandwich ELISA, the test kit of myohaemoglobin also should within protection scope of the present invention.
In addition, to the sequence of gained antibody and structural analysis known, the variable region of heavy chain of described monoclonal antibody 3M4 is by 348 based compositions, and 116 amino acid of encoding, 3 CDR(complementary determining region are contained in variable region) district.CDR1 encodes 6 amino acid, and CDR2 encodes 19 amino acid, and CDR3 encodes 8 amino acid (as shown in Figure 1).The homology of the framework region of variable region and other murine antibody is up to 91.8%, and 3 CDR districts are then specific sequences, variant with other CDR districts, murine antibody variable region of heavy chain.
The variable region of light chain of described monoclonal antibody 3M4 is by 309 based compositions, and 103 amino acid of encoding, 3 CDR(complementary determining region are contained in variable region) district.CDR1 encodes 12 amino acid, and CDR2 encodes 7 amino acid, and CDR3 encodes 7 amino acid (as shown in Figure 2).The homology of the framework region of variable region and other murine antibody is 95.7%, 3 CDR districts is then specific sequence, variant with other CDR districts, murine antibody variable region of light chain.
The variable region of heavy chain of described monoclonal antibody 5M7 is by 357 based compositions, and 119 amino acid of encoding, 3 CDR(complementary determining region are contained in variable region) district.CDR1 encodes 5 amino acid, and CDR2 encodes 17 amino acid, and CDR3 encodes 16 amino acid (as shown in Figure 3).The homology of the framework region of variable region and other murine antibody is up to 83.3%, and 3 CDR districts are then specific sequences, variant with other CDR districts, murine antibody variable region of heavy chain.
The variable region of light chain of described monoclonal antibody 5M7 is by 324 based compositions, and 108 amino acid of encoding, 3 CDR(complementary determining region are contained in variable region) district.CDR1 encodes 15 amino acid, and CDR2 encodes 7 amino acid, and CDR3 encodes 6 amino acid (as shown in Figure 4).The homology of the framework region of variable region and other murine antibody is 92.9%, 3 CDR districts is then specific sequence, variant with other CDR districts, murine antibody variable region of light chain.
The function of above-mentioned two strain anti-human myohaemoglobin monoclonal antibody protein is light by antibody, variable region of heavy chain antigen is complementary determines that race (complementarity determing regions CDRs) CDR1, CDR2, CDR3(are functionally active districts of the present invention) middle specific nucleotide sequences decision, its corresponding aminoacid sequence constitutes antibodies specific in conjunction with the different epi-positions on human muscle hemoglobin.
This research is by large quantifier elimination and exploration, obtain the cell strain of 13 strain energy stably excreting monoclonal antibodies, wherein the antibody capable of this two plant heights specificity of 3M4 and 5M7, high-affinity forms pairing, and this combinations of pairs of 5M7/HRP-3M4 has higher sensitivity and larger linearity range; Best pairing is utilized to combine 5M7/HRP-3M4 Criterion curve, using 3M4 as detection antibody, 5M7 establishes double-antibody sandwich Elisa method as capture antibody, and its linearity range is 25ng/ml-1000ng/ml, is better than the linearity range 25ng/ml-500ng/ml of import reagent box.
Pattern detection result shows, double-antibody sandwich Elisa method of the present invention is 95%(19 example/20 examples to the positive rate of human muscle hemoglobin), negative recall rate is 100%(40 example/40 examples), therefore can be clear and definite, the present invention obtains 2 plant height specificitys, the anti-human myohaemoglobin monoclonal antibody of high-affinity, can be used for the development of sandwich Elisa test kit.
In addition, the present invention's application mainly detects index, i.e. a myohaemoglobin in serum, and the direct result of detection is that the presence or absence of myohaemoglobin in serum or content are how many, is not directly involved in the diagnosis of disease.
The present invention has following beneficial effect:
The invention discloses mouse monoclonal antibody 3M4 and 5M7 of a pair energy specific binding human muscle hemoglobin, the different epi-positions of this antagonism physical efficiency specific binding human muscle hemoglobin, form double-antibody sandwich pattern, realize the detection by quantitative of the myohaemoglobin in human serum, clinically for the detection of myohaemoglobin in human serum, important directive significance can be had to the early diagnosis of myocardial infarction.
Using 3M4 as detection antibody, the double-antibody sandwich Elisa method that 5M7 sets up as capture antibody has good detection specificity and sensitivity.Detection sensitivity is more close with import reagent box, and the double-antibody sandwich Elisa method that the present invention sets up has larger linearity range, has the commercial value applied.
Accompanying drawing explanation
Fig. 1 is 3,3M4 antibody heavy chain variable region CDR(complementary determining region) district
Fig. 2 is 3,3M4 antibody chain variable region CDR(complementary determining region) district.
Fig. 3 is 3,5M7 antibody heavy chain variable region CDR(complementary determining region) district.
Fig. 4 is 3,5M7 antibody chain variable region CDR(complementary determining region) district.
