CN102417920A - Method for preparing anti-human chorionic gonadotrophin (hCG) monoclonal antibody fragment - Google Patents
Method for preparing anti-human chorionic gonadotrophin (hCG) monoclonal antibody fragment Download PDFInfo
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Abstract
The invention discloses a method for preparing an anti-human chorionic gonadotrophin (hCG) monoclonal antibody fragment. The method comprises the following steps of: putting activated immobilized papain into a 0.1mol/L acetic acid buffer solution with pH of 5.0, adding an anti-hCG monoclonal antibody, performing water bath reaction for 4 to 24 hours at the temperature of between 30 and 60 DEG C, and filtering the reaction solution, wherein filter cakes B are immobilized papain and recycled, and filtrate B is a hydrolysate containing the monoclonal antibody fragment; and separating and purifying the hydrolysate containing the monoclonal antibody fragment by adopting weak anion exchange chromatography, and thus obtaining the anti-hCG monoclonal antibody fragment. The method is easy to implement and has a few purification steps, and the obtained antibody fragment has high purity; and the antibody fragment prepared in the method can reach the purity of over 90 percent by one-step purification.
Description
(1) technical field
The present invention relates to a kind of preparation method of monoclonal antibody fragment, particularly a kind of preparation method of anti-human chorionic gonad-stimulating hormone monoclonal antibody fragment.
(2) background technology
(human chorionic gonadotrophin is a kind of glycoprotein hormones of placenta plamoditrophoblast emiocytosis after the mankind become pregnant hCG) to pregnancy urine extract, and molecular weight is about 38000D, and synthetic and secretion and the gestation of hCG are closely-related.The inspection of hCG can be used as the important indicator that early pregnancy detects, and adopts anti-hCG monoclonal antibody to carry out early pregnancy and detects, and can further improve the susceptibility and the specificity of diagnosis of early gestation.And the content of hCG and many neoplastic diseases have close getting in touch, and can regard hCG one of as tumor markers, so the inspection of hCG also can provide foundation to the Clinics and Practices monitoring of trophocyte's tumour and non-trophocyte's tumour.Discovered in recent years; HCG also can raise in the blood such as malignant tumour such as carcinoma of the pancreas, cancer of the stomach, liver cancer, mammary cancer, lung cancer; In the serum of the individuality of suffering from bladder cancer, carcinoma of the pancreas, colorectal carcinoma, often can detect and obtain hCG β protein subunit and have significantly and improve, and in tumor tissues, also can detect through Goodpasture's staining.
With respect to monoclonal antibody, monoclonal antibody fragment (comprising Fab and F (ab ') 2 fragments) has the influence that more advantages (1) are not positioned at the Fc acceptor on the cells such as scavenger cell, B cell, T cell, neutrophil leucocyte and giant cells in addition; (2) Precipitation Antigen not; (3) decomposition radio-labeling fragment is faster than entire I gG conjugate in healthy tissues; (4) reduce immunogenicity (Fc district disappearance), the anti-rat immune globulin of people (HAMA) reaction is minimized; (5) more not being subject to phagocytic cell attacks; (6) molecular weight is little, and it is rapid to arrive tumor locus.
Therefore, if can develop high purity, weak pregnancy urine extract (hCG) monoclonal antibody fragment of effective, immunogenicity, and the exploitation of application and diagnostic reagent, will bring considerable society and economy benefit.
According to present domestic and foreign literature and patent report; The preparation of antibody fragment; Separation and purification makes through multistep more all to adopt free papoid or pepsin hydrolysis antibody, and these class methods exist resolvase to reuse, and removes resolvase and can reduce problems such as the product recovery simultaneously.
(3) summary of the invention
The object of the invention provides a kind of preparation method of anti-human chorionic gonad-stimulating hormone monoclonal antibody fragment, and this method is easy and simple to handle, purification step is few, gained antibody fragment purity high.
