CN110954706B - Method for measuring coupling rate of GnRH paralog dimer artificial antigen - Google Patents
Method for measuring coupling rate of GnRH paralog dimer artificial antigen Download PDFInfo
- Publication number
- CN110954706B CN110954706B CN201911285131.8A CN201911285131A CN110954706B CN 110954706 B CN110954706 B CN 110954706B CN 201911285131 A CN201911285131 A CN 201911285131A CN 110954706 B CN110954706 B CN 110954706B
- Authority
- CN
- China
- Prior art keywords
- gnrh
- dimer
- protein
- coupling
- mobile phase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 title claims abstract description 138
- 239000000539 dimer Substances 0.000 title claims abstract description 80
- 230000008878 coupling Effects 0.000 title claims abstract description 67
- 238000010168 coupling process Methods 0.000 title claims abstract description 67
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000000427 antigen Substances 0.000 title claims abstract description 14
- 102000036639 antigens Human genes 0.000 title claims abstract description 14
- 108091007433 antigens Proteins 0.000 title claims abstract description 14
- NMJREATYWWNIKX-UHFFFAOYSA-N GnRH Chemical compound C1CCC(C(=O)NCC(N)=O)N1C(=O)C(CC(C)C)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)CNC(=O)C(NC(=O)C(CO)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C(CC=1NC=NC=1)NC(=O)C1NC(=O)CC1)CC1=CC=C(O)C=C1 NMJREATYWWNIKX-UHFFFAOYSA-N 0.000 title claims abstract 26
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 98
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 98
- 239000003446 ligand Substances 0.000 claims abstract description 43
- 230000009467 reduction Effects 0.000 claims abstract description 18
- 238000004925 denaturation Methods 0.000 claims abstract description 8
- 230000036425 denaturation Effects 0.000 claims abstract description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 33
- 238000001514 detection method Methods 0.000 claims description 32
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 22
- 239000000523 sample Substances 0.000 claims description 18
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 14
- 102000014914 Carrier Proteins Human genes 0.000 claims description 13
- 108010078791 Carrier Proteins Proteins 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 108010058846 Ovalbumin Proteins 0.000 claims description 10
- 229940092253 ovalbumin Drugs 0.000 claims description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 9
- 229940098773 bovine serum albumin Drugs 0.000 claims description 9
- 239000003638 chemical reducing agent Substances 0.000 claims description 7
- 239000003398 denaturant Substances 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 4
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 4
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 4
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 claims description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- VHJLVAABSRFDPM-ZXZARUISSA-N dithioerythritol Chemical compound SC[C@H](O)[C@H](O)CS VHJLVAABSRFDPM-ZXZARUISSA-N 0.000 claims description 3
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 3
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 3
- 102000009843 Thyroglobulin Human genes 0.000 claims description 2
- 108010034949 Thyroglobulin Proteins 0.000 claims description 2
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 claims description 2
- 229960003983 diphtheria toxoid Drugs 0.000 claims description 2
- 239000000178 monomer Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 239000012488 sample solution Substances 0.000 claims description 2
- 238000002798 spectrophotometry method Methods 0.000 claims description 2
- 229960000814 tetanus toxoid Drugs 0.000 claims description 2
- 229960002175 thyroglobulin Drugs 0.000 claims description 2
- 238000010268 HPLC based assay Methods 0.000 claims 1
- 229940079593 drug Drugs 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 230000001850 reproductive effect Effects 0.000 abstract description 2
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 112
- XLXSAKCOAKORKW-AQJXLSMYSA-N gonadorelin Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 XLXSAKCOAKORKW-AQJXLSMYSA-N 0.000 description 112
- 229940035638 gonadotropin-releasing hormone Drugs 0.000 description 112
- 235000018102 proteins Nutrition 0.000 description 83
- 239000000243 solution Substances 0.000 description 17
- 238000006722 reduction reaction Methods 0.000 description 14
- 239000012086 standard solution Substances 0.000 description 12
- 238000002347 injection Methods 0.000 description 10
- 239000007924 injection Substances 0.000 description 10
- 108091028732 Concatemer Proteins 0.000 description 8
- 239000008367 deionised water Substances 0.000 description 8
- 229910021641 deionized water Inorganic materials 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000012417 linear regression Methods 0.000 description 6
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 150000001718 carbodiimides Chemical class 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- XXMFJKNOJSDQBM-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;hydrate Chemical compound [OH3+].[O-]C(=O)C(F)(F)F XXMFJKNOJSDQBM-UHFFFAOYSA-N 0.000 description 3
- 241000282887 Suidae Species 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 2