Fig. 5 is that the SDS-PAGE of the anti-human myohaemoglobin antibody purification of 9 strain analyzes, and M is albumen Marker, and swimming lane 1 is 2M1, and 2 is 3M4, and 3 is 5M7, and 4 is 10D4, and 5 is 2M5, and 6 is 5M11, and 7 is 2M11, and 8 is 10M1, and 9 is 3M7.
Fig. 6 is the SDS-PAGE detected result figure of the anti-human myohaemoglobin monoclonal antibody 3M4 in mouse source and 5M7 purifying; No. 1 swimming lane is Marker, and No. 2 swimming lanes are 3M4 monoclonal antibody after purifying under Denaturing, and No. 3 swimming lanes are 5M7 monoclonal antibody after purifying under Denaturing.
Fig. 7 is the bioactivity ELISA result figure after the anti-human myohaemoglobin monoclonal antibody 3M4 in mouse source and 5M7 affinity purification.
Fig. 8 is the avidity curve of anti-human myohaemoglobin monoclonal antibody 3M4 and 5M7.
Fig. 9 is the specificity identification Western Blot result figure after the two anti-human myohaemoglobin monoclonal antibody 3M4 in strain mouse source and 5M7 affinity purification.
Figure 10 be the double-antibody sandwich elisa test kit set up based on anti-human myohaemoglobin monoclonal antibody 3M4 and 5M7 of the present invention and import reagent box typical curve comparing result figure.
Figure 11 is the ELISA detected result figure that the anti-human myohaemoglobin monoclonal antibody 3M4 in mouse source tires after HRP is labeled.
Embodiment
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but embodiment does not limit in any form to the present invention.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.
Unless stated otherwise, agents useful for same of the present invention and material are commercial.
the preparation of embodiment 1 antigen
Human muscle hemoglobin (Human Myoglobin), buy from ABCam company, article No. ab77876, its concentration is 2.4mg/mL, and be natural human muscle hemoglobin molecule, molecular weight is 16.7kDa, is made up of 154 amino-acid residues.The major function of human muscle hemoglobin is transhipment and store oxygen in myocyte, when myocardial infarction occurs, is occur one of mark in blood the earliest.
the preparation of embodiment 2 monoclonal antibody
1, the present embodiment material therefor
(1) reagent and animal: human muscle hemoglobin standard antigen, anti-human myohaemoglobin standard antibody, human muscle hemoglobin Elisa detection kit are all purchased from ABCam company; Freund's complete adjuvant, Freund's incomplete adjuvant purchased from American Sigma company, PRMI1640 substratum, foetal calf serum, HAT, HT, polyoxyethylene glycol (PEG, Mw4000) are U.S. Gibco Products; Low cross reaction diluent and enzyme labelled antibody protection liquid are all purchased from German Candor Bioscience company; Murine myeloma cell (SP2/0) is preserved for this laboratory passage; SPF level BALB/c is sheerly female mice, 6 ~ 8 weeks ages of mouse, purchased from Nanfang Medical Univ's animal center; Horseradish peroxidase-labeled test kit (A type, Type B) is purchased from safe sky and Bioisystech Co., Ltd; Normal human serum and patients serum take from Guangzhou overseas Chinese's hospital laboratory.
(2) instrument Multiskan MK3 microplate reader, BB15 type CO2 incubator are U.S. Thermo Fisher Products; Milli-Q AdvantageA10 ultrapure water Yi Shi U.S. Millipore Products; High speed freezing centrifuge is eppendorf is Products; Protein G affinity column is GE Products.
2, animal immune
(1) initial immunity: 30 μ g antigens add the fully emulsified rear subcutaneous multi-point injection of isopyknic Freund's complete adjuvant;
(2) second immunisation: after 2 weeks, the antigen amount same with initial immunity adds the fully emulsified rear subcutaneous multi-point injection of equivalent Freund's incomplete adjuvant;
(3) three immunity: after 2 weeks, the antigen amount same with initial immunity adds the fully emulsified rear subcutaneous multi-point injection of equivalent Freund's incomplete adjuvant (after 10 days tail vein blood survey it tire);
(4) booster immunization: after three times immunizing potency reaches cytogamy requirement, do not add adjuvant by the antigen amount that initial immunity is same and carry out abdominal injection;
(5) get spleen after 72h to merge.
3, cytogamy
(1) preparation of feeder cell: get a healthy Balb/c mouse, plucks eyeball blood sampling, treat blood put clean after, neck dislocation is put to death, body surface sterilization and fixing after, cut off skin from thigh, expose peritonaeum, cotton ball soaked in alcohol sterilization peritonaeum.With 5mL injector to inject 10 ml RPMI1640 basic medium (RPMI Medium 1640 basic, gibco buys, article No. 8114056, penicillin and Streptomycin sulphate is added before using, add-on be every 100ml substratum add 1ml containing the penicillin of 100U and Streptomycin sulphate dual anti-) to abdominal cavity, the right hand fixes syringe, left hand holds cotton ball soaked in alcohol abdomen massage gently, draw back intraperitoneal liquid, inject off-the-shelf aseptic 10ml centrifuge tube, centrifugal 7 minutes of 1000rmp, with 10% foetal calf serum RPMI 1640(containing HAT) perfect medium is resuspended, dilution is about 2 × 105/ml, join in 96 orifice plates subsequently, every hole 100 μ l, be placed in cell culture incubator (37 DEG C, for subsequent use 5%CO2).
