CN104862283B

CN104862283B - The monoclonal antibody of a pair of of high specific high-affinity combination human muscle hemoglobin and its application

Info

Publication number: CN104862283B
Application number: CN201510157324.0A
Authority: CN
Inventors: 向军俭; 杨全利; 黄建芳
Original assignee: Jinan University
Current assignee: Guangzhou Purui Biotechnology Co.,Ltd.
Priority date: 2015-04-03
Filing date: 2015-04-03
Publication date: 2018-05-01
Anticipated expiration: 2035-04-03
Also published as: CN104862283A

Abstract

The invention discloses the hybridoma cell strain 3M4 and 5M7 of the monoclonal antibody of a pair of of secretion high specific high-affinity combination human muscle hemoglobin（China typical culture collection center is preserved on March 25th, 2015, deposit number is respectively CCTCC C201529 and CCTCC C201530）, and the monoclonal antibody 3M4 and 5M7 of its secretion.This has high specific and high-affinity to antibody, can specifically bind the different epitopes of human muscle hemoglobin, forms double-antibody sandwich pattern, realizes the quantitative detection of myoglobins in human serum, has detection specificity and sensitivity well.Detection sensitivity is close with import reagent box, and the double-antibody sandwich Elisa methods that the present invention establishes have the range of linearity of bigger, the detection of myoglobins in human serum can be used clinically for, the early diagnosis to miocardial infarction has important directive significance, has commercial application value.

Description

The monoclonal antibody of a pair of of high specific high-affinity combination human muscle hemoglobin and its Using

Technical field

The invention belongs to biological technical field.More particularly, to a pair of of high specific high-affinity combination people's flesh red eggs White monoclonal antibody and its application.

Background technology

All the time, angiocardiopathy, particularly acute myocardial infarction（AMI）It is the chief-criminal's misfortune for seriously affecting human health Head, once miocardial infarction is broken out, if cannot clearly diagnose and effectively treat in a short time, Autopsy Cases will be because of Hypoxic-ischemic and cause irreversible necrosis.Therefore, acute myocardial infarction early stage, correctly diagnosis came to reducing the death rate in time Say and be particularly important.The diagnosis for being found to be acute myocardial infarction of serum cardiac damage markers provides important foundation.

There is the history of more than 50 years using the diagnosis of myocardial injury markers progress acute myocardial infarction, substantially experienced Three important developing stage.In 50 to the sixties of eighties of last century, L-aminobutanedioic acid amino invertase（AST), lactic dehydrogenase （LDH）And creatine kinase（CK）It is the leading indicator of acute myocardial infarction detection, is then found that heart spy 70 to the eighties The isoenzymes of creatine kinase of different in nature higher（CK-MB）, to the initial stage nineties, myoglobins, Troponin I (TnI), flesh calcium egg White T（TnT）Discovery with application then become the Testing index that Diagnosis of AMI is more special, more sensitive.As one kind Preferable myocardial injury markers should possess following some advantages：（1）The high concentration in cardiac muscle cell, and it is thin in its hetero-organization It is not present in born of the same parents or a small amount of existing material；（2）Blood can be just released at once when cardiac muscle cell has slight damage, and mesh can be used Preceding detection means detects；（3）Early stage myocardial damage can be detected, and window phase is longer；（4）It can estimate infarction size Size judging prognosis, and thrombolytic effect etc. can be assessed.Myoglobins is due to being present in cardiac muscle cell's cytoplasm, and molecular weight It is small, be to be discharged into myocardial injury markers in blood earliest after acute myocardial infarction occurs so that the timely diagnosis of disease into For possibility.Additionally, due to its in blood half-life period it is very short, so being to speculate myocardial infarct size and judge the important finger of efficiency of thrombolysis Mark.If 8h after pectoralgia, myoglobins level is still within normal value, then can exclude the possibility of AMI, negative elimination factor For 100%.Although since it also largely exists in skeletal muscle, the disease of kidney excretion dysfunction may also cause flesh red eggs The rise of Bai Hanliang, its specificity are not especially high, but other specificity are higher with reference to troponin, creatine kinase etc. Index can then significantly improve its diagnostic value.Therefore, consider, myoglobins is an extraordinary acute myocardial infarction Diagnosis marker.

At present, the immunological detection method based on monoclonal antibody mainly clinically is used to detect flesh red eggs In vain, double-antibody sandwich Elisa due to its high sensitivity, high specificity, suitable for detecting multiple samples at the same time, and is grasped among these Make simple, it is not necessary to be used widely the advantages that special instrument and equipment.Foreign countries successfully have developed and can use at present In the double-antibody sandwich Elisa kits of detection human muscle hemoglobin, but develop the domestic examination that can be matched in excellence or beauty with import reagent box Agent box, the monopolization for breaking external product, are still the striving direction of domestic reagent box research and development.For double-antibody sandwich Elisa's Antibody will can also identify the different epitopes of same antigen in addition to possessing high specific, high-affinity.Other two strain antibody removes With being immunized outside original reaction, can also react with the native antigen in sample.Therefore, the matter of the monoclonal antibody of preparation Amount is successfully to research and develop the key of double-antibody sandwich Elisa kits.