The technical scheme that the present invention adopts is:
A kind of preparation method of anti-human chorionic gonad-stimulating hormone monoclonal antibody fragment, said method is: the 0.1mol/L hac buffer of pH5.0～9.0 that will contain 0.1～1mmol/L DTT and 0.1～2.5mmol/L EDTA adds immobilized papain at 30～50 ℃ of following water-bath 5～10min; 30～50 ℃ of water-bath 20～30min; Suction filtration discards filtrating A and promptly removes DTT, obtains filter cake A and is the activatory immobilized papain; With the activatory immobilized papain in the pH5.00.1mol/L acetate buffer solution; Add anti-human chorionic gonad-stimulating hormone monoclonal antibody, 30～60 ℃ of water-bath 4～24h, reaction solution suction filtration; Filter cake B is that immobilized papain is recycled; Liquor B is the hydrolyzed solution that contains monoclonal antibody fragment, adopts the weak anionic displacement chromatography that the hydrolyzed solution that contains monoclonal antibody fragment is carried out separation and purification, obtains said anti-human chorionic gonad-stimulating hormone monoclonal antibody fragment; Said immobilized papain is a carrier with ammonification paper fiber; With glutaraldehyde as cross linker; With the papoid is substrate, in the 0.1mol/L acetate buffer solution of the pH5.0 that contains 1mmol/L DTT and 2mmol/L EDTA, stirs 8～24h under the room temperature; Suction filtration gets filter cake C and is immobilized papain; Said ammonification paper fiber is paper fiber NaOH solution-treated 2～3h of warp 0.1～0.2mol/L earlier; Use epoxy chloropropane in 60～70 ℃ of activation 0.5～1h again, use Ursol D at last, use the zero(ppm) water thorough washing in 40～50 ℃ of ammonification 0.5h; Vacuum-drying obtains ammonification paper fiber to constant weight; Said immobilized papain quality consumption is counted 0.1～0.2g/ml antibody with the antibody volume; It is the 0.002g/g immobilized enzyme that said immobilized papain carries the enzyme amount, and said anti-human chorionic gonad-stimulating hormone monoclonal antibody is utilized sad-ammonium sulfate method that mouse ascites is carried out purifying and obtained.
Said immobilized papain prepares as follows: with ammonification paper fiber dispersion in the 0.1mol/L hac buffer A of pH2.0～6.5 that contain 1mmol/L DTT and 2mmol/L EDTA; The glutaraldehyde water solution that adds papoid and mass concentration 25%; Stir 8～24h under the room temperature, suction filtration, filter cake C are used the acetate buffer solution B thorough washing identical with described hac buffer A; Filter, filter cake promptly obtains immobilized papain; The mass ratio of said ammonification paper fiber and papoid is 1: 0.006; Said 25% glutaraldehyde water solution is counted 0.317ml/g with ammonification paper fiber quality; The volumetric usage of said hac buffer A is to not influence of the present invention; Can make the paper fiber fully disperse to get final product, volumetric usage is counted 33～40ml/g with ammonification paper quality of fiber usually, and the volumetric usage of said acetate buffer solution B can get final product by thorough washing usually.
Said ammonification paper fiber prepares as follows: with filter paper shred, poach is to mashed, filtered through gauze, filter cake a is the paper fiber, the paper fiber is added in the NaOH solution A of 0.1～0.2mol/L to stir 2h; Wash to pH7.0, suction filtration is got the NaOH solution B of filter cake b by mass volume ratio adding in 1: 9 0.5～1mol/L, presses and filter cake b mass volume ratio adding in 1: 1 epoxy chloropropane; 60～70 ℃ of stirring in water bath reaction 30min, suction filtration, filter cake c drain after with the deionized water thorough washing; Obtain filter cake d, add Ursol D at 0.2: 1 with mass volume ratio again, react 30min down in 45 ℃; Use the zero(ppm) water thorough washing, vacuum-drying obtains ammonification paper fiber to constant weight.
Said weak anionic displacement chromatography is the DEAE-Sephorose-FF chromatography; The Tris-HCl of 20mmol/L that contains 1mol/LNaCl with pH8.0 is as elutriant; Linear gradient elution is collected first elution peak, obtains said anti-human chorionic gonad-stimulating hormone monoclonal antibody fragment.
Year enzyme amount of immobilized papain according to the invention is the 0.002g/g immobilized enzyme, enzyme 1940U/g alive, and enzyme work is defined as under these conditions, and the enzyme amount that PM discharges the tyrosine of 1ug is an enzyme activity unit U.
Immobilized papain enzyme activity determination method according to the invention is: the acetate buffer solution (pH5.0 that in test tube, adds the 0.1mol/L of 2ml; Include 0.005mol/L DTT and 0.001mol/LEDTA), 8ml 2% casein solution (pH7.0), 37 ℃ of water-bath preheating 5min; Add 0.5g (weight in wet base then; The 1g weight in wet base is equivalent to the 0.4g dry weight) immobilized enzyme, behind 37 ℃ of reaction 30min, add 5ml 10% trichoroacetic acid(TCA) solution, 10min is placed in 37 ℃ in the concussion back; Filter, survey the absorbancy of filtrating at the 280nm place.
Tire (unit/g)=A/As*Cs*12/2* extension rate/w
A is the absorbancy that the absorbancy of need testing solution deducts blank solution in the formula;
As is the absorbancy of tyrosine reference substance solution;
Cs is the concentration ug/ml of tyrosine reference substance solution;
W is the trial-product quality, g.
Mouse ascites according to the invention source is normally injected hCG antigen in the mouse body; Immunoreation takes place in the mouse body; Obtain hCG antigen, extract mouse ascites, promptly obtain said thick anti-; The present invention adopts the mouse ascites of the anti-human chorionic gonad-stimulating hormone monoclonal antibody of Jinake Biotechnology ltd production to resist as thick, obtains anti-human chorionic gonad-stimulating hormone monoclonal antibody through sad-ammonium sulfate method purifying.
Paper fiber according to the invention is taken from common filter paper.
DTT is DL-Dithiothreitol, Chinese WR 34678 by name.Molecular formula is C
4H
10O
2S
2, molecular weight is 154.25, purity＞99%.
Filter cake A according to the invention, filter cake B, filter cake C, filter cake a, filter cake b, filter cake c are different step and filter the gained filter cake, and filtrating A is filtrating, names for ease of differentiation.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
Among the present invention is carrier with ammonification paper fiber; With glutaraldehyde as cross linker, be that the immobilized papain that substrate obtains can be recycled with the papoid, reclaim simple; Along with production-scale expansion; Can reduce production costs greatly, and need not consider the residue problem of resolvase in antibody fragment, thereby can save the yield of the step raising antibody fragment of separation and purification; The inventive method is easy and simple to handle in addition, purification step is few, gained antibody fragment purity high: the antibody fragment for preparing among the present invention only need pass through single step purification just can reach the purity more than 90%.
(4) description of drawings
Fig. 1 immobilized papain suspension-s photo
Fig. 2 DEAE-Sephorose-FF column purification antibody fragment color atlas: A is for penetrating the peak, the B elution peak
The polyacrylamide gel electrophoresis of Fig. 3 antibody fragment is identified figure: 1 is the antibody fragment electrophorogram, and 2 is the standard protein electrophorogram
(5) embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
1) immobilization of papoid: filter paper is shredded, add tap water, boil, well-done back is with 8 layers of filtered through gauze; Get the NaOH solution that filter cake adds 1000ml 0.1mol/L, stir 2h, wash to pH nearly 7.0, suction filtration; Get the NaOH solution of 1.2g filter cake (dry weight), again in adding epoxy chloropropane with the ratio of filter cake mass volume ratio 1: 1 (g/ml), 60 ℃ of stirring in water bath reaction 30min in the ratio adding 1mol/L of 1: 9 (g/ml); Suction filtration, the cakes with deionized water thorough washing is drained; Filter cake by adding Ursol D (0.24g Ursol D/1.2g filter cake carrier) at 0.2: 1 with 1.2g filter cake mass ratio, reacts 30min, filtration down in 45 ℃ again; Filter cake is used the zero(ppm) water thorough washing, and vacuum-drying obtains ammonification paper fiber 1.2g to constant weight.With ammonification paper fiber is carrier; Prepare immobilized papain with glutaraldehyde as cross linker: with acetate buffer solution (include 1mmol/L DTT and the 2mmol/L EDTA) 40ml of above-mentioned 1.2g ammonification paper fiber dispersion in the 0.1mol/L of pH5.0, add 7.2mg papoid (giving birth to worker's biotechnology Shanghai ltd) and mass concentration 25% glutaraldehyde water solution 0.387ml, room temperature (25 ℃) stirs 12h down; Suction filtration; Filter cake filters with the acetate buffer solution thorough washing of pH5.0,0.1mol/L, and filter cake is an immobilized papain; Be suspended in the acetate buffer solution of pH5.0,0.1mol/L subsequent use, as shown in Figure 1.
2) MONOCLONAL ANTIBODIES SPECIFIC FOR: utilize sad-ammonium sulfate method that mouse ascites is carried out purifying: with the acetate buffer solution of 4 times of volumes (60mmol/L, pH4.0) dilution contains the mouse ascites (Jinake Biotechnology ltd) of anti-human chorionic gonad-stimulating hormone monoclonal antibody, with 0.1mmol/L NaOH accent pH to 4.5; Obtain the 150ml mixed solution, dropwise add 1.875ml sad (25ul/ml mixed solution), stir while dripping; Drip off continued and stir 30min, the centrifugal 30min of 8000r/min gets supernatant 10: 1 by volume and mixes with PBS; NaOH with 0.1mol/L transfers pH to 7.4; After adding 35.0658g ammonium sulfate (0.227g/ml mixed solution) stirring 30min, the centrifugal 15min of 6000r/min abandons supernatant; Deposition is suspended among the PBS (initial ascites volume 1/5); Suspension-s obtains anti-human chorionic and promotes gonadal hormone monoclonal antibody (HCG) 5ml, in-20 ℃ of preservations with the PBS dialysed overnight of 100 times of volumes.In order to prevent that antibody is destroyed in the purification process, more than all stir and operating process is all carried out under 4 ℃ of conditions.
3) enzymolysis of monoclonal antibody: in test tube, add 3.5ml 0.1mol/L, the acetate buffer solution of pH5.0 (including 0.5ml DTT and 1mlEDTA), 30 ℃ of water-bath preheating 5min; Add 1g immobilized papain (weight in wet base is carried enzyme amount 0.002g, enzyme 1940U/g alive) then; 30 ℃ of reaction 30min, suction filtration discards filtrating and promptly removes DTT; Obtaining filter cake is the activatory immobilized papain, places the 15ml hac buffer of 0.1mol/L, pH5.0 to add the above-mentioned HCG antibody of 5ml, 37 ℃ of water-bath 8h the immobilized papain 1g after the activation; Suction filtration is got filtrating, is to contain the segmental hydrolyzed solution of HCG; Filter cake is an immobilized papain, recycles and reuses.
4) purifying of monoclonal antibody fragment: get the segmental hydrolyzed solution of the above-mentioned HCG of containing, (electrophoresis of irreducibility is not add DTT in the sample, thereby guarantees that the inner disulfide linkage of antibody is not destroyed with the degree of the SDS-PAGE electrophoresis detection antibody of irreducibility digestion; Electrophoresis can intuitively show segmental molecular weight and purity, if digestion is not fully, then except segmental band is arranged; Also have complete antibody band), adopt weak anionic displacement chromatography (DEAE-Sephorose-FF, 2cm * 18cm; Pharmacia company) the gained antibody fragment is carried out separation and purification; (pH8.0 is 20mmol/L) as sample-loading buffer (A liquid), with the Tris-HCl (pH8.0 of 1mol/LNaCl to utilize Tris-HCl; 20mmol/L) as elutriant (B liquid); Adopt linear gradient elution, flow velocity 2ml/min collects first elution peak and is antibody fragment (referring to Fig. 2).
1) immobilization of papoid: filter paper is shredded, add tap water, boil, well-done back is with 8 layers of filtered through gauze; The NaOH solution that in the paper fiber, adds 1000ml 0.2mol/L stirs 3h, washes to pH nearly 7.0; Suction filtration is got the NaOH solution of 1.2g filter cake (dry weight) in the ratio adding 1mol/L of 1: 9 (g/ml), again in adding epoxy chloropropane with the ratio of filter cake mass volume ratio 1: 1 (g/ml); 60 ℃ of stirring in water bath reaction 30min, suction filtration is drained behind the cakes with deionized water thorough washing; Obtain filter cake, by adding Ursol D (0.24g Ursol D/1.2g filter cake carrier) at 0.2: 1, react 30min down again in 45 ℃ with 1.2g filter cake mass volume ratio; Use the zero(ppm) water thorough washing, vacuum-drying obtains ammonification paper fiber to constant weight.
With ammonification paper fiber is carrier; With glutaraldehyde as cross linker, be that substrate prepares immobilized papain: 1.2g ammonification paper fibrous carrier is scattered in acetate buffer solution (including 1mmol/L DTT and the 2mmol/L EDTA) 40ml of the 0.1mol/L of pH5.0, adds 7.2mg papoid and 0.387ml mass concentration 25% glutaraldehyde water solution with the papoid; Stir 12h under the room temperature; Suction filtration, filter cake filter with the acetate buffer solution thorough washing of 0.1mol/L; Filter cake is immobilized papain, is suspended in the acetate buffer solution of pH5.0,0.1mol/L subsequent use.