(2) take the logarithm the murine myeloma cell SP2/0 grown, and with the washing of RPMI1640 basic medium, counts after blowing outstanding dilution;
(3) mouse spleen is got, the washing of RPMI1640 basic medium, the single splenocyte suspension of preparation of milling, counting;
(4) myeloma cell and splenocyte are mixed in the ratio of 1:10, the centrifugal 7min of 1000rpm;
(5) abandon supernatant, exhaust residual liquid with dropper, under 37 DEG C of water bath condition, in 1min, dropwise add the polyoxyethylene glycol (PEG) of 1mL, leave standstill 90 seconds, in 2 ~ 4min, dropwise add 15mL RPMI 1640 basic medium termination reaction;
(6) the centrifugal 7min of 1000rpm, abandons supernatant, with 100mL 10% foetal calf serum RPMI 1640(containing HAT) suspendible gently; Drip in 96 orifice plates being covered with feeder cell in advance, 100 μ l/ holes; 37 DEG C, cultivate in 5%CO2 incubator.
4, the screening of fused cell and cloning
(1) within about the 7th day after cytogamy, get cells and supernatant, carry out indirect ELISA detection with the elisa plate being coated with 30ng/ hole human muscle hemoglobin, screen positive hole; With the serum got before immune mouse fusion as positive control, with SP2/0 supernatant as negative control.Finishing screen selects 13 strain positive cell strains, respectively called after 2M1,3M4,5M7,10M4,2M5,5M11,2M11,10M1,3M7,10M6,4M8,10M8,1M3.
(2) the positive hole hybridoma screened is carried out limiting dilution assay first time cloning, after indirect ELISA is detected, the picking positive is worth high mono-clonal hole again through repeatedly subclone screening, until all mono-clonal holes are the positive, namely obtain 13 strain of hybridoma strains of the anti-human myohaemoglobin monoclonal antibody of stably excreting, it is frozen obtained cell strain to be built strain.
Choose supernatant in 13 strain positive cell strains to tire 9 higher strain cells, adopt in Mice Body and induce the preparation that method carries out ascitic type antibody, adopt saturated ammonium sulphate method antagonist carry out slightly pure after be further purified with Protein G affinity column again, concrete grammar is as follows.
5, the preparation of ascitic type monoclonal antibody
(1) female Balb/c mouse peritoneal injection in 10 week age 0.5mL Freund's incomplete adjuvant is got;
Inoculate an about 5*105 hybridoma in mouse peritoneal after (2) 1 weeks, cell inoculation can induce ascites after 7 ~ 12 days;
(3) when ascites is many as far as possible, ascites is extracted;
(4) 1 ~ 2 day, interval, after ascites regeneration is gathered, take out with method, the centrifugal 10min of ascites 3000rpm after extracting, gets supernatant and is placed in-20 DEG C of preservations again.
6, the purifying of monoclonal antibody
(1) get ascites 4 DEG C of centrifugal 30min of 12000rpm, get supernatant;
(2) equal-volume saturated ammonium sulphate solution is dropwise added under Keep agitation, 4 DEG C of hold over night;
(3) liquid after spending the night is in 7500rpm, and 4 DEG C of centrifugal 30min, abandon supernatant, and precipitation 0.1M PBS redissolves;
(4) redissolution liquid desalting column is carried out desalting treatment, concrete operation step is as follows:
1. pillar is balanced: with 0.1M PBS(5 ~ 10 times column volume), balance pillar, goes out 20% ethanol in post, is returned to zero by nucleic acid-protein instrument;
2. loading: before loading, first closes constant flow pump, slower loading, continues to pass into 0.1M PBS after loading, when A value starts to rise, collects liquid (target protein), stops collecting when A value drops to below 10.The sample of collection proceeds next step purifying.
3. pillar is balanced: when A value is for " 0 ", cross post with 0.1M PBS (more than 5 times column volumes);
4. preserve: cross pillar with 20% ethanol (5 times of column volumes), preserve pillar.
(5) albumen after the desalination of Protein G affinity chromatography column purification, purification step is as follows:
1. column equilibration pillar is filled: with 0.1M PBS(5 ~ 10 times column volume), balance pillar, goes out 20% ethanol in post, is returned to zero by nucleic acid-protein instrument;
2. loading: before loading, first closes constant flow pump, slower loading, when A value starts to rise, collects liquid (foreign protein) and prevents protein in conjunction with upper pillar, add 0.1M PBS dilute when sample is complete by loading, makes protein almost complete upper prop;
3. wash-out: when A value is down to " 0 ", use elution target protein, collects liquid (due to protein belt negative electricity, in weakly alkaline, add a certain amount of neutralizer in advance in collection tube, make collection liquid pH remain on more than 7.0) when A value starts to rise;
4. pillar is balanced: when A value is for " 0 ", cross post with 0.1M PBS (more than 5 times column volumes);
5. preserve: cross pillar with 20% ethanol (5 times of column volumes), preserve pillar.