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 2
- 102000009151 Luteinizing Hormone Human genes 0.000 description 2
- 108010073521 Luteinizing Hormone Proteins 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229940028334 follicle stimulating hormone Drugs 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000000506 liquid--solid chromatography Methods 0.000 description 2
- 229940040129 luteinizing hormone Drugs 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 230000001568 sexual effect Effects 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108010060123 Conjugate Vaccines Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 239000000589 Siderophore Substances 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 230000007012 clinical effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229940031670 conjugate vaccine Drugs 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000006334 disulfide bridging Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Endocrinology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Reproductive Health (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical field of reproductive drugs, and particularly relates to a method for measuring coupling rate of GnRH paralog dimer artificial antigen, which comprises the following steps: (1) quantitatively determining the coupling protein and the residual GnRH parallel body dimer in the coupling protein sample to obtain CConjugated proteinsAnd CResidual GnRH ligand dimer(ii) a (2) Performing denaturation reduction treatment on the coupled protein to ensure that the GnRH paralleling body dimer is subjected to reduction breakage to free a GnRH paralleling body single chain, and quantitatively measuring the free GnRH paralleling body single chain to obtain CFree GnRH paralogue single chain(ii) a (3) Calculating the coupling ratio:
Description
Technical Field
The invention belongs to the technical field of reproductive drugs, and particularly relates to a method for measuring coupling rate of GnRH paralog dimer artificial antigen.
Background
Gonadotropin releasing hormone (GnRH), which was first isolated by Schally et al in 1971 from the hypothalamus of pigs and sheep, has the function of stimulating the release of Luteinizing Hormone (LH) and the secretion of Follicle Stimulating Hormone (FSH), and plays an important role in the regulation of animal reproduction. The immune castration is to utilize GnRH or its analog as antigen to immunize animals, so that the organism generates GnRH resisting antibody, which is combined with endogenous GnRH to make it lose biological activity, thus inhibiting the development of sexual organs to achieve the goal of castration. Compared with the traditional operation castration, the immune castration can effectively prevent the occurrence of operation infection, has the advantages of safety, convenience, small stress and the like, promotes the growth of animals, improves the meat quality, and has wide application prospect in animal production.
Scientists have conducted extensive research into GnRH castration vaccines since the last 70 s. The GnRH decapeptide or an analogue thereof is coupled on carrier proteins such as Bovine Serum Albumin (BSA), Keyhole Limpet Hemocyanin (KLH), Ovalbumin (OVA) and the like, and immune experiments are carried out on animals such as rabbits, mice, monkeys, sheep, pigs, horses, cows and the like, and the experimental result shows that the GnRH coupling protein has obvious effect on inhibiting the development of animal sexual organs. In the 90 s, Meloen et al (U.S. patent: US5484592) developed a 20 peptide GnRH siderophore with enhanced immunogenicity relative to GnRH monomers. Subsequently, they found that disulfide bonding of GnRH juxtaposition to a dimer via a thiol group in the inserted Cys can produce greater immunogenicity than GnRH juxtaposition (US 5885966). Patents CN 103554265a and CN103554228A also disclose a GnRH ligand dimer, which is coupled to ovalbumin, and experiments show that the coupled protein has good clinical effect in pigs.
For conjugated proteins, such as conjugate vaccines, the conjugation rate has an important influence on the immunogenicity strength, i.e. the biological activity of the conjugated protein, and is one of the most important quality attributes of the product. The polypeptide antigen-coupled protein coupling rate method adopted in the literature includes amino acid analysis contrast method, electrophoresis method, mass spectrometry method and the like. The amino acid analysis method has low detection precision on the amino acid composition ratio, and the coupling rate is difficult to obtain an accurate result by calculating the difference of the amino acid composition ratio of the coupling protein and the carrier protein. The coupling protein is difficult to obtain accurate molecular weight by an electrophoresis method, and the carrier protein is subjected to artificial chemical modification, or is dispersive, or has certain migration deviation, so that the judgment of the molecular weight is influenced. Moreover, compared with the natural protein, the ionization capacity of the coupled protein in MALDI-TOF-MS is changed a little, and ideal results are difficult to obtain.
Disclosure of Invention
Aiming at the defect of low accuracy of the coupling rate determination method of the coupling protein in the prior art, the invention provides a method capable of accurately determining the coupling rate of GnRH concatemer dimer (abbreviated as GnRH-TD) on carrier protein.
The above object of the present invention is achieved by the following technical solutions:
a method for measuring the coupling rate of GnRH paralog dimer artificial antigen is characterized by comprising the following steps:
(1) quantitatively determining the coupling protein and the residual GnRH parallel body dimer in the coupling protein sample to obtain CConjugated proteinsAnd CResidual GnRH ligand dimer；
(2) Performing denaturation reduction treatment on the coupled protein to ensure that the GnRH paralleling body dimer is subjected to reduction breakage to free a GnRH paralleling body single chain, and quantitatively measuring the free GnRH paralleling body single chain to obtain CFree GnRH paralogue single chain；
(3) The coupling ratio was calculated as follows:
where n represents the amount of substance in mol, C represents the mass concentration in mg/ml, MW represents the molar mass in g/mol.
CConjugated proteins、CResidual GnRH ligand dimer、CFree GnRH paralogue single chain、CCoupled protein-bound GnRH juxtapomer dimers、CCarrier proteinsRespectively representing the mass concentrations of the coupling protein, residual GnRH parallel body dimer, free GnRH parallel body single chain, coupling protein combined GnRH parallel body dimer and carrier protein in the coupling protein sample; n isCoupled protein-bound GnRH juxtapomer dimers、nCarrier proteinsThe amounts of substances representing GnRH ligand dimer bound to the conjugate protein and carrier protein, respectively; MWCarrier proteins、MWGnRH ligand dimerRespectively represent the molar masses of the carrier protein and the GnRH ligand dimer.
The coupling protein is a coupling compound of a carrier protein and GnRH ligand dimer, and the carrier protein is preferably selected from one or more of the following: bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, thyroglobulin, diphtheria toxoid, and tetanus toxoid; the GnRH parallels dimer is obtained by connecting GnRH parallels through-s-s-bonds of cysteine Cys, and the GnRH parallels are formed by connecting at least 2 GnRH in series.
Preferably, the quantitative determination of the conjugated protein is selected from one or more of the following methods: high Performance Liquid Chromatography (HPLC), ultraviolet spectrophotometry, Coomassie brilliant blue binding, Folin-phenol reagent, biuret, BCA. The methods are common methods in protein quantitative determination, and can effectively determine the mass concentration of the coupled protein in a sample.
Further preferably, the quantitative determination of the conjugated protein is performed by high performance liquid chromatography, more preferably by liquid solid chromatography and size exclusion. In the liquid-solid chromatography, the chromatographic column is a C4 or C8 or C18 column, acetonitrile aqueous solution containing 0.05-0.1% (v/v) trifluoroacetic acid (TFA) is used as a mobile phase for gradient elution, and the detection wavelength is 210-280 nm. Obtaining the area of the coupling protein chromatographic peak by HPLC, and calculating the mass concentration C of the coupling protein according to a protein standard curveConjugated proteins。
The mass concentration of residual GnRH concatamer dimer in the conjugated protein sample is also requiredThis is because residual GnRH ligand dimer is converted to GnRH ligand single chain under subsequent reducing conditions, thereby interfering with the quantification of GnRH ligand single chain shedding from the conjugate protein. The quantitative determination of the residual GnRH ligand dimer preferably adopts high performance liquid chromatography, the chromatographic column is a C4 or C8 or C18 column, acetonitrile aqueous solution containing 0.05-0.1% (v/v) trifluoroacetic acid is used as a mobile phase for gradient elution, and the detection wavelength is 210-280 nm. Obtaining the area of the chromatographic peak of GnRH paralog dimer by HPLC, and calculating the mass concentration C of the residual GnRH paralog dimer in the coupled protein sample according to the standard curve of GnRH paralog dimerResidual GnRH ligand dimer。
During the quantitative determination of the coupled protein, the mass concentration of the residual GnRH concatamer dimer in the coupled protein sample can also be determined simultaneously: under the HPLC chromatographic condition, 2 chromatographic peaks are obtained by separating the GnRH parallel body dimer and the coupling protein, and C is obtained according to the chromatographic peak areas of the coupling protein and the GnRH parallel body dimer and the standard curve of the coupling protein and the GnRH parallel body dimer respectivelyConjugated proteinsAnd CResidual GnRH ligand dimer。
More preferably, in the high performance liquid chromatography quantitative determination process of the coupled protein, C4 is used as a chromatographic column, and the detection wavelength is 210 nm. The background interference peak generated under the detection wavelength of 210nm is less, and the accurate quantification of the coupled protein is facilitated.
Further preferably, the detection wavelength is 280nm in the quantitative measurement process by high performance liquid chromatography of the residual GnRH ligand dimer. Under the detection wavelength of 280nm, the baseline noise is lower, the number of the miscellaneous peaks is less, and the detection sensitivity is higher.
And (3) performing denaturation reduction treatment on the coupled protein, so that all GnRH paralog dimers on the coupled protein are subjected to reduction fragmentation, and half of free GnRH paralogs are released. Preferably, the step of performing denaturation reduction treatment on the coupling protein comprises: adding a protein reducing agent and a protein denaturant into the coupled protein sample solution, wherein the reaction temperature is 20-50 ℃, and the reaction time is 0.5-2 h.
Preferably, the amount concentration of the protein reducing agent substance is 10-100mmol/L, the amount concentration of the protein denaturant substance is 3-10mol/L, and a high-concentration protein denaturant and a reducing agent are adopted, so that the amino acid residues in the coupled protein are fully exposed, and the reduction reaction is more thorough.
Further preferably, the protein denaturant is selected from one or two of the following: urea, guanidine hydrochloride; the protein reducing agent is selected from one or more of the following: dithiothreitol (DTT), beta-mercaptoethanol (BME), tris (2-carboxyethyl) phosphine (TCEP), and Dithioerythritol (DTE).
The quantitative determination of the free GnRH ligand single strand obtained by the denaturation reduction treatment of the coupled protein preferably adopts high performance liquid chromatography, the chromatographic column is a C4 or C8 or C18 column, acetonitrile aqueous solution containing 0.05-0.1% (v/v) trifluoroacetic acid is adopted as a mobile phase for gradient elution, and the detection wavelength is 210-280 nm. Obtaining the chromatographic peak area of the free GnRH parallel body single chain by HPLC, and calculating the mass concentration C of the free GnRH parallel body single chain according to the standard curve of the GnRH parallel body single chainFree GnRH paralogue single chain。
More preferably, the detection wavelength is 280 nm. Under the detection wavelength of 280nm, the baseline noise is lower, the number of the miscellaneous peaks is less, and the detection sensitivity is higher.
Calculating the coupling ratio:
because the residual GnRH ligand dimer in the coupled protein sample can be converted into a GnRH ligand single chain under the reducing condition, the mass concentration of the GnRH ligand single chain actually dropped from the coupled protein is as follows:
Cconjugated protein split GnRH juxtaposition＝CFree GnRH paralogue single chain－CResidual GnRH ligand dimer；
Since still one molecule of GnRH ligand remains bound to the conjugate protein, the mass concentration of GnRH ligand dimer bound to the conjugate protein is:
Ccoupled protein-bound GnRH juxtapomer dimers＝2×CConjugated protein split GnRH juxtaposition＝2×(CFree GnRH paralogue single chain－CResidual GnRH ligand dimer)；
The mass concentration of the carrier protein before coupling is as follows:
Ccarrier proteins＝CConjugated proteins-CCoupled protein-bound GnRH juxtapomer dimers
The coupling mass ratio (mg/mg) between GnRH ligand dimer and carrier protein is then:
mcoupled protein-bound GnRH juxtapomer dimers/mCarrier proteins＝CCoupled protein-bound GnRH juxtapomer dimers/CCarrier proteins，
And the coupling molar ratio, namely the coupling rate, between the GnRH ligand dimer and the carrier protein is as follows:
compared with the prior art, the invention has the beneficial effects that:
the invention carries out denaturation reduction treatment on the coupled protein through a protein denaturant and a reducing agent, so that the GnRH concatemer coupled on the protein is subjected to reduction breakage, one part of GnRH concatemer is released, and the concentration of the GnRH concatemer in a free state and the concentration of the coupled protein are detected through HPLC (high performance liquid chromatography), thereby obtaining the coupling rate of the coupled protein. In addition, the concentration of the residual GnRH ligand dimer is detected by HPLC, and the concentration is deducted, so that the measurement of the coupling rate is more accurate.
The method for measuring the coupling rate of the GnRH parallel body dimer artificial antigen is simpler and more accurate.
Drawings
FIG. 1 is a BSA standard curve according to example 1 of the present invention;
FIG. 2 is a GnRH concatamer dimer standard curve of example 1 of the present invention;
FIG. 3 is a GnRH parallel body standard curve of example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be further described and illustrated below by means of specific examples and drawings, however, these embodiments are exemplary, the disclosure of the present invention is not limited thereto, and the drawings used herein are only for better illustrating the disclosure of the present invention and do not have a limiting effect on the scope of protection. Unless otherwise specified, the raw materials used in the following specific examples of the present invention are those commonly used in the art, and the methods used in the examples are those conventional in the art.
In the following examples, the coupling buffer was PBS buffer comprising 100mM sodium phosphate and 150mM NaCl, pH 7.2; GnRH concatamer dimer was obtained by the preparation method of synthetic example 1 of patent CN103554228A, and the structure is shown below:
example 1
1. Preparation of conjugated proteins
The samples were divided into A, B two groups, each containing 2mg of bovine serum albumin dissolved in 200. mu.L of the coupling buffer, and each containing 2mg of GnRH concatamer dimer dissolved in 500. mu.L of the same coupling buffer, and the two solutions were mixed. Carbodiimide (EDC) was dissolved in a small amount of deionized water, and 1mg and 2mg of EDC were added to the A, B mixed solution, respectively, and reacted at room temperature for 2 hours. And then, carrying out centrifugal ultrafiltration for multiple times by adopting an ultrafiltration centrifugal tube with the molecular weight cut-off of 10kD, wherein the displacement solution is still the coupling buffer solution, and obtaining GnRH-TD-BSA A and GnRH-TD-BSA B.
2. Quantitative detection of conjugated proteins
High performance liquid chromatography is adopted, and the conditions are as follows: c4 reverse phase chromatography column; mobile phase: phase A is 0.05% (v/v) aqueous TFA, phase B is acetonitrile; flow rate: 1 ml/min; column temperature: 40 ℃; gradient: 15% B at 0min, 25% B at 10min, 50% B at 20min, 80% B at 30min, 15% B at 30.1min, and 15% B at 40 min; detection wavelength: 210 nm.
Drawing a protein standard curve
Deionized water is used as a solvent to prepare BSA standard solutions of 0.0mg/ml, 0.1mg/ml, 0.2mg/ml, 0.5mg/ml, 1.0mg/ml, 2.0mg/ml and 5.0mg/ml respectively, detection is carried out according to the chromatographic conditions in turn,the sample injection amount is 20 uL; taking the concentration of the standard solution as an abscissa and the corresponding chromatographic peak area as an ordinate, drawing a standard curve, wherein fig. 1 is a BSA standard curve, a linear regression equation is that y is 17047x +681.53, and a correlation coefficient R2Is 0.9988.
The coupling protein concentrations of GnRH-TD-BSA A and GnRH-TD-BSA B are shown in Table 1.
3. Detection of residual GnRH ligand dimer
High performance liquid chromatography is adopted, and the conditions are as follows: c4 reverse phase chromatography column; mobile phase: phase A is 0.05% (v/v) aqueous TFA, phase B is acetonitrile; flow rate: 1 ml/min; column temperature: 40 ℃; gradient: 15% B at 0min, 25% B at 10min, 50% B at 20min, 80% B at 30min, 15% B at 30.1min, and 15% B at 40 min; detection wavelength: 280 nm.
Drawing GnRH paralogue dimer standard curve
Deionized water is used as a solvent, 0.0mg/ml, 0.1mg/ml, 0.2mg/ml, 0.5mg/ml, 1.0mg/ml, 2.0mg/ml and 5.0mg/ml GnRH concatemer dimer standard solutions are prepared respectively, and detection is carried out according to the chromatographic conditions in sequence, wherein the sample injection amount is 5 uL. Taking the concentration of the standard solution as an abscissa and the corresponding chromatographic peak area as an ordinate, drawing a standard curve, as shown in fig. 2, wherein the linear regression equation of the GnRH parallel body dimer is that y is 11018.5x +6.8008, and the correlation coefficient R is2Is 0.9994.
The mass concentrations of residual GnRH ligand dimer in GnRH-TD-BSA A and GnRH-TD-BSA B are shown in Table 1.
4. Denatured reduction of conjugated proteins
50uL of the coupled protein solution obtained in the step 1 is taken, 130uL of 9M urea solution is added respectively, 20uL of 1mol/L DTT solution is added, and the mixture is placed in a water bath at 50 ℃ for 30 min. And cooling to room temperature to be tested.
5. Detection of free GnRH parallels after reduction
High performance liquid chromatography is adopted, and the conditions are as follows: c4 reverse phase chromatography column; mobile phase: phase A is 0.05% (v/v) aqueous TFA, phase B is acetonitrile; flow rate: 1 ml/min; column temperature: 40 ℃; gradient: 15% B at 0min, 25% B at 10min, 50% B at 20min, 80% B at 30min, 15% B at 30.1min, and 15% B at 40 min; detection wavelength: 280 nm. And (4) carrying out sample injection analysis on the solution obtained in the step (4), wherein the sample injection amount is 20 uL.
Drawing GnRH paralogue standard curve
Deionized water is used as a solvent, 0.0mg/ml, 0.1mg/ml, 0.2mg/ml, 0.5mg/ml, 1.0mg/ml, 2.0mg/ml and 5.0mg/ml GnRH parallel body standard solutions are prepared respectively, and detection is carried out according to the chromatographic conditions in sequence, wherein the sample injection amount is 5 uL. Taking the concentration of the standard solution as an abscissa and the corresponding chromatographic peak area as an ordinate, drawing a standard curve, as shown in fig. 3, wherein the linear regression equation of the GnRH parallel body is that y is 1118x +15.083, and the correlation coefficient R is2Is 0.9997.
The mass concentrations of free GnRH ligand single chains in the solutions after denaturation reduction of GnRH-TD-BSA A and GnRH-TD-BSA B are shown in Table 1.
TABLE 1 Mass concentrations of coupling proteins GnRH-TD-BSA A and GnRH-TD-BSA B, residual GnRH concatemer dimer and free GnRH concatemer Single chain
6. Calculation of coupling ratio
The molecular weight of BSA was 66.4kDa, the molecular weight of GnRH ligand dimer was 5332Da, and the results of calculating the coupling ratio are shown in Table 2 below.
TABLE 2 coupling ratio of GnRH-TD-BSA A and GnRH-TD-BSA B
Example 2
1. Preparation of conjugated proteins
Two groups of C, D were divided, each 2mg Ovalbumin (OVA) was dissolved in 200. mu.L of coupling buffer, each 2mg GnRH concatamer dimer was dissolved in 500. mu.L of the same coupling buffer, and the two solutions were mixed. Carbodiimide (EDC) was dissolved in a small amount of deionized water, and 5mg and 10mg of EDC were added to the A, B mixed solution, respectively, and reacted at room temperature for 2.5 hours. And then, carrying out centrifugal ultrafiltration for multiple times by adopting an ultrafiltration centrifugal tube with the molecular weight cut-off of 10kD, wherein the replacement solution is still the coupling buffer solution, and obtaining GnRH-TD-OVA C and GnRH-TD-OVA D.
2. Quantitative detection of conjugated proteins
High performance liquid chromatography is adopted, and the conditions are as follows: c4 reverse phase chromatography column; mobile phase: phase A is 0.08% (v/v) TFA water solution, and phase B is acetonitrile; flow rate: 0.8 ml/min; column temperature: 40 ℃; gradient: 0min 20% B, 30% B at 10min, 55% B at 20min, 85% B at 30min, 20% B at 30.1min, and 20% B at 40 min; detection wavelength: 210 nm.
Drawing a protein standard curve
Using deionized water as a solvent, respectively preparing 0.0mg/ml, 0.1mg/ml, 0.2mg/ml, 0.5mg/ml, 1.0mg/ml, 2.0mg/ml and 5.0mg/ml OVA standard solutions, and sequentially detecting according to the chromatographic conditions, wherein the sample injection amount is 20 uL; and (4) drawing a standard curve by taking the concentration of the standard solution as an abscissa and taking the corresponding chromatographic peak area as an ordinate. The linear regression equation of the OVA standard curve is that y is 17283x +878.57, and the correlation coefficient R2Is 0.9991.
The coupling protein concentrations of GnRH-TD-OVA C and GnRH-TD-OVA D are shown in Table 3.
3. Detection of residual GnRH ligand dimer
High performance liquid chromatography is adopted, and the conditions are as follows: c8 reverse phase chromatography column; mobile phase: phase A is 0.08% (v/v) TFA water solution, and phase B is acetonitrile; flow rate: 0.8 ml/min; column temperature: 40 ℃; gradient: 0min 20% B, 30% B at 10min, 55% B at 20min, 85% B at 30min, 20% B at 30.1min, and 20% B at 40 min; detection wavelength: 280 nm.
Drawing GnRH paralogue dimer standard curve
Deionized water is used as a solvent, 0.0mg/ml, 0.1mg/ml, 0.2mg/ml, 0.5mg/ml, 1.0mg/ml, 2.0mg/ml and 5.0mg/ml GnRH concatemer dimer standard solutions are prepared respectively, and detection is carried out according to the chromatographic conditions in sequence, wherein the sample injection amount is 5 uL. Taking the concentration of the standard solution as an abscissa and the corresponding chromatographic peak area as an ordinate, drawing a standard curve, wherein the linear regression equation of GnRH parallel body dimer is that y is 11003x +195.2, and the correlation coefficient R2Is 0.9994.
The mass concentrations of residual GnRH side-by-side dimer in GnRH-TD-OVA C and GnRH-TD-OVA D are shown in Table 3.
4. Denatured reduction of conjugated proteins
50uL of the coupled protein solution obtained in the step 1 is taken, 150uL of 10M guanidine hydrochloride solution is respectively added, 30uL of 0.7mol/L beta-mercaptoethanol solution is added, and the mixture is placed in a water bath at the temperature of 45 ℃ for 1 h. And cooling to room temperature to be tested.
5. Detection of free GnRH parallels after reduction
High performance liquid chromatography is adopted, and the conditions are as follows: c8 reverse phase chromatography column; mobile phase: phase A is 0.08% (v/v) TFA water solution, and phase B is acetonitrile; flow rate: 0.8 ml/min; column temperature: 40 ℃; gradient: 0min 20% B, 30% B at 10min, 55% B at 20min, 85% B at 30min, 20% B at 30.1min, and 20% B at 40 min; detection wavelength: 280 nm. And (4) carrying out sample injection analysis on the solution obtained in the step (4), wherein the sample injection amount is 20 uL.
Drawing GnRH paralogue standard curve
Deionized water is used as a solvent, 0.0mg/ml, 0.1mg/ml, 0.2mg/ml, 0.5mg/ml, 1.0mg/ml, 2.0mg/ml and 5.0mg/ml GnRH parallel body standard solutions are prepared respectively, and detection is carried out according to the chromatographic conditions in sequence, wherein the sample injection amount is 5 uL. Taking the concentration of the standard solution as an abscissa and the corresponding chromatographic peak area as an ordinate, drawing a standard curve, setting the linear regression equation of the GnRH parallel body as y to be 1157x +38.618, and setting the correlation coefficient R2Is 0.9991.
The mass concentration of free GnRH ligand single chains in the solution after the GnRH-TD-OVA C and the GnRH-TD-OVA D are denatured and reduced is shown in Table 3.
TABLE 3 Mass concentrations of conjugated proteins, residual GnRH paralog dimers and free GnRH paralog single chains of GnRH-TD-OVA C and GnRH-TD-OVA D
6. Calculation of coupling ratio
The molecular weight of OVA is 44.3KDa, the molecular weight of GnRH ligand dimer is 5332Da, and the calculated coupling rate results are shown in the following table 4.
TABLE 4 coupling ratio of GnRH-TD-OVA C and GnRH-TD-OVA D
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (9)
1. A method for measuring the coupling rate of GnRH paralog dimer artificial antigen is characterized by comprising the following steps:
(1) quantitatively determining the coupling protein and the residual GnRH parallel body dimer in the coupling protein sample to obtain CConjugated proteinsAnd CResidual GnRH ligand dimer；
(2) Performing denaturation reduction treatment on the coupled protein to ensure that the GnRH paralleling body dimer is subjected to reduction breakage to free a GnRH paralleling body single chain, and quantitatively measuring the free GnRH paralleling body single chain to obtain CFree GnRH paralogue single chain；
(3) The coupling ratio was calculated as follows:
coupling ratio =
=
=
，
Where n represents the amount of substance, C represents the mass concentration and MW represents the molar mass.
2. The method of claim 1, