CN109320608A - A kind of preparation method of hemoglobin antibodies - Google Patents
A kind of preparation method of hemoglobin antibodies Download PDFInfo
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- CN109320608A CN109320608A CN201811108661.0A CN201811108661A CN109320608A CN 109320608 A CN109320608 A CN 109320608A CN 201811108661 A CN201811108661 A CN 201811108661A CN 109320608 A CN109320608 A CN 109320608A
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- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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Abstract
The present invention relates to a kind of preparation methods of hemoglobin antibodies, comprising the following steps: from separation and purification of hemoglobin in people's whole blood, with liposome hemoglobin, then immune animal prepares hemoglobin antibodies.This method is simple to operation, and the antibody titer being prepared is high.
Description
Technical field
The present invention relates to Antibody preparation fields, and in particular to a kind of preparation method of hemoglobin antibodies.
Background technique
Colorectal cancer (CRC) is a kind of common malignant tumour, and disease incidence occupies the 5th in China's malignant tumour, one
As developed by benign adenoma, early detection to improve colorectal cancer patients cure rate and life cycle have valuable help.Knot
The most commonly used is occult blood tests for the methods for screening of the carcinoma of the rectum, compared with traditional fecal occult blood detection method, are immunized
Chemical method is directly detected (Fecal Immunochemical detection method, FIT) with many advantages to the hemoglobin in excrement.Exempt from
Epidemic disease method occult blood test is the new method of colorectal cancer screening, using the antibody for the globin ingredient in hemoglobin,
To detect the hemoglobin in excrement.FDA is annual to check side FIT it has been proposed that 60 years old or more crowd.FIT is to utilize Dan Ke
Grand antibody or polyclonal antibody directly detect the hemoglobin in human faecal mass, are not influenced by feed food.Qualitative FIT is in excrement
Just middle content of hemoglobin is more than that can generate visual color change after certain threshold value, and quantitative FIT then can measure numerical value, when super
It is defined as the positive after crossing certain normal range (NR), therefore, preparation needs the hemoglobin antibodies of high-titer.
During preparing hemoglobin antibodies, need extraction purification hemoglobin, hemoglobin be a kind of conformation not
Stable protein molecule is a kind of tetramer protein of binding protein ferroheme, in the extraction process of hemoglobin by
Loss in 2,3- diphosphoglyceric acid (2,3-DPG) is detached from ferroheme from globin subunit, while tetrameric hemoglobin
Body is cracked into dimer, this has manufactured very big obstacle to the preparation purifying of hemoglobin and Antibody preparation.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of preparation methods of hemoglobin antibodies being simple and efficient.
The technical scheme to solve the above technical problems is that a kind of preparation method of hemoglobin antibodies, including
Following steps:
S1: from separation and purification of hemoglobin in people's whole blood；
S2: liposome hemoglobin is used, the hemoglobin of liposome is obtained；
S3: the hemoglobin of liposome is used to prepare hemoglobin antibodies as antigen-immunized animal.
Based on the above technical solution, the present invention can also be improved as follows:
Further, the specific steps of the step S1 are as follows: take Whole Blood of Healthy, 4000rpm centrifugation removal supernatant, precipitating is used
Brine 2-4 times, and be with normal saline erythroblast suspension, the physiological saline of addition and the volume ratio of whole blood
1.5~2.5:1, the sodium phosphate buffer that the 10mMPH of addition 10-15 times of volume of red cell suspension is 7.4, hypotonic haemolysis 1h,
15000rpm4 DEG C of 35~45min of centrifugation, takes supernatant, the supernatant is added in the super filter tube of 100kd, and 4000rpm centrifugation 20~
30min collects the liquid in collecting pipe, as containing the solution of hemoglobin.
Further, the specific steps of the step S2 are as follows: chloroform and ether are mixed to get mixing with the volume ratio of 2:3
Soybean lecithin and cholesterol are added in mixed liquor according to mass ratio for 2:1 and obtain lipid solution, in the lipid solution by liquid
The mass fraction of soybean lecithin and cholesterol is 8~10mg/mL, and it is 6~7mg/mL that the hemoglobin, which is configured to concentration,
Solution, and be slowly dropped in lipid solution with syringe, the volume ratio of hemoglobin solutions and the lipid solution is 3:
10, water bath sonicator 5min form w/o type lotion, lotion are placed 30min, prepare again if layering, if not stratified will be newborn
Liquid is placed in 37 DEG C of reduction vaporization in Rotary Evaporators and removes organic solvent, after reaching bonding state, adds water to continue vacuum distillation straight
It falls off to gel from bottle wall, the volume ratio of the water of addition and the lipid solution is 2~5:1, and gel is crossed 0.45 μm of micropore
Filter membrane obtains milky liposome turbid liquor, the as hemoglobin of liposome.
Further, the hemoglobin antibodies be polyclonal antibody, the step S3 the following steps are included:
S31: with the coated hemoglobin immune animal of the liposome, animal antihuman hemoglobin antiserum is obtained；
S32: being coupled to affinity column for hemoglobin, and coupling, which is added, in the animal antihuman hemoglobin antiserum has
The affinity column of hemoglobin, is then eluted with neutralized eluate, the hemoglobin antibodies purified.
Optionally, the hemoglobin antibodies are monoclonal antibody, the specific steps of the step S3 are as follows: with the lipid
Body coated hemoglobin immune mouse takes spleen cell and myeloma cell to merge and obtains multiple hybridomas after culture,
It chooses positive colony and expands culture, and be injected to Balb/c mouse peritoneal, extract ascites after 7~10 days, purify to obtain hemoglobin
Antibody.
10. further, the specific steps of the S32 are as follows: it is 1mg/ that hemoglobin, which is configured to concentration with coupling buffer,
The hemoglobin solutions of mL weigh the agarose gel microsphere of hydrogen bromide activation, fill column, activate in 4 DEG C 25 with the HCl of 1mM~
35min balances chromatographic column with coupling buffer, the hemoglobin solutions, the hemoglobin solutions of addition and institute is added
The volume ratio for stating agarose gel microsphere is 5:2, is incubated at room temperature 3~4h, cleans chromatography with the coupling buffer of 5 times of column volumes
Column, the Tris-Hcl that the 1MpH that 5 times of column volumes are added is 8.0 close chromatographic column, 4 DEG C of 8~16h of placement, then with coupling acid, alkali
Washing lotion alternately cleaning chromatographic column 3 times, finally use equilibration buffer chromatographic column, obtain affinity column, contain blood for described
The animal body fluid of Lactoferrin antibody dilutes 2~3 times with equilibration buffer, is incubated at room temperature 2~3h, slow with the balance of 5 times of column volumes
Fliud flushing washes away the antibody of non-specific binding, and neutral elution buffer is then added and is eluted, collection eluent obtains blood red
Protein antibodies.
Further, the equilibration buffer is the PBS buffer solution for the 150mM that pH is 7.4.
Further, the pH value of the middle elution buffer is 7.2, including 3M MgCl2, 0.075M 4- hydroxyethyl piperazine second
Sulfonic acid (Hepes) and 25vol% ethylene glycol.
Beneficial effect using above-mentioned further scheme is that animal is immunized after being wrapped up hemoglobin with liposome,
Hemoglobin can be prevented to be degraded, the antibody titer being prepared is than directly with the hemoglobin immune without liposome
The antibody titer that animal obtains is high, and during the polyclonal antibody affinity purification being prepared, neutralized eluate can be more
Good protects antigen and antibody, obtains the antibody of more high-affinity.
The present invention also provides the hemoglobin antibodies that above-mentioned preparation method is prepared, antibody titer is high.
Detailed description of the invention
Fig. 1 is the DAB colour developing figure that ELISA identifies antiserum titre in the embodiment of the present invention 3, and wherein lan1 and lan2 is real
Group antiserum prepared with the hemoglobin immune mouse of liposome is tested, lan3 and lan4 are control group without liposome packet
The antiserum for the hemoglobin immune mouse preparation wrapped up in；
Fig. 2 is that ELISA purification Identification obtains the DAB colour developing of hemoglobin polyclonal antibody potency in the embodiment of the present invention 3
Figure, wherein lan1, lan2 and lan3 are respectively that serum, neutral elution flow through liquid and neutral afford experimental group before purification
Antibody, lan4, lan5 and lan6 are respectively that serum, acidic elution flow through liquid to control group before purification and that acidic elution obtains is anti-
Body；
Specific embodiment
Principles and features of the present invention are described below in conjunction with drawings and the specific embodiments, example is served only for solving
The present invention is released, is not intended to limit the scope of the present invention.
The present invention provides a kind of preparation method of hemoglobin antibodies, will be with the hemoglobin immune animal of liposome
It is prepared, specific embodiment is as follows.
1 hemoglobin of embodiment isolates and purifies
Whole Blood of Healthy 20mL is taken, 4000rpm centrifugation removes the blood plasma and leukocytic cream on upper layer, and precipitating uses physiological saline
Cleaning three times, is added physiological saline 40mL and is configured to red cell suspension, and the sodium phosphate buffer that 600mL pH is 7.4 is added, low
Vadose solution blood 1h, with 4 DEG C of supercentrifuge, 15000rpm is centrifuged 40min, takes supernatant, and the ultrafiltration that supernatant is added to 100kd is centrifuged
Guan Zhong, 4000rpm are centrifuged 30min, the liquid in collecting pipe are collected, as containing the solution of hemoglobin.
The hemoglobin of the preparation liposome of embodiment 2
It takes 4mL chloroform and 6mL ether to be mixed to get mixed liquor, weighs 60mg soybean lecithin and 30mg cholesterol obtains class
Hemoglobin is configured to the solution that concentration is 6.67mg/mL with ultrapure water by lipoprotein solution, and the hemoglobin after taking 3mL to dilute is molten
Liquid is simultaneously slowly dropped in lipid solution with syringe, water bath sonicator 5min, forms w/o type lotion, lotion is stood half an hour,
It prepares if layering, if not stratified be placed in 37 DEG C of reduction vaporization removing organic solvents in Rotary Evaporators for lotion, reaches again
To after bonding state, be added 30mL ultrapure water, continue vacuum distillation except until gel fall off from bottle wall, gel is crossed 0.45 μm
Miillpore filter obtains milky liposome turbid liquor, the as hemoglobin of liposome.
The preparation of 3 hemoglobin polyclonal antibody of embodiment
Experimental group takes the test at the 2-3 monthly age month with rabbit, by the hemoglobin of above-mentioned liposome and isometric Freund
After Freund's complete adjuvant is sufficiently mixed, according to every subcutaneous multi-point injection of experimental rabbit 400ug antigen, serum titer is measured after 15 days, is selected
The experimental rabbit being immunoreacted booster immunization again is selected, the hemoglobin and isometric incomplete Freund's adjuvant of liposome are taken
After being sufficiently mixed, according to every 125 subcutaneous multi-point injection of μ g antigen of experimental rabbit, the number of booster immunization is 3 times, every minor tick 15
It, arteria carotis takes blood, obtains rabbit-anti human hemoglobin antiserum.
The hemoglobin that control group is directly used is the hemoglobin without liposome, the same experimental group of other steps.
Indirect ELISA identifies antiserum titre: 96 microwell plates is coated with freshly prepared hemoglobin, according to every hole
4 DEG C of amount of 100ng coatings overnight, are washed version 3 times with PBST (PBS+0.2%Teween 20), and serum is with the degreasing ox of PBST+5%
Milk dilution, thinner ratio is since 1:10000,1/2 doubling dilution, and every hole is loaded 100 μ L, 37 DEG C of incubation 1h and washes version three with PBST
DAB colour developing after secondary, as a result as shown in Figure 1, wherein lan1 and lan2 is that the hemoglobin immune of experimental group liposome is small
The antiserum of mouse preparation, it is blood red egg of the control group without liposome that serum titer, which reaches 1:64 ten thousand, lan3 and lan4,
The antiserum of white immune mouse preparation, serum titer are lower than 1:32 ten thousand, and animal is immunized in hemoglobin after liposome can be with
Improve the sero-fast potency of preparation.