2) MONOCLONAL ANTIBODIES SPECIFIC FOR: utilize sad-ammonium sulfate method that mouse ascites is carried out purifying, the acquisition of mouse ascites is with embodiment 1.With the acetate buffer solution of 4 times of volumes (60mmol/L, pH4.0) dilution ascites is transferred pH to 4.5 with 0.1mmom/LNaOH; Obtain the 150ml mixed solution, 1.875ml sad (25ul/ml mixed solution) is dropwise added in the sample, stir while dripping; Drip off continued and stir 30min, the centrifugal 30min of 8000r/min gets supernatant 10: 1 by volume and mixes with PBS; NaOH with 0.1mol/L transfers pH to 7.4, after adding ammonium sulfate 35.0658g (0.227g/ml mixed solution) stirs 30min, and the centrifugal 15min of 6000r/min; Abandon supernatant, deposition is suspended among the PBS (initial ascites volume 1/5).Suspension-s obtains anti-human chorionic and promotes gonadal hormone monoclonal antibody (HCG) 5ml, in-20 ℃ of preservations with the PBS dialysed overnight of 100 times of volumes.Trace prevents that antibody is destroyed in the purification process, more than all stir and operating process is all carried out under 4 ℃ of conditions.
3) enzymolysis of monoclonal antibody: in test tube, add 3.5ml 0.1mol/L; The phosphoric acid buffer of pH8.0 (including 0.5ml DTT and 1mlEDTA); 50 ℃ of water-bath preheating 5min, (weight in wet base is carried enzyme amount 0.002g to add the 0.5g immobilized papain then; Enzyme 1940U/g alive), 50 ℃ of reaction 30min.Suction filtration discards filtrating and promptly removes DTT, and filter cake is the immobilized papain of reactivate.Immobilized papain 0.5g after the activation places the 15ml hac buffer of 0.1mol/L, pH5.0 to add 5ml HCG antibody; 50 ℃ of water-bath 12h; Suction filtration, getting filtrates to be contains the segmental hydrolyzed solution of HCG, and filter cake is that immobilized enzyme recycles and reuses.
4) purifying of monoclonal antibody fragment: get the segmental hydrolyzed solution of the above-mentioned HCG of containing,, adopt the weak anionic displacement chromatography that the gained antibody fragment is carried out separation and purification with the degree of the SDS-PAGE electrophoresis detection antibody of irreducibility digestion; (pH9.0 is 20mmol/L) as sample-loading buffer (A liquid, the Tris-HCl (pH9.0 of 1mol/LNaCl to utilize Tris-HCl; 20mmol/L) as elutriant (B liquid); Adopt linear gradient elution, flow velocity 2ml/min collects first elution peak and is antibody fragment.The gained antibody fragment identifies that through polyacrylamide gel electrophoresis purity reaches 95% (referring to Fig. 3).
Claims (4)
1. the preparation method of an anti-human chorionic gonad-stimulating hormone monoclonal antibody fragment; It is characterized in that said method is: the 0.1mol/L hac buffer of pH5.0～9.0 that will contain 0.1～1mmol/L DTT and 0.1～2.5mmol/L EDTA is at 30～50 ℃ of following water-bath 5～10min; Add immobilized papain, 30～50 ℃ of water-bath 20～30min, suction filtration; Discard filtrating A, obtain filter cake A and be the activatory immobilized papain; With the activatory immobilized papain in pH5.0,0.1mol/L acetate buffer solution; Add anti-human chorionic gonad-stimulating hormone monoclonal antibody; 30～60 ℃ of water-bath 4～24h; Reaction solution suction filtration, filter cake B are that immobilized papain is recycled, and liquor B is the hydrolyzed solution that contains monoclonal antibody fragment; Adopt the weak anionic displacement chromatography that the said hydrolyzed solution that contains monoclonal antibody fragment is carried out separation and purification, obtain said anti-human chorionic gonad-stimulating hormone monoclonal antibody fragment; Said immobilized papain is a carrier with ammonification paper fiber; With glutaraldehyde as cross linker; With the papoid is substrate, in the 0.1mol/L acetate buffer solution of the pH5.0 that contains 1mmol/LDTT and 2mmol/L EDTA, stirs 8～24h under the room temperature; Suction filtration gets filter cake C and is immobilized papain; Said ammonification paper fiber is paper fiber NaOH solution-treated 2～3h of warp 0.1～0.2mol/L earlier; Use epoxy chloropropane in 60～70 ℃ of activation 0.5～1h again, use Ursol D at last, use the zero(ppm) water thorough washing in 40～50 ℃ of ammonification 0.5h; Vacuum-drying obtains ammonification paper fiber to constant weight; Said immobilized papain quality consumption is counted 0.1～0.2g/ml antibody with the antibody volume, and carrying the enzyme amount is the 0.002g/g immobilized enzyme, and said anti-human chorionic gonad-stimulating hormone monoclonal antibody is utilized sad-ammonium sulfate method that mouse ascites is carried out purifying and obtained.
2. the preparation method of anti-human chorionic gonad-stimulating hormone monoclonal antibody fragment as claimed in claim 1; It is characterized in that said immobilized papain prepares as follows: with ammonification paper fiber dispersion in the 0.1mol/L hac buffer A of pH2～6.5 that contain 1mmol/L DTT and 2mmol/L EDTA; Add papoid and mass concentration 25% glutaraldehyde water solution; Stir 8～24h under the room temperature, suction filtration, filter cake C are used the acetate buffer solution B thorough washing identical with described hac buffer A; Filter, filter cake promptly obtains immobilized papain; The mass ratio of said ammonification paper fiber and papoid is 1: 0.006, and the volumetric usage of said 25% glutaraldehyde water solution is counted 0.317ml/g with ammonification paper fiber quality.
3. according to claim 1 or claim 2 the preparation method of anti-human chorionic gonad-stimulating hormone monoclonal antibody fragment is characterized in that said ammonification paper fiber prepares as follows: with filter paper shred, poach is to mashed, filtered through gauze; Filter cake a is the paper fiber, stirs 2h in the NaOH solution A with paper fiber adding 0.1～0.2mol/L, washes to pH7.0; Suction filtration is got the NaOH solution B of filter cake b by mass volume ratio adding in 1: 9 0.5～1mol/L, presses and filter cake b mass volume ratio adding in 1: 1 epoxy chloropropane; 60～70 ℃ of stirring in water bath reaction 30min, suction filtration, filter cake c drain after with the deionized water thorough washing; Obtain filter cake d, by adding Ursol D at 0.2: 1, react 30min down again in 45 ℃ with filter cake b mass ratio; Use the zero(ppm) water thorough washing, vacuum-drying obtains ammonification paper fiber to constant weight.
4. the preparation method of anti-human chorionic gonad-stimulating hormone monoclonal antibody fragment as claimed in claim 1; It is characterized in that said weak anionic displacement chromatography is the DEAE-Sephorose-FF chromatography; The Tris-HCl of 20mmol/L that contains 1mol/LNaCl with pH8.0 is as elutriant; Linear gradient elution is collected first elution peak, obtains said anti-human chorionic gonad-stimulating hormone monoclonal antibody fragment.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103275961A (en) * | 2013-06-05 | 2013-09-04 | 浙江工业大学 | Temperature-sensitive immobilized papain, and application thereof in preparing monoclonal antibody |
| CN104313091A (en) * | 2014-09-27 | 2015-01-28 | 浙江工业大学 | Method for separating human chorionic gonadotropin monoclonal antibody segment |
| CN115820652A (en) * | 2022-12-27 | 2023-03-21 | 中国人民解放军军事科学院军事医学研究院 | Nucleic acid aptamer group for specifically recognizing human chorionic gonadotropin and application thereof |
-
2011
- 2011-12-07 CN CN2011104030074A patent/CN102417920A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103275961A (en) * | 2013-06-05 | 2013-09-04 | 浙江工业大学 | Temperature-sensitive immobilized papain, and application thereof in preparing monoclonal antibody |
| CN104313091A (en) * | 2014-09-27 | 2015-01-28 | 浙江工业大学 | Method for separating human chorionic gonadotropin monoclonal antibody segment |
| CN115820652A (en) * | 2022-12-27 | 2023-03-21 | 中国人民解放军军事科学院军事医学研究院 | Nucleic acid aptamer group for specifically recognizing human chorionic gonadotropin and application thereof |
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Application publication date: 20120418 |