The content of the invention

The technical problem to be solved in the present invention is overcome in the prior art myoglobins detection technique there are the defects of and not Foot, there is provided a pair of targeting human muscle hemoglobin（Human Myoglobin）The mouse monoclonal antibody of different epitopes, this is to antibody Can high-affinity, high specific combination human muscle hemoglobin different epitopes, and double-antibody sandwich pattern is formed, so as to right Myoglobins carries out quantitative detection.

The object of the present invention is to provide the monoclonal antibody of a pair of of high specific high-affinity combination human muscle hemoglobin.

The present invention is another object is that the application of the monoclonal antibody of the high specific high-affinity combination human muscle hemoglobin.

Above-mentioned purpose of the present invention is achieved through the following technical solutions：

A kind of hybridoma cell strain 3M4 for the monoclonal antibody for secreting high specific high-affinity combination human muscle hemoglobin, The China typical culture collection center of Chinese Wuhan Wuhan Universitys is preserved on March 25th, 2015, deposit number is CCTCC C201529。

A kind of hybridoma cell strain 5M7 for the monoclonal antibody for secreting high specific high-affinity combination human muscle hemoglobin, The China typical culture collection center of Chinese Wuhan Wuhan Universitys is preserved on March 25th, 2015, deposit number is CCTCC C201530。

The monoclonal antibody 3M4 and 5M7 of a pair of of high specific high-affinity combination human muscle hemoglobin, respectively by above-mentioned hybridization Tumor cell strain 3M4 and hybridoma cell strain 5M7 secrete to obtain.

Wherein, the variable region gene of the monoclonal antibody 3M4 and 5M7 includes heavy chain variable region gene and light chain variable Area's gene；The gene order of the heavy chain variable region of monoclonal antibody 3M4 is as shown in SEQ ID NO.1, the gene of light chain variable region Sequence is as shown in SEQ ID NO.2；The gene order of the heavy chain variable region of monoclonal antibody 5M7 is as shown in SEQ ID NO.3, gently The gene order of chain variable region is as shown in SEQ ID NO.4.

The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3M4 is as shown in SEQ ID NO.5, light chain variable region Amino acid sequence as shown in SEQ ID NO.6；The amino acid sequence such as SEQ of the heavy chain variable region of the monoclonal antibody 5M7 Shown in ID NO.7, the amino acid sequence of light chain variable region is as shown in SEQ ID NO.8.

Applications of the said monoclonal antibody 3M4 and 5M7 in terms of myoglobins in detecting human serum, and detected preparing Application in human serum in terms of the reagent or kit of myoglobins, within protection scope of the present invention.

The double-antibodies sandwich ELISA of myoglobins in a kind of detection human serum, is made with said monoclonal antibody 3M4 To detect antibody, established using monoclonal antibody 5M7 as capture antibody.

Preferably, the step of double-antibodies sandwich ELISA is as follows：

S1. it is 2.5 μ g/ml to concentration is coated with the carbonate buffer solution dilution capture antibody 5M7 of pH9.6,4 DEG C were coated with Night；

S2. after washing 3 times with PBST, closed using 5% skimmed milk power or 2% BSA, sealing condition is 37 DEG C, 1h；

S3. 80 μ l, twice of diluted serum sample to be detected of Sample dilution is added, then adds 120 μ l HRP marks Detection antibody 3M4（The detection antibody 3M4 low cross reaction diluteds to 0.5 μ g/ml of working concentration of HRP marks）, 37 DEG C of reaction 1h；

S4. to be washed five times, each 3min with PBST, nitrite ion is added after patting dry, room temperature lucifuge terminates after being incubated 10min, Microplate reader reads OD450 values.

Wherein, Sample dilution and low cross reaction dilution used in the embodiment of the present invention are purchased from Germany Candor Bioscience companies.

Meanwhile the reagent for detecting myoglobins in human serum established based on above-mentioned double-antibodies sandwich ELISA Box also should be within protection scope of the present invention.

In addition, sequence and structural analysis to gained antibody are understood, the heavy chain variable region of the monoclonal antibody 3M4 by 348 base compositions, encode 116 amino acid, 3 CDR are contained in variable region（Complementary determining region）Area.CDR1 encodes 6 amino Acid, CDR2 encode 19 amino acid, and CDR3 encodes 8 amino acid（As shown in Figure 1）.The framework region of variable region and other mouse sources The homology of property antibody is up to 91.8%, and 3 CDR regions are then specific sequences, with other murine antibody heavy chain variable regions CDR region is variant.

The light chain variable region of the monoclonal antibody 3M4 encodes 103 amino acid, variable region contains by 309 base compositions There are 3 CDR（Complementary determining region）Area.CDR1 encodes 12 amino acid, and CDR2 encodes 7 amino acid, and CDR3 encodes 7 amino acid （As shown in Figure 2）.The framework region of variable region is 95.7% with the homology of other murine antibodies, and 3 CDR regions are then spy Different in nature sequence, it is variant with other murine antibody light chain variable region CDR regions.