6. take a morsel after protein ultrafiltration and concentration and carry out SDS-PAGE gel electrophoresis qualification purity.
As shown in Figure 5, visible heavy chain and light chain bands, respectively at about 50KD and 25KD, and have no significantly other band, illustrate that purification effect is better, may be used for follow-up experiment result.
Wherein, carried out purifying (subsequent experimental finds, it is best that this antagonist carries out pairing) to monoclonal antibody 3M4 and 5M7 purifying in addition, after purifying, SDS-PAGE detected result as shown in Figure 6, and other is sub-packed in-20 DEG C of preservations.
7, to the sequence of gained antibody and structural analysis known, the variable region gene of preparation-obtained monoclonal antibody 3M4 and 5M7 includes heavy chain variable region gene and chain variable region gene; The gene order of the variable region of heavy chain of monoclonal antibody 3M4 is as shown in SEQ ID NO.1, and the gene order of variable region of light chain is as shown in SEQ ID NO.2; The gene order of the variable region of heavy chain of monoclonal antibody 5M7 is as shown in SEQ ID NO.3, and the gene order of variable region of light chain is as shown in SEQ ID NO.4.
The aminoacid sequence of the variable region of heavy chain of described monoclonal antibody 3M4 is as shown in SEQ ID NO.5, and the aminoacid sequence of variable region of light chain is as shown in SEQ ID NO.6; The aminoacid sequence of the variable region of heavy chain of described monoclonal antibody 5M7 is as shown in SEQ ID NO.7, and the aminoacid sequence of variable region of light chain is as shown in SEQ ID NO.8.
The variable region of heavy chain of described monoclonal antibody 3M4 is by 348 based compositions, and 116 amino acid of encoding, 3 CDR(complementary determining region are contained in variable region) district.CDR1 encodes 6 amino acid, and CDR2 encodes 19 amino acid, and CDR3 encodes 8 amino acid (as shown in Figure 1).The homology of the framework region of variable region and other murine antibody is up to 91.8%, and 3 CDR districts are then specific sequences, variant with other CDR districts, murine antibody variable region of heavy chain.
The variable region of light chain of described monoclonal antibody 3M4 is by 309 based compositions, and 103 amino acid of encoding, 3 CDR(complementary determining region are contained in variable region) district.CDR1 encodes 12 amino acid, and CDR2 encodes 7 amino acid, and CDR3 encodes 7 amino acid (as shown in Figure 2).The homology of the framework region of variable region and other murine antibody is 95.7%, 3 CDR districts is then specific sequence, variant with other CDR districts, murine antibody variable region of light chain.
The variable region of heavy chain of described monoclonal antibody 5M7 is by 357 based compositions, and 119 amino acid of encoding, 3 CDR(complementary determining region are contained in variable region) district.CDR1 encodes 5 amino acid, and CDR2 encodes 17 amino acid, and CDR3 encodes 16 amino acid (as shown in Figure 3).The homology of the framework region of variable region and other murine antibody is up to 83.3%, and 3 CDR districts are then specific sequences, variant with other CDR districts, murine antibody variable region of heavy chain.
The variable region of light chain of described monoclonal antibody 5M7 is by 324 based compositions, and 108 amino acid of encoding, 3 CDR(complementary determining region are contained in variable region) district.CDR1 encodes 15 amino acid, and CDR2 encodes 7 amino acid, and CDR3 encodes 6 amino acid (as shown in Figure 4).The homology of the framework region of variable region and other murine antibody is 92.9%, 3 CDR districts is then specific sequence, variant with other CDR districts, murine antibody variable region of light chain.
the CHARACTERISTICS IDENTIFICATION of embodiment 3 monoclonal antibody
1, Ig class and subclass measure
Adopt commercial mouse mAb parting kit to carry out Ig class and subgroup identification to the anti-human myohaemoglobin monoclonal antibody of 10 strain through affinity purification, all operations all strictly operates according to test kit specification sheets.
Result is as shown in table 1, and wherein, 2M1,5M7,10M4,2M5 and 5M11 are IgG1,3M4 and 2M11 is IgG2b, 10M1 and 3M7 is IgG3.
2, monoclonal antibody titration
Titration is carried out with 9 strain antibodies of indirect Elisa to affinity purification.
Being diluted by human muscle hemoglobin standard antigen coating buffer is 300ng/ml, and every hole adds 100 μ l, 4 DEG C of overnight incubation.Be 0.05% with PBST(tween content) washing three times, each 3min, then use skim-milk 37 DEG C of closed 1h, PBST of 5% to wash three times, each 3min, pat dry rear placement-20 DEG C for subsequent use.During titration, antibody is carried out two doubling dilutions from 1:10 000.After dilution, every hole adds 100 μ l, and with package amount be the human hemoglobin of 1 μ g/ml in contrast, hatch 1h for 37 DEG C.PBST washs 3 times, each 3min, then the sheep anti mouse two of the HRP mark adding 8 000 times of dilutions resists 100 μ l, hatches 40 min for 37 DEG C.PBST washs 5 times, adds AB nitrite ion, and room temperature lucifuge stops after reacting 10 min, and microplate reader reads OD450 value, get OD450 value about 1.0 time antibody dilution multiple as antibody titer.
Result is as shown in table 1, and tiring of 2M1,3M4,5M7 and 10M4 has has all met or exceeded 106, wherein 2M1 tire the highest, reach 2.6 × 10
6, and all antibody and mechanism and intimate human hemoglobin there is no obvious cross reaction, and display specificity is good.
Table 1 antibody characteristic is identified
Wherein, the bioactivity ELISA result figure after monoclonal antibody 3M4 and 5M7 affinity purification as shown in Figure 7.
the double-antibody sandwich pairing experiment of embodiment 4 monoclonal antibody
1, double-antibody sandwich tentatively matches experiment
(1) 6 higher strain antibodies of tiring after choosing affinity purification carry out HRP mark and bag quilt respectively, respectively as detection antibody and capture antibody, adopt chessboard method double-antibody sandwich pairing experiment to carry out the preliminary screening of pairing antibody.The bag of capture antibody is 10 μ g/ml by concentration, bag quilt is carried out with carbonate buffer solution (pH9.6), 0.05% tween is contained with PBST(after 4 DEG C of overnight incubation) wash three times, 3min/ time, three times are washed with PBST after adding skim-milk 37 DEG C of closed 1h of 5%, 3min/ time, the detection antibody of human muscle hemoglobin standard antigen (300ng/ml) and HRP mark is added successively after patting dry, 37 DEG C hatch 1h after, PBST washs five times, 3min/ time, adds nitrite ion after patting dry, room temperature lucifuge stops after hatching 10min, and microplate reader reads OD
450value.
(2) respectively bag quilt is carried out for 6 strain antibodies (10M4,2M11,2M5,5M7,3M4,2M1) that titration result is higher and HRP marks, test for antibody conjugates.In double-antibody sandwich pairing process, antibody cannot form sandwich pairing for identical or close epi-position, only for two epi-positions different and relatively far apart, just likely form pairing.When adding antigen amount and being identical, OD
450be worth higher, illustrate that pairing effect is better.In this experiment, as long as OD
450namely value is thought can carry out sandwich pairing higher than 0.3.
Pairing result is as shown in table 2, in 36(6 × 6) plant in antibody conjugates combination, have 10 kinds of combinations can form double-antibody sandwich pairing, wherein, what have better pairing effect has 3 kinds of combinations: 2M1/HRP-3M4,5M7/HRP-3M4,10M4/HRP-5M7.
Table 2 anti-human myohaemoglobin monoclonal antibody pairing antibody screening
Note: "+" represents can carry out matching (OD
450>=0.3), "-" represents and can not carry out matching (OD
450< 0.3).
No matter in addition, find in follow-up experiment, be that in linearity range or sensitivity, this combinations of pairs of 5M7/HRP-3M4 is all better than other combinations of pairs, so follow-up experiment mainly adopts, this matched group is incompatible to carry out.
2, antibody affinity costant of matching measures
(1) select best two strain antibodies (5M7/HRP-3M4) of pairing effect according to preliminary pairing the selection result and follow-up experiment, adopt non-competing enzyme immunoassay to measure the affinity costant Ka value of 3M4 and 5M7.With reference to the method [5] of G.P.S.Raghava, according to formula Ka=(n-1)/2, (n ［ Ab' ］ t-［ Ab ］ t) calculates affinity constant Ka value.The antigen coated gradient of 5M7 is 300,75,18.75ng/ml, the antigen coated gradient of 3M4 is 300,150,75ng/ml, with human muscle hemoglobin standard antigen bag by Elisa plate, add the 5M7/3M4 of two doubling dilutions again, 37 DEG C hatch 1h after, PBST washs 3 times, 3min/ time, the sheep anti mouse two adding the HRP mark of 1: 8 000 dilution after patting dry resists, and hatch 40min for 37 DEG C, PBST washs 5 times, 3min/ time, add nitrite ion after patting dry, room temperature lucifuge stops after hatching 10min, and microplate reader reads OD450 value.Using antibody concentration as X-coordinate, OD450 value is ordinate zou mapping, and the maximum OD value of OD450 value of curve upper planar section, calculates the antibody concentration corresponding to maximum OD value half by matching, then go out affinity costant Ka value according to above-mentioned formulae discovery.
(2) test the curve that obtains as shown in Figure 8, according to formula Ka=(n-1)/2, (n ［ Ab' ］ t-［ Ab ］ t) calculates affinity constant Ka value.
3, antibodies specific of matching is identified
(1) immunoblotting (Western blot) is adopted to carry out the qualification of pairing antibodies specific.To identify the specificity of this 2 strain of 3M4 and 5M7 pairing antibody, namely identify that whether this 2 strain pairing antibody have with certain composition in serum and intersect, whether can identify in serum the antigen with native conformation; First carry out SDS-PAGE electrophoresis to human serum, applied sample amount is 20 μ l, and loading order is followed successively by human muscle hemoglobin positive serum (Cmyo > 500ng/ml), normal human serum.Stop electrophoresis after 100V electrophoresis 80min, transferring film adopts wet turning, by protein delivery on the 0.2 μm of PVDF film activated through methyl alcohol.Transfer printing condition is 120V, 50min.After transferring film, with PBST(containing 0.05% tween) wash 3 times, 5min/ time, the skim-milk 4 DEG C adding 5% subsequently closedly spends the night, then 3M4,5M7 and anti-human myohaemoglobin standard antibody to be diluted to after 1ug/mL as primary antibodie respectively at incubated at room 2h.After hatching, wash 3 times with PBST, 5min/ time; The sheep anti mouse two adding HRP mark again resists, and adopts 1:10 000 to dilute, in incubated at room 1h.Wash 3 times with PSBT, 5min/ time, add enhanced chemical luminescence (ECL) substrate after draining, develop.
(2) result as shown in Figure 9, as can be seen from the results, standard antibody, 3M4 and 5M7 all only have single band, and only have band at positive serum swimming lane, in negative serum swimming lane, do not have band to occur, this two strain antibodies high specificity of 3M4 and 5M7 is described, there is no cross reaction with other complicated ingredient in serum, and can react with natural myohaemoglobin in human serum.
embodiment 5 monoclonal antibody is to the detection of 3M4 and 5M7 to myohaemoglobin in human serum
1, typical curve is set up
(1) by a series of condition optimizing, using 5M7 as capture antibody, using 3M4 as detection antibody, the double-antibody sandwich Elisa detection method detecting human muscle hemoglobin is established.
Operate as follows:
S1. capture antibody 5M7 adopts carbonate buffer solution (pH9.6) 4 DEG C of bags to be spent the night, and wrapping by concentration is 2.5 μ g/ml;
S2. adopt 2% BSA to close after washing 3 times with PBST, sealing condition is 37 DEG C, 1h;
S3. the human muscle hemoglobin standard antigen with the dilution of Sample dilution twice of different concns and serum sample to be detected is added, add-on 80 μ l, add the detection antibody 3M4 of HRP mark subsequently, adopt HRP labelling kit (safe sky and Glue Type B activation horseradish peroxidase HRP labelling kit) mark, by low cross reaction diluted to working concentration 0.5 μ g/ml, add-on is 120 μ l, 37 DEG C of reaction 1h;
S4. wash five times, 3min/ time with PBST, add nitrite ion after patting dry, room temperature lucifuge stops after hatching 10min, and microplate reader reads OD450 value.
Wherein, Sample dilution used and low cross reaction diluent are purchased from German Candor Bioscience company.
(2) typical curve of import reagent box (purchased from ABCam company, article No. 3108652) then operates in strict accordance with test kit specification sheets.
(3) obtain the contrast of typical curve as shown in Figure 10.The linear detection range of the typical curve of self-control test kit is at 25 ~ 1000ng/ml, and the sensing range of import Elisa test kit is not desirable especially in 25 ~ 1000ng/ml scope internal linear, linearity range is about 25 ~ 500ng/ml, therefore relatively can find, the homemade test kit of the present invention has better linear dependence and linearity range.
2, simultaneously, detect monoclonal antibody 3M4 tiring after HRP is labeled, detected result as shown in Figure 11.
3, the detection of serum sample
Carry out the detection of serum sample with the typical curve set up, and contrast with import reagent box, the accuracy of checking pattern detection.
Have collected 20 parts of positive serums and 40 parts of negative serums altogether from hospital, respectively with method and the test kit (double-antibodies sandwich ELISA set up based on anti-human myohaemoglobin monoclonal antibody 3M4 and 5M7) of the present invention's foundation, and commercial import reagent box is (purchased from ABCam company, article No. 3108652) measure, statistics positive rate and negative rate, the accuracy of comparative result.
Result is as shown in table 3,20 positive sample, and commercial kit can all detect, and the method oneself set up can detect 19, and positive rate is 95%, there will be false negative situation for indivedual weak positive sample.For 40 negative sample, all do not have the appearance of false positive situation, negative rate is 100%.
The detected result of table 3 serum sample
In sum, compared with import reagent box, remolding sensitivity is more close.In addition, the double-antibody sandwich Elisa method that the present invention sets up has larger linearity range, has the commercial value applied.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
SEQUENCE LISTING
<110> Ji'nan University
<120> a pair high specific high-affinity is in conjunction with the monoclonal antibody of human muscle hemoglobin and application thereof
<130>
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 348
<212> DNA
The gene order of the variable region of heavy chain of <213> monoclonal antibody 3M4
<400> 1
gtcaaactgc aggagtctgg aggaggcttg gtgcaacctg gaggatccat gaaactctcc 60
tgtgtagcct ctggatttaa tttcagtagt tactggatgt cttgggtccg ccagtctcca 120
gagaaggggc ttgagtgggt cgctgaaatt agattgaaat ctgataatta cgcaacacat 180
tacgcggagt ctgtgaaagg gaagttcacc atctcaaggg atgattccaa aagtagtctc 240
tacctgcaaa tgaacaactt aagaactgag gacactggaa tttatttttg tgcagatcac 300
ggacttacgg ggcactgggg ccaagggacc acggtcaccg tctcctca 348
<210> 2
<211> 309
<212> DNA
The gene order of the variable region of light chain of <213> monoclonal antibody 3M4
<400> 2
ctgacccagt ctccagcaat catgtctgca tctcctgggg agaaggtcac cttgacctgc 60
agtgccagct cgagtgtaag ttccagctac ttgtactggt accagcagaa gccaggatcc 120
tcccccaaaa tctggattta tagcacatcc aacctggctt ttggagtccc tgatcgcttc 180
agtggcagtg ggtctgggac ctcttactct ctcacaatca gcaccatgga ggctgaagat 240
gctgcctctt atttctgcca tcagtggagt agttacccgt tcacgttcgg tgctgggacc 300
aagctggag 309
<210> 3
<211> 360
<212> DNA
The gene order of the variable region of heavy chain of <213> monoclonal antibody 5M7
<400> 3
aggtcaaact gcagcagtct ggggctgagc tggcaagacc tggggcttca gtgaagttgt 60
cctgcaaggc ttctggctac acctttacta tctactggat gcagtgggta aaacagaggc 120
ctggacaggg tctggaatgg attgggtcta tttatcctgg agatggtgat actaggtata 180
atcagaagtt caagggcaag gccacattca ctgcagataa atcctccagc acagcctaca 240
tgcaactcat cagtttggca tctgaggact ctgcggtcta ttactgtgca accggtctct 300
actatggtta cgacgagggc cactggtact tcgatgtctg gggccaaggg accacggtca 360
<210> 4
<211> 329
<212> DNA
The gene order of the variable region of light chain of <213> monoclonal antibody 5M7
<400> 4
gacattgtga tgtcacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60
atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120
caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180
ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240
cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300
tcggaggggg gaccaagctg gaaataaaa 329
<210> 5
<211> 116
<212> PRT
The aminoacid sequence of the variable region of heavy chain of <213> monoclonal antibody 3M4
<400> 5
Val Lys Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
1 5 10 15
Met Lys Leu Ser Cys Val Ala Ser Gly Phe Asn Phe Ser Ser Tyr Trp
20 25 30
Met Ser Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val Ala
35 40 45
Glu Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His Tyr Ala Glu Ser
50 55 60
Val Lys Gly Lys Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser Leu
65 70 75 80
Tyr Leu Gln Met Asn Asn Leu Arg Thr Glu Asp Thr Gly Ile Tyr Phe
85 90 95
Cys Ala Asp His Gly Leu Thr Gly His Trp Gly Gln Gly Thr Thr Val
100 105 110
Thr Val Ser Ser
115
<210> 6
<211> 103
<212> PRT
The aminoacid sequence of the variable region of light chain of <213> monoclonal antibody 3M4
<400> 6
Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val
1 5 10 15
Thr Leu Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu Tyr
20 25 30
Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ile Trp Ile Tyr Ser
35 40 45
Thr Ser Asn Leu Ala Phe Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
50 55 60
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Thr Met Glu Ala Glu Asp
65 70 75 80
Ala Ala Ser Tyr Phe Cys His Gln Trp Ser Ser Tyr Pro Phe Thr Phe
85 90 95
Gly Ala Gly Thr Lys Leu Glu
100
<210> 7
<211> 119
<212> PRT
The aminoacid sequence of the variable region of heavy chain of <213> monoclonal antibody 5M7
<400> 7
Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser
1 5 10 15
Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ile Tyr Trp
20 25 30
Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly
35 40 45
Ser Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Asn Gln Lys Phe Lys
50 55 60
Gly Lys Ala Thr Phe Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met
65 70 75 80
Gln Leu Ile Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
85 90 95
Thr Gly Leu Tyr Tyr Gly Tyr Asp Glu Gly His Trp Tyr Phe Asp Val
100 105 110
Trp Gly Gln Gly Thr Thr Val
115
<210> 8
<211> 108
<212> PRT
The aminoacid sequence of the variable region of light chain of <213> monoclonal antibody 5M7
<400> 8
Asp Ile Val Met Ser Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg
85 90 95
Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys
100 105
Claims (10)
1. secrete the hybridoma cell strain 3M4 of high specific high-affinity in conjunction with the monoclonal antibody of human muscle hemoglobin for one kind, it is characterized in that, this hybridoma cell strain 3M4 is preserved in China typical culture collection center on March 25th, 2015, and deposit number is CCTCC C201529.
2. secrete the hybridoma cell strain 5M7 of high specific high-affinity in conjunction with the monoclonal antibody of human muscle hemoglobin for one kind, it is characterized in that, this hybridoma cell strain 5M7 is preserved in China typical culture collection center on March 25th, 2015, and deposit number is CCTCC C201530.
3. a pair high specific high-affinity is in conjunction with the monoclonal antibody 3M4 of human muscle hemoglobin and 5M7, it is characterized in that, is secreted respectively obtain by hybridoma cell strain 5M7 described in hybridoma cell strain 3M4 described in claim 1 and claim 2.
4. monoclonal antibody 3M4 and 5M7 according to claim 3, it is characterized in that, the variable region gene of described monoclonal antibody 3M4 and 5M7 includes heavy chain variable region gene and chain variable region gene; The gene order of the variable region of heavy chain of monoclonal antibody 3M4 is as shown in SEQ ID NO.1, and the gene order of variable region of light chain is as shown in SEQ ID NO.2; The gene order of the variable region of heavy chain of monoclonal antibody 5M7 is as shown in SEQ ID NO.3, and the gene order of variable region of light chain is as shown in SEQ ID NO.4.
5. monoclonal antibody 3M4 and 5M7 according to claim 4, it is characterized in that, the aminoacid sequence of the variable region of heavy chain of described monoclonal antibody 3M4 is as shown in SEQ ID NO.5, and the aminoacid sequence of variable region of light chain is as shown in SEQ ID NO.6; The aminoacid sequence of the variable region of heavy chain of described monoclonal antibody 5M7 is as shown in SEQ ID NO.7, and the aminoacid sequence of variable region of light chain is as shown in SEQ ID NO.8.
6. the arbitrary described monoclonal antibody 3M4 and 5M7 of claim 3 ~ 5 is detecting the application in human serum in myohaemoglobin.
7. the arbitrary described monoclonal antibody 3M4 and 5M7 of claim 3 ~ 5 is preparing the application in the reagent or test kit detecting myohaemoglobin in human serum.
8. detect a double-antibodies sandwich ELISA for myohaemoglobin in human serum, it is characterized in that, the method using monoclonal antibody 3M4 described in claim 3 as detection antibody, using monoclonal antibody 5M7 as capture antibody.
9. double-antibodies sandwich ELISA according to claim 8, it is characterized in that, step is as follows:
S1. extremely wrapping by concentration with the carbonate buffer solution dilution capture antibody 5M7 of pH9.6 is 2.5 μ g/ml, and 4 DEG C of bags are spent the night;
S2., after washing 3 times with PBST, adopt 5% skim-milk or 2% BSA to close, sealing condition is 37 DEG C, 1h;
S3. add serum sample to be detected, add the detection antibody 3M4 of HRP mark subsequently, 37 DEG C of reaction 1h;
S4. with PBST washing 3 ~ 5 times, each 3min, adds nitrite ion after patting dry, and room temperature lucifuge stops after hatching 10min, and microplate reader reads OD
450value.
10. detect a double-antibody sandwich elisa test kit for myohaemoglobin in human serum, it is characterized in that, set up based on double-antibodies sandwich ELISA described in claim 8.
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| CN114957457A (en) * | 2022-05-27 | 2022-08-30 | 中国科学院武汉病毒研究所 | anti-EV 71 virus neutralizing antibody and preparation method and application thereof |
| WO2023238821A1 (en) * | 2022-06-09 | 2023-12-14 | デンカ株式会社 | Antimyoglobin monoclonal antibody |
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| CN109725151A (en) * | 2018-12-28 | 2019-05-07 | 江苏众红生物工程创药研究院有限公司 | Human muscle hemoglobin Test paper card and its clinical application |
| CN109721655A (en) * | 2018-12-28 | 2019-05-07 | 江苏众红生物工程创药研究院有限公司 | Anti-human myoglobins antibody and its application in detection kit |
| CN109721655B (en) * | 2018-12-28 | 2020-09-22 | 江苏众红生物工程创药研究院有限公司 | Anti-human myoglobin antibody and application thereof in detection kit |
| CN109725151B (en) * | 2018-12-28 | 2021-08-27 | 江苏众红生物工程创药研究院有限公司 | Human myoglobin detection test paper card and clinical application thereof |
| CN112521497A (en) * | 2020-12-17 | 2021-03-19 | 杭州贤至生物科技有限公司 | Preparation and application of myoglobin monoclonal antibody |
| CN112521497B (en) * | 2020-12-17 | 2022-05-20 | 杭州贤至生物科技有限公司 | Preparation and application of myoglobin monoclonal antibody |
| CN112778419A (en) * | 2021-02-01 | 2021-05-11 | 重庆中元汇吉生物技术有限公司 | anti-CK-MB antibodies or antigen-binding portions thereof and uses thereof |
| CN113980125A (en) * | 2021-10-15 | 2022-01-28 | 中国科学院武汉病毒研究所 | Neutralizing monoclonal antibody for resisting SFTSV and application thereof |
| CN113980125B (en) * | 2021-10-15 | 2024-03-26 | 中国科学院武汉病毒研究所 | Neutralizing monoclonal antibody for resisting SFTSV and application thereof |
| CN114957457A (en) * | 2022-05-27 | 2022-08-30 | 中国科学院武汉病毒研究所 | anti-EV 71 virus neutralizing antibody and preparation method and application thereof |
| WO2023238821A1 (en) * | 2022-06-09 | 2023-12-14 | デンカ株式会社 | Antimyoglobin monoclonal antibody |
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