Using the hemoglobin polyclonal antibody of the above-mentioned preparation of affinitive layer purification, the buffer used is respectively as follows:
Coupling buffer: 0.1M NaHCO3,0.5M NaCl,PH8.3；
Equilibration buffer: 150mM NaCl, 10mM Na2HPO4, 1.8mM NaH2PO4, pH7.4；
Neutral elution buffer: 3mM MgCl2, 0.075mM Hepes, 25vol% ethylene glycol, pH7.2；
It is coupled pickling solution: 0.1M HCl, 0.5M NaCl, PH4.0；
It is coupled alkali wash water: 0.1M Tris-HCl, 0.5M NaCl, pH 8.0
It takes 5mg hemoglobin to be added in 5mL coupling buffer and is prepared into hemoglobin solutions, weigh the activation of 2mL hydrogen bromide
Agarose gel microsphere, be packed into chromatographic column balanced with the HCl of 1mM in 4 DEG C of activation 30min with coupling buffer, addition blood
Hemoglobin solution is incubated at room temperature 4h, cleans pillar with the coupling buffer of 5 times of column volumes, and the Tris-HCl envelope of 10mL 1M is added
Pillar is closed, 4 DEG C overnight, then with alternately cleaning pillar 3 times of coupling soda acid washing lotion, finally uses equilibration buffer pillar, obtains
To affinity purification column.Antiserum obtained above equilibration buffer is diluted 3 times, crosses 0.45 μm of filter, and be added to affine
In chromatographic column, 3h is incubated at room temperature with the Equilibration buffer wash pillar of 5 times of column volumes and washes away the antibody of non-specific binding, so
After elution buffer is added dropwise, be in charge of collection eluent.The bag filter pre-processed is taken out, is rinsed well with PBS, one end is used
Dialysis clamp clamps, and the eluent containing hemoglobin card antibody of collection is added, the other end is also clamped with dialysis clamp, is put into 1L
In PBS, 4 DEG C are slowly stirred, and every 3-4 hours is changed a PBS, are concentrated after dialysis 3 times with polyethylene glycol 2000, the blood purified
Lactoferrin polyclonal antibody.
Acidic elution buffer is used when the elution of control group, the formula of acidic elution buffer is 150mM NaCl/
HCl, PH 2.0-2.5, it is 7.4 that the eluent containing hemoglobin antibodies afforded, which is first neutralized to pH with phosphoric acid, other
Step is consistent with above-mentioned steps.
By the concentration of SDS-PAGE electrophoretic determination antibody, use the neutral hemoglobin antibodies afforded for 0.9mg/
ML antiserum, the hemoglobin antibodies for using acidic elution buffer to afford in control group obtain for 1.0mg/mL antiserum
Rate is low compared to the control group；The potency for the hemoglobin polyclonal antibody that indirect ELISA purification Identification obtains, DAB develop the color result such as
Shown in Fig. 2, wherein lan1, lan2 and lan3 are respectively that serum, neutral elution flow through liquid and neutral elution to experimental group before purification
Obtained antibody titer, lan4, lan5 and lan6 are respectively that serum, acidic elution flow through liquid to control group before purification and acidity is washed
De- obtained antibody titer, the antibody titer that neutrality affords can achieve 1:32 ten thousand, and the antibody effect that acidic elution obtains
Valence only has 1:8 ten thousand, it is seen that the vigor of hemoglobin antibodies is protected more preferably in neutrality elution, and what is obtained has more the anti-of affinity
Body has higher sensitivity and broader detection range in clinical detection.
The preparation of 3 hemoglobin monoclonal antibody of embodiment
The Balb/C mouse 3 of 8 week old is taken to be only used as antigen injection, the hemoglobin and Freund of enough liposomes are helped
Agent mixes, and uses Freund's complete adjuvant for the first time, and later booster shots use incomplete Freund's adjuvant, fill with isometric antigen
Divide and mixes back part multi-point injection, main injection 100 μ g antigens/mouse, 50 μ g antigen of booster shots/mouse, after main injection,
Every 15 days booster immunization 1 time, after total booster immunization 3 times, tail vein takes blood, detects antiserum titre, takes potency highest small
Mouse is merged.
It merges the last week, recovery SP2/0 cell, normal culture to logarithmic phase.The selected mouse to be merged, the fusion same day are used
Cervical dislocation is put to death, and spleen is taken, and normal process is collected splenocyte and counted.In 1:3-1:10 ratio mixing myeloma cell and
Splenocyte, normal process carry out cell fusion operation, then use the culture of HATDMEM complete medium, can see within 3 days after fusion
To hybridoma, 1/2HAT complete medium is changed within the 7th day, changes 1/2HT culture medium within the 8th day.Start within 10 days or so after fusion into
Row selective mechanisms.The culture of HAT selective medium, microscopically observation are used after fusion, it is seen that the hybridoma of multiple growths,
Prove mixing operation success.
It draws the hole cell conditioned medium 100ul/ and carries out indirect ELISA detection.According to ELISA as a result, judging positive hole.Use single track
Pipettor chooses the positive hole that inspection whole plate detects, carries out second and rechecks, further confirms that positive hole.
Two-wheeled subclone is done to the positive hole cell of secondary screening.(because being subcloned obtained positive hole cell strain for the first time still
It is unstable, it is possible to it include multiple hybridomas, hybridoma is individual cells strain after generally believing second of subclone,
And it is determined as the positive).Cell in subclone limiting dilution positive hole for the first time, until adding HT DMEM culture medium to train in multiple holes
It supports, observes under the microscope within 7 days or so, indirect ELISA detects the hole for having clonal growth, and the hole for taking OD value high is positive hole；It chooses
It takes second of cell progress of positive hole and is subcloned, detect the hybridoma cell strain for stablizing the positive, prepare monoclonal antibody as final
Cell, and expand culture.
Above-mentioned positive cell is expanded to the abdominal cavity for cultivating and being injected to Balb/C mouse (through incomplete Freund's adjuvant to quick),
General 7-10 days visible mouse web portion protuberances, which represent, ascites generation.Abdomen is extracted in time when mouse has the generation of obvious ascites
Water.It by the ascites of above-mentioned cell, is purified, purified antibodies purity is greater than 90%.Purification process is as follows:
Sad ammonium sulfate+DEAE ion column method purifies (IgG1, IgG2a, IgG2b, IgG3 subclass antibodies):
Ascites centrifugation is sucked out weak yellow liquid and calculates volume, and 60mM acetate buffer solution (pH4.0) 1:3 with 4 times of volumes is dilute
It releases, octanoic acid (final concentration of 25 μ l/ml dilutes ascites) is added dropwise, 30min is stirred at room temperature, then 4 DEG C of standing 2h or more, make it
Sufficiently precipitating.10000r/min 4 DEG C, 20min, collects supernatant, the 10*PBS (0.1M pH7.4) of 1/10 volume is added.According to
Every above-mentioned mixed liquor of ml adds 0.277g solid ammonium sulfate (under the conditions of 0 DEG C, 45% saturated ammonium sulfate is 0.291g/ml), continues quiet
Set at least 60min or more.10000r/min, abandons supernatant, will be precipitated and dissolved in a small amount of PBS by 4 DEG C, 20min.It dialyses to PBS, 4
DEG C dialysed overnight.
After detectable concentration purity, adjustment concentration to 2mg/ml, indirect ELISA verifies antibody titer.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of preparation method of hemoglobin antibodies, which comprises the following steps:
S1: from separation and purification of hemoglobin in people's whole blood；
S2: liposome hemoglobin is used, the hemoglobin of liposome is obtained；
S3: the hemoglobin of liposome is used to prepare hemoglobin antibodies as antigen-immunized animal.
2. a kind of preparation method of hemoglobin antibodies according to claim 1, which is characterized in that the tool of the step S1
Body step are as follows: take Whole Blood of Healthy, 4000rpm centrifugation removal supernatant, precipitating is used brine 2-4 times, and uses physiology salt
Water is configured to red cell suspension, and the physiological saline of addition and the volume ratio of whole blood are 1.5~2.5:1, and red cell suspension 10- is added
The sodium phosphate buffer that the 10mM PH of 15 times of volumes is 7.4,4 DEG C of 35~45min of centrifugation of hypotonic haemolysis 1h, 15000rpm take
Supernatant the supernatant is added in the super filter tube of 100kd, and 4000rpm is centrifuged 20~30min, collects the liquid in collecting pipe, i.e.,
For the solution containing hemoglobin.
3. a kind of preparation method of hemoglobin antibodies according to claim 1, which is characterized in that the tool of the step S2
Body step are as follows: chloroform and ether are mixed to get mixed liquor with the volume ratio of 2:3, by soybean lecithin and cholesterol according to quality
Lipid solution is obtained than being added in mixed liquor for 2:1, the mass fraction of soybean lecithin and cholesterol is 8 in the lipid solution
The hemoglobin is configured to concentration and is the solution of 6~7mg/mL, and is slowly dropped to lipoid with syringe by~10mg/mL
In solution, the volume ratio of hemoglobin solutions and the lipid solution is 3:10, water bath sonicator 5min, forms w/o type lotion, will
Lotion places 30min, prepares again if layering, lotion is placed in 37 DEG C of reduction vaporization in Rotary Evaporators removes if not stratified
Remove organic solvent, after reaching bonding state, add water continue vacuum distillation until gel fall off from bottle wall, the water of addition with it is described
The volume ratio of lipid solution is 2~5:1, and gel is crossed 0.45 μm of miillpore filter, obtains milky liposome turbid liquor, as rouge
The hemoglobin of plastid package.
4. a kind of preparation method of hemoglobin antibodies according to claim 1, which is characterized in that the hemoglobin is anti-
Body is polyclonal antibody, the step S3 the following steps are included:
S31: with the coated hemoglobin immune animal of the liposome, animal antihuman hemoglobin antiserum is obtained；
S32: being coupled to affinity column for hemoglobin, animal antihuman hemoglobin antiserum addition coupling is had blood red
The affinity column of albumen, is then eluted with neutralized eluate, the hemoglobin antibodies purified.
5. a kind of preparation method of hemoglobin antibodies according to claim 1, which is characterized in that the hemoglobin is anti-
Body is monoclonal antibody, the specific steps of the step S3 are as follows: with the coated hemoglobin immune mouse of the liposome, culture
After take spleen cell and myeloma cell to merge to obtain multiple hybridomas, choose positive colony and expand culture, and be injected to
Balb/c mouse peritoneal extracted ascites after 7~10 days, purified to obtain hemoglobin antibodies.
6. a kind of preparation method of hemoglobin antibodies according to claim 4, which is characterized in that the S32's is specific
Step are as follows: hemoglobin is configured to the hemoglobin solutions that concentration is 1mg/mL with coupling buffer, weighs hydrogen bromide activation
Agarose gel microsphere, fill column, added in 4 DEG C of 25~35min of activation with coupling buffer balance chromatographic column with the HCl of 1mM
Enter the hemoglobin solutions, the hemoglobin solutions of addition and the volume ratio of the agarose gel microsphere are 5:2, room
Temperature is incubated for 3~4h, cleans chromatographic column with the coupling buffer of 5 times of column volumes, and the Tris- that the 1MpH of 5 times of column volumes is 8.0 is added
Hcl closes chromatographic column, and 4 DEG C of 8~16h of placement are finally slow with balance then with alternately cleaning chromatographic column 3 times of coupling acid, alkali wash water
Fliud flushing balances chromatographic column, obtains affinity column, and the animal body fluid equilibration buffer containing hemoglobin antibodies is dilute
2~3 times are released, 2~3h is incubated at room temperature, the antibody of non-specific binding is washed away with the equilibration buffer of 5 times of column volumes, is then added
Neutral elution buffer is eluted, and is collected eluent and is obtained hemoglobin antibodies.
7. a kind of preparation method of hemoglobin antibodies according to claim 6, which is characterized in that the equilibration buffer
For the PBS buffer solution of the pH 150mM for being 7.4.
8. a kind of preparation method of hemoglobin antibodies according to claim 6, which is characterized in that the middle elution buffer
The pH value of liquid is 7.2, including 3M MgCl2, 0.075M 4- hydroxyethyl piperazineethanesulfonic acid (Hepes) and 25vol% ethylene glycol.
9. a kind of hemoglobin antibodies, which is characterized in that be prepared by the described in any item preparation methods of claim 1-8
It arrives.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112979799A (en) * | 2019-12-18 | 2021-06-18 | 东莞市朋志生物科技有限公司 | Binding protein containing hemoglobin antigen structural domain |
| CN113461979A (en) * | 2021-07-19 | 2021-10-01 | 吉林大学 | Preparation method of simulated mussel hydrogel catalytically crosslinked by hemoglobin |
| CN113004402B (en) * | 2019-12-18 | 2022-11-04 | 东莞市朋志生物科技有限公司 | Binding protein containing hemoglobin antigen structural domain |
| CN116046506A (en) * | 2022-07-26 | 2023-05-02 | 南京立顶医疗科技有限公司 | Separation and purification method of natural high-purity glycosylated hemoglobin HbA1c and hemoglobin HbA0 |
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| CN113461979A (en) * | 2021-07-19 | 2021-10-01 | 吉林大学 | Preparation method of simulated mussel hydrogel catalytically crosslinked by hemoglobin |
| CN116046506A (en) * | 2022-07-26 | 2023-05-02 | 南京立顶医疗科技有限公司 | Separation and purification method of natural high-purity glycosylated hemoglobin HbA1c and hemoglobin HbA0 |
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