The heavy chain variable region of the monoclonal antibody 5M7 encodes 119 amino acid, variable region contains by 357 base compositions There are 3 CDR（Complementary determining region）Area.CDR1 encodes 5 amino acid, and CDR2 encodes 17 amino acid, and CDR3 encodes 16 amino Acid（As shown in Figure 3）.The framework region of variable region is up to 83.3% with the homology of other murine antibodies, and 3 CDR regions are then It is specific sequence, it is variant with other murine antibody heavy chain variable region CDR regions.

The light chain variable region of the monoclonal antibody 5M7 encodes 108 amino acid, variable region contains by 324 base compositions There are 3 CDR（Complementary determining region）Area.CDR1 encodes 15 amino acid, and CDR2 encodes 7 amino acid, and CDR3 encodes 6 amino acid （As shown in Figure 4）.The framework region of variable region is 92.9% with the homology of other murine antibodies, and 3 CDR regions are then spy Different in nature sequence, it is variant with other murine antibody light chain variable region CDR regions.

The function of above-mentioned two plants of anti-human myoglobins monoclonal antibody proteins is determined by the antigen complementation of antibody light and heavy chain variable region Determine race（complementarity determing regions CDRs）CDR1、CDR2、CDR3（It is the functional activity of the present invention Area）What middle specific nucleotide sequences determined, its corresponding amino acid sequence constitutes antibody specificity combination human muscle hemoglobin On different epitopes.

This research is by largely research and explores, and has obtained the cell line of 13 plants of energy stably excreting monoclonal antibodies, its Middle 3M4 and 5M7 this two plant height specificity, the antibody of high-affinity can form pairing, and 5M7/HRP-3M4 this combinations of pairs has Higher sensitivity and the larger range of linearity；Standard curve is established using best pairing combination 5M7/HRP-3M4, is made with 3M4 To detect antibody, 5M7 establishes double-antibody sandwich Elisa methods as capture antibody, its range of linearity is 25ng/ml- 1000ng/ml, better than the range of linearity 25ng/ml-500ng/ml of import reagent box.

Pattern detection the results show that the double-antibody sandwich Elisa methods of the present invention to the positive rate of human muscle hemoglobin For 95%（19/20）, negative recall rate is 100%（40/40）, therefore can be clear and definite, the present invention has obtained 2 plant heights spy The opposite sex, the anti-human myoglobins monoclonal antibody of high-affinity, the development available for sandwich Elisa kits.

In addition, an index in present invention application mainly detection serum, i.e. myoglobins, the direct result of detection are How much are the presence or absence of myoglobins or content in serum, are not directed to the diagnosis of disease.

The invention has the advantages that：

The invention discloses the mouse monoclonal antibody 3M4 and 5M7 that a pair can specifically bind human muscle hemoglobin, this is right Antibody can specifically bind the different epitopes of human muscle hemoglobin, form double-antibody sandwich pattern, realize the flesh red eggs in human serum White quantitative detection, can be used clinically for the detection of myoglobins in human serum, and the early diagnosis to miocardial infarction has weight The directive significance wanted.

Using 3M4 as detection antibody, 5M7 has good as the double-antibody sandwich Elisa methods that capture antibody is established Detection specificity and sensitivity.Detection sensitivity is more close with import reagent box, and the double-antibody sandwich that the present invention establishes Elisa methods have the range of linearity of bigger, have the commercial value promoted and applied.

Brief description of the drawings

Fig. 1 is the CDR of 3M4 heavy chain of antibody variable region 3（Complementary determining region）Area

Fig. 2 is the CDR of 3M4 antibody light chains variable region 3（Complementary determining region）Area.

Fig. 3 is the CDR of 5M7 heavy chain of antibody variable region 3（Complementary determining region）Area.

Fig. 4 is the CDR of 5M7 antibody light chains variable region 3（Complementary determining region）Area.

Fig. 5 is the SDS-PAGE analyses of 9 plants of anti-human myoglobins antibody purifications, and M is albumen Marker, and swimming lane 1 is 2M1,2 It is 5M7 for 3M4,3,4 be 10D4, and 5 be 2M5, and 6 be 5M11, and 7 be 2M11, and 8 be 10M1, and 9 be 3M7.

Fig. 6 is the SDS-PAGE testing result figures of the anti-human myoglobins monoclonal antibody 3M4 and 5M7 purifying in mouse source；No. 1 swimming Road is Marker, and No. 2 swimming lanes are 3M4 monoclonal antibodies after purification under Denaturing, and No. 3 swimming lanes are 5M7 monoclonal antibodies after purification under Denaturing.

Fig. 7 is the bioactivity ELISA results after anti-human myoglobins monoclonal antibody 3M4 and the 5M7 affinity purification in mouse source Figure.

Fig. 8 is the affinity curve of anti-human myoglobins monoclonal antibody 3M4 and 5M7.

Fig. 9 is the specificity identification after anti-human myoglobins monoclonal antibody 3M4 and the 5M7 affinity purification in two plants of mouse sources Western Blot result figures.

Figure 10 is the double-antibody sandwich that the present invention is established based on anti-human myoglobins monoclonal antibody 3M4 and 5M7 ELISA kit and the standard curve comparing result figure of import reagent box.

Figure 11 is the ELISA testing results of the anti-human myoglobins monoclonal antibody 3M4 in mouse source potency after HRP is labeled Figure.

Embodiment

The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention Limit in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus routinely try for the art Agent, method and apparatus.

Unless stated otherwise, agents useful for same and material of the present invention are purchased in market.

The preparation of 1 antigen of embodiment

Human muscle hemoglobin（Human Myoglobin）, bought from ABCam companies, article No. ab77876, its concentration is 2.4mg/mL, is natural human muscle hemoglobin molecule, molecular weight 16.7kDa, is made of 154 amino acid residues.People's flesh is red The major function of albumen is to be transported in myocyte and store oxygen, is to occur earliest in blood when miocardial infarction occurs One of marker.

The preparation of 2 monoclonal antibody of embodiment

1st, the present embodiment material therefor

（1）Reagent and animal：Human muscle hemoglobin standard antigen, anti-human myoglobins standard antibody, human muscle hemoglobin Elisa Detection kit is purchased from ABCam companies；Freund's complete adjuvant, incomplete Freund's adjuvant are purchased from Sigma Co., USA, PRMI1640 culture mediums, hyclone, HAT, HT, polyethylene glycol (PEG, Mw4000) are U.S.'s Gibco Products；It is low Cross reaction dilution and enzyme labelled antibody protection liquid are purchased from German Candor Bioscience companies；Murine myeloma cell (SP2/0) preserved for this laboratory passage；SPF grades of BALB/c are sheerly female mice, 6~8 weeks mouse ages, purchased from Nanfang Medical Univ Animal center；Horseradish peroxidase-labeled kit（A types, Type B）Purchased from safe day and Bioisystech Co., Ltd；Normal person's blood Cleer and peaceful patients serum is derived from Guangzhou overseas Chinese's hospital laboratory.

（2）Instrument Multiskan MK3 microplate reader, BB15 type CO2 incubators are U.S. Thermo Fisher public affairs Take charge of product；Milli-Q AdvantageA10 ultra-pure water instrument is U.S.'s Millipore Products；High speed freezing centrifuge is Eppendorf is Products；Protein G affinity columns are GE Products.

2nd, animal immune

（1）Initial immunity：Subcutaneous multi-point injection after 30 μ g antigens add isometric Freund's complete adjuvant fully emulsified；

（2）Secondary immunity：After 2 weeks, the amount of antigen same with initial immunity adds equivalent incomplete Freund's adjuvant fully emulsified Subcutaneous multi-point injection afterwards；

（3）It is immunized three times：After 2 weeks, the amount of antigen same with initial immunity adds equivalent incomplete Freund's adjuvant fully emulsified Subcutaneous multi-point injection afterwards（Tail vein blood surveys its potency after 10 days）；

（4）Booster immunization：After immunizing potency reaches cell fusion requirement three times, it is not added with the same amount of antigen of initial immunity Adjuvant is injected intraperitoneally；

（5）Spleen is taken to merge after 72h.

3rd, cell fusion

（1）The preparation of feeder cells：A healthy Balb/c mouse is taken, plucks eyeball blood sampling, after blood is put it clean, neck Dislocation is put to death, and after body surface sterilizes and is fixed, skin, exposure peritonaeum, cotton ball soaked in alcohol disinfection peritonaeum are cut off from thigh.Noted with 5mL Emitter injects 10 ml RPMI1640 basal mediums（RPMI Medium 1640 basic, gibco purchases, article No. 8114056, using preceding addition penicillin and streptomysin, addition be added per 100ml culture mediums penicillin of the 1ml containing 100U with Streptomysin is dual anti-）To abdominal cavity, the right hand fixes syringe, and left hand holds cotton ball soaked in alcohol gently abdomen massage, draws back intraperitoneal liquid, notes Enter in the sterile 10ml centrifuge tubes being ready for, 1000rmp is centrifuged 7 minutes, with 10% hyclone RPMI 1640（Containing HAT）It is complete Full culture medium is resuspended, and dilution is about 2 × 105/ml, is subsequently added in 96 orifice plates, per 100 μ l of hole, is placed in cell incubator （37 DEG C, 5%CO2）In it is spare.

（2）Take the logarithm the murine myeloma cell SP2/0 of growth, is washed with RPMI1640 basal mediums, blows outstanding dilution After count；

（3）Mouse spleen is taken, the washing of RPMI1640 basal mediums, milling prepares single splenocyte suspension, counts；

（4）Myeloma cell and splenocyte are pressed 1：10 ratio mixes, 1000rpm centrifugations 7min；

（5）Supernatant is abandoned, residual liquid is exhausted with dropper, the poly- second of 1mL is added dropwise in 1min under 37 DEG C of water bath conditions Glycol（PEG）, 90 seconds are stood, 1640 basal mediums of 15mL RPMI are added dropwise in 2~4min and terminate reaction；

（6）1000rpm centrifuges 7min, supernatant is abandoned, with 10% hyclone RPMI 1640 of 100mL（Containing HAT）Gently mix It is outstanding；It is added dropwise in being covered with advance in 96 orifice plates of feeder cells, 100 μ l/ holes；37 DEG C, the interior culture of 5%CO2 incubators.

4th, the screening and cloning of fused cell

（1）Cells and supernatant is taken within the 7th day or so after cell fusion, with being coated with 30ng/ holes human muscle hemoglobin Elisa plate carries out indirect ELISA detection, screens positive hole；By the use of the serum taken before immune mouse fusion as positive control, use SP2/0 supernatants are as negative control.Finally filter out 13 plants of positive cell strains, be respectively designated as 2M1,3M4,5M7,10M4, 2M5、5M11、2M11、10M1、3M7、10M6、4M8、10M8、1M3。

（2）The positive hole hybridoma screened is subjected to limiting dilution assay first time cloning, was detected through indirect ELISA Afterwards, picking is positive is worth high monoclonal hole and is screened again by repeatedly subclone, until all monoclonal holes are the positive, to obtain the final product To 13 strain of hybridoma strains of the anti-human myoglobins monoclonal antibody of stably excreting, obtained cell line is built into strain and is frozen.

9 plants of cells that supernatant potency is higher in 13 plants of positive cell strains are chosen, ascites is carried out using method is induced in Mice Body The preparation of type antibody, antibody is carried out using saturated ammonium sulfate method it is thick it is pure after again with Protein G affinity columns into advancing one Step purifying, specific method are as follows.

5th, the preparation of ascitic type monoclonal antibody

（1）10 week old female Balb/c mouse peritoneals are taken to inject 0.5mL incomplete Freund's adjuvants；

（2）About 5*105 hybridoma is inoculated with after mouse peritoneal within 1 week, cell inoculation can induce abdomen after 7~12 days Water；

（3）Ascites is extracted when ascites is as more as possible；

（4）Interval 1~2 day, after ascites regeneration accumulation, takes out again with method, and ascites 3000rpm centrifuges 10min after extraction, Supernatant is taken to be placed in -20 DEG C of preservations.

6th, the purifying of monoclonal antibody

（1）4 DEG C of 12000rpm centrifugation 30min of ascites are taken, take supernatant；

（2）Isometric saturated ammonium sulfate solution is added dropwise under lasting stirring, 4 DEG C stand overnight；

（3）Liquid after overnight abandons supernatant, precipitation 0.1M PBS redissolve in 7500rpm, 4 DEG C of centrifugation 30min；

（4）Liquid will be redissolved and carry out desalting processing with desalting column, concrete operation step is as follows：

1. balance pillar：With 0.1M PBS（5~10 times of column volumes）, pillar is balanced, 20% ethanol in column is gone out, by nucleic acid Albumen instrument returns to zero；

2. loading：Before loading, first constant flow pump is closed, then slow loading, 0.1M PBS are continually fed into after loading, when When A values are begun to ramp up, liquid is collected（Destination protein）, stop collecting when A values drop to below 10.The sample of collection continue into The purifying of row next step.

3. balance pillar：When A values are " 0 ", with 0.1M PBS（More than 5 times column volumes）Cross column；

4. preserve：With 20% ethanol（5 times of column volumes）Pillar is crossed, preserves pillar.

（5）Albumen after Protein G affinity chromatography column purification desalinations, purification step are as follows：

1. fill column equilibration pillar：With 0.1M PBS（5~10 times of column volumes）, pillar is balanced, goes out 20% ethanol in column, will Nucleic acid-protein instrument returns to zero；

2. loading：Before loading, first constant flow pump is closed, then slow loading, when A values are begun to ramp up, collect liquid（Miscellaneous egg In vain）Prevent protein to be not associated with pillar, 0.1M PBS dilutions are added when sample finishes loading, make protein almost complete Full upper prop；

3. elute：When A values are down to " 0 ", with elution destination protein, liquid is collected when A values are begun to ramp up（By In protein belt negative electricity, in alkalescent, a certain amount of neutralizer is previously added in collecting pipe, make collection liquid pH be maintained at 7.0 with On）；

4. balance pillar：When A values are " 0 ", with 0.1M PBS（More than 5 times column volumes）Cross column；

5. preserve：With 20% ethanol（5 times of column volumes）Pillar is crossed, preserves pillar.

6. protein takes a small amount of progress PAGE gel electroresis appraisal purity after being concentrated by ultrafiltration.

As a result as shown in Figure 5, it is seen that heavy chain and light chain bands, respectively in 50KD and 25KD or so, and have no obvious Other bands, illustrate that purification effect is preferable, can be used for follow-up experiment.

Wherein, in addition monoclonal antibody 3M4 and 5M7 are purified（Subsequent experimental finds that this carries out antibody Pairing is optimal）, SDS-PAGE testing results are as shown in Figure 6, other to be sub-packed in -20 DEG C of preservations after purification.

7th, the sequence to gained antibody and structural analysis understand that preparation-obtained monoclonal antibody 3M4's and 5M7 is variable Area's gene includes heavy chain variable region gene and chain variable region gene；The gene sequence of the heavy chain variable region of monoclonal antibody 3M4 Row are as shown in SEQ ID NO.1, and the gene order of light chain variable region is as shown in SEQ ID NO.2；The heavy chain of monoclonal antibody 5M7 The gene order of variable region is as shown in SEQ ID NO.3, and the gene order of light chain variable region is as shown in SEQ ID NO.4.

The heavy chain variable region of the monoclonal antibody 3M4 encodes 116 amino acid, variable region contains by 348 base compositions There are 3 CDR（Complementary determining region）Area.CDR1 encodes 6 amino acid, and CDR2 encodes 19 amino acid, and CDR3 encodes 8 amino acid （As shown in Figure 1）.The framework region of variable region is up to 91.8% with the homology of other murine antibodies, and 3 CDR regions are then Specific sequence, it is variant with other murine antibody heavy chain variable region CDR regions.

The CHARACTERISTICS IDENTIFICATION of 3 monoclonal antibody of embodiment

1st, Ig classes and subclass measure

Using mouse mAb parting kits purchased in market to 10 plants of anti-human myoglobins monoclonal antibodies by affinity purification Ig classes and subgroup identification are carried out, all operations are strictly operated according to kit specification.

The results are shown in Table 1, wherein, 2M1,5M7,10M4,2M5 and 5M11 are that IgG1,3M4 and 2M11 are IgG2b, 10M1 and 3M7 is IgG3.

2nd, monoclonal antibody titration

Titration is carried out to 9 strain antibodies of affinity purification with indirect Elisa.

Human muscle hemoglobin standard antigen is diluted to 300ng/ml with coating buffer, 100 μ l, 4 DEG C of overnight incubations are added per hole. Use PBST（0.05%) tween content is washs three times, each 3min, then closes 1h, PBST washings with 5% 37 DEG C of skimmed milk power Three times, each 3min, -20 DEG C of placement is spare after patting dry.During titration, by antibody from 1：10 000 to proceed by two multiple proportions dilute Release.After dilution, 100 μ l are added per hole, and control is used as by the use of the human hemoglobin that package amount is 1 μ g/ml, 37 DEG C are incubated 1h. PBST is washed 3 times, each 3min, adds the 100 μ l of sheep anti mouse secondary antibody of 8 000 times of diluted HRP marks, 37 DEG C are incubated 40 min.PBST is washed 5 times, adds AB nitrite ions, and room temperature lucifuge terminates after reacting 10 min, and microplate reader reads OD450 values, takes OD450 values at 1.0 or so antibody extension rate as antibody titer.

The results are shown in Table 1, and the potency of 2M1,3M4,5M7 and 10M4 have met or exceeded 106, the wherein potency of 2M1 Highest, has reached 2.6 × 10⁶, and all antibody has no obvious intersect with mechanism and intimate human hemoglobin Reaction, shows that specificity is good.

1 antibody characteristic of table is identified

Wherein, the bioactivity ELISA result figures after monoclonal antibody 3M4 and 5M7 affinity purifications are as shown in Figure 7.

The double-antibody sandwich pairing experiment of 4 monoclonal antibody of embodiment

1st, double-antibody sandwich tentatively matches experiment

（1）6 higher strain antibodies of potency carry out HRP marks and coating respectively after selection affinity purification, respectively as detection Antibody and capture antibody, the preliminary screening of pairing antibody is carried out using the pairing experiment of chessboard method double-antibody sandwich.Capture antibody Coating concentration is 10 μ g/ml, uses carbonate buffer solution（pH9.6）It is coated with, PBST is used after 4 DEG C of overnight incubations（Spat containing 0.05% Temperature）Washing three times, 3min/ times, is washed three times, 3min/ times, after patting dry after adding 5% 37 DEG C of closing 1h of skimmed milk power with PBST Sequentially add human muscle hemoglobin standard antigen（300ng/ml）With the detection antibody of HRP marks, after 37 DEG C are incubated 1h, PBST washings Five times, 3min/ times, nitrite ion is added after patting dry, room temperature lucifuge terminates after being incubated 10min, and microplate reader reads OD₄₅₀Value.

（2）For 6 higher strain antibodies of titration result（10M4、2M11、2M5、5M7、3M4、2M1）Wrapped respectively Quilt and HRP marks, are tested for antibody conjugates.During double-antibody sandwich pairing, antibody is directed to same or similar table Position can not form sandwich pairing, and two be only directed to epitope is different and relatively far apart, is possible to form pairing.Adding In the case that amount of antigen is identical, OD₄₅₀Value is higher, illustrates that pairing effect is better.In this experiment, as long as OD₄₅₀Value is recognized higher than 0.3 For sandwich pairing can be carried out.

The results are shown in Table 2 for pairing, 36（6×6）In kind antibody conjugates combination, there are 10 kinds of combinations to form double antibody folder The heart matches, wherein, have preferable pairing effect shares 3 kinds of combinations：2M1/HRP-3M4、5M7/HRP-3M4、10M4/HRP- 5M7。

The anti-human myoglobins monoclonal antibody pairing antibody screening of table 2

Note："+" represents to be matched（OD₄₅₀≥0.3）, "-" represent cannot be matched（OD₄₅₀＜ 0.3）.

In addition, follow-up it was found that, either in the range of linearity or sensitivity, 5M7/HRP-3M4 this matches somebody with somebody Other combinations of pairs are superior to combination, so follow-up experiment is mainly carried out using this combinations of pairs.

2nd, antibody affinity costant measure is matched

（1）Two best strain antibody (5M7/ of pairing effect are selected according to preliminary pairing the selection result and follow-up experiment HRP-3M4), using the affinity costant Ka values of non-competing enzyme immunoassay measure 3M4 and 5M7.With reference to G.P.S.Raghava's Method【5】, affinity constant Ka values are calculated according to formula Ka=(n-1)/2 (n ［ Ab' ］ t- ［ Ab ］ t).The antigen bag of 5M7 Be 300 by gradient, 75, the antigen coat gradient of 18.75ng/ml, 3M4 be 300,150,75ng/ml, with human muscle hemoglobin standard Antigen coat Elisa plates, add the diluted 5M7/3M4 of two multiple proportions, after 37 DEG C are incubated 1h, PBST washings 3 times, and 3min/ times, The sheep anti mouse secondary antibody of 1: 8 000 diluted HRP marks is added after patting dry, 37 DEG C are incubated 40min, and PBST is washed 5 times, 3min/ It is secondary, nitrite ion is added after patting dry, room temperature lucifuge terminates after being incubated 10min, and microplate reader reads OD450 values.Using antibody concentration as Abscissa, OD450 values are mapped for ordinate, the OD450 values maximum OD values of curve upper planar section, and maximum OD is calculated by fitting It is worth the antibody concentration corresponding to half, then calculates affinity costant Ka values according to above-mentioned formula.

（2）Test obtained curve as shown in Figure 8, counted according to formula Ka=(n-1)/2 (n ［ Ab' ］ t- ［ Ab ］ t) Calculate affinity constant Ka values.

3rd, antibody specificity identification is matched

（1）Using Western blot（Western blot）Carry out pairing antibody specificity identification.To identify 3M4 and 5M7 The specificity of this 2 plants pairing antibody, that is, identify whether this 2 plants pairing antibody have with certain component in serum and intersect, if can know There is the antigen of native conformation in other serum；SDS-PAGE electrophoresis is first carried out to human serum, applied sample amount is 20 μ l, and loading is sequentially It is followed successively by human muscle hemoglobin positive serum（Cmyo ＞ 500ng/ml）, normal human serum.Stop electrophoresis after 100V electrophoresis 80min, Transferring film uses wet turn, by protein delivery to the 0.2 μm of PVDF film activated by methanol.Transfer condition is 120V, 50min. After transferring film, PBST is used（Containing 0.05% tween）Washing 3 times, 5min/ times, 4 DEG C of skimmed milk power for then adding 5% was closed Night, then after 3M4,5M7 and anti-human myoglobins standard antibody are diluted to 1ug/mL as primary antibody respectively in incubation at room temperature 2h. After incubation, washed 3 times, 5min/ times with PBST;The sheep anti mouse secondary antibody of HRP marks is added, using 1:10 000 is dilute Release, in incubation at room temperature 1h.3 times are washed with PSBT, 5min/ times, enhanced chemical are added after draining and is shone (ECL) substrate, into Row development.

（2）As a result as shown in Figure 9, from the results, it was seen that standard antibody, 3M4 and 5M7 only have single band, and Only there is band in positive serum swimming lane, there is no band appearance in negative serum swimming lane, illustrate that this two strain antibody of 3M4 and 5M7 is special It is different in nature strong, there is no cross reaction with other complicated ingredients in serum, and can occur with natural myoglobins in human serum anti- Should.

5 monoclonal antibody of embodiment is to detections of the 3M4 and 5M7 to myoglobins in human serum

1st, standard curve is established

（1）By a series of condition optimizing, using 5M7 as capture antibody, using 3M4 as detection antibody, inspection is established Survey the double-antibody sandwich Elisa detection methods of human muscle hemoglobin.

Operation is as follows：

S1. capture antibody 5M7 uses carbonate buffer solution（pH9.6）Overnight, coating concentration is 2.5 μ g/ml to 4 DEG C of coatings；

S2. closed after washing 3 times with PBST using 2% BSA, sealing condition is 37 DEG C, 1h；

S3. add various concentrations uses twice of diluted human muscle hemoglobin standard antigen of Sample dilution and serum to be detected Sample, 80 μ l of addition, then add the detection antibody 3M4 that HRP is marked, using HRP labelling kits（Safe day and Glue B Type activates horseradish peroxidase HRP labelling kits）Mark, with low cross reaction diluted to 0.5 μ g/ of working concentration Ml, addition are 120 μ l, 37 DEG C of reaction 1h；

S4. washed five times with PBST, 3min/ times, nitrite ion is added after patting dry, room temperature lucifuge terminates after being incubated 10min, enzyme Mark instrument and read OD450 values.

Wherein, Sample dilution used and low cross reaction dilution are purchased from Candor Bioscience companies of Germany.

（2）Import reagent box（Purchased from ABCam companies, article No. 3108652）Standard curve then said in strict accordance with kit Bright book is operated.

（3）The contrast of resulting standard curve is as shown in Figure 10.Make the linearity test model of the standard curve of kit by oneself 25~1000ng/ml is trapped among, and it is not special that the detection range of import Elisa kits is linear in the range of 25~1000ng/ml Not preferable, the range of linearity compares it can be found that the homemade kit of present invention institute has more preferably about in 25~500ng/ml Linear dependence and the range of linearity.

2nd, at the same time, potency of the monoclonal antibody 3M4 after HRP is labeled is detected, testing result such as attached drawing 11 It is shown.

3rd, the detection of serum sample

The detection of serum sample is carried out with the standard curve of foundation, and is compareed with import reagent box, verification sample inspection The accuracy of survey.

20 parts of positive serums and 40 parts of negative serums are have collected from hospital altogether, the method and reagent established respectively with the present invention Box（The double-antibodies sandwich ELISA established based on anti-human myoglobins monoclonal antibody 3M4 and 5M7）, and it is purchased in market Import reagent box（Purchased from ABCam companies, article No. 3108652）It is measured, counts positive rate and negative rate, comparative result Accuracy.

The results are shown in Table 3, and 20 positive samples, commercial kit can be detected all, the method that oneself is established It can detect 19, positive rate 95%, occurs false negative situation for indivedual weakly positive samples.For 40 feminine genders Sample, the appearance without false positive situation, negative rate 100%.

The testing result of 3 serum sample of table

In conclusion compared with import reagent box, remolding sensitivity is more close.In addition, the double-antibody sandwich that the present invention establishes Elisa methods have the range of linearity of bigger, have the commercial value promoted and applied.

Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention and from above-described embodiment Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

SEQUENCE LISTING

<110>Ji'nan University

<120>The monoclonal antibody of a pair of of high specific high-affinity combination human muscle hemoglobin and its application

<130>

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 348

<212> DNA

<213>The gene order of the heavy chain variable region of 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

<213>The gene order of the light chain variable region of 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

<213>The gene order of the heavy chain variable region of 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

<213>The gene order of the light chain variable region of 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

<213>The amino acid sequence of the heavy chain variable region of 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

<213>The amino acid sequence of the light chain variable region of 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

<213>The amino acid sequence of the heavy chain variable region of 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

<213>The amino acid sequence of the light chain variable region of 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

1. a kind of hybridoma cell strain 3M4 for the monoclonal antibody for secreting high specific high-affinity combination human muscle hemoglobin, its It is characterized in that, hybridoma cell strain 3M4 is preserved in China typical culture collection center on March 25th, 2015, and preservation is compiled Number it is CCTCC No. C201529.

2. a kind of hybridoma cell strain 5M7 for the monoclonal antibody for secreting high specific high-affinity combination human muscle hemoglobin, its It is characterized in that, hybridoma cell strain 5M7 is preserved in China typical culture collection center on March 25th, 2015, and preservation is compiled Number it is CCTCC No. C201530.

3. the monoclonal antibody 3M4 and 5M7 of a pair of of high specific high-affinity combination human muscle hemoglobin, it is characterised in that respectively Hybridoma cell strain 5M7 described in hybridoma cell strain 3M4 and claim 2 as described in claim 1 secretes to obtain.

4. monoclonal antibody 3M4 and 5M7 according to claim 3, it is characterised in that the monoclonal antibody 3M4's and 5M7 Variable region gene includes heavy chain variable region gene and chain variable region gene；The base of the heavy chain variable region of monoclonal antibody 3M4 Because sequence is as shown in SEQ ID NO.1, the gene order of light chain variable region is as shown in SEQ ID NO.2；Monoclonal antibody 5M7's The gene order of heavy chain variable region is as shown in SEQ ID NO.3, and the gene order of light chain variable region is as shown in SEQ ID NO.4.

5. monoclonal antibody 3M4 and 5M7 according to claim 4, it is characterised in that the heavy chain of the monoclonal antibody 3M4 The amino acid sequence of variable region is as shown in SEQ ID NO.5, and the amino acid sequence of light chain variable region is as shown in SEQ ID NO.6； The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5M7 is as shown in SEQ ID NO.7, the amino acid of light chain variable region Sequence is as shown in SEQ ID NO.8.

6. any monoclonal antibody 3M4 and 5M7 of claim 3~5 detects the reagent of myoglobins in human serum preparing Or the application in terms of kit.

7. a kind of double-antibody sandwich elisa kit for detecting myoglobins in human serum, it is characterised in that be to detect people's blood Established in clear based on the double-antibodies sandwich ELISA of myoglobins；The method is with monoclonal described in claim 3 Antibody 3M4 is used as capture antibody as detection antibody using monoclonal antibody 5M7.

8. kit according to claim 7, it is characterised in that the method step is as follows：

S1. the carbonate buffer solution dilution capture antibody 5M7 with pH9.6 is to concentration is coated with as 2.5 μ g/ml, and 4 DEG C of coatings are overnight；

S3. serum sample to be detected is added, then adds detection the antibody 3M4,37 DEG C of reaction 1h of HRP marks；

S4. washed 3~5 times, each 3min with PBST, nitrite ion is added after patting dry, room temperature lucifuge terminates after being incubated 10min, enzyme Mark instrument and read OD₄₅₀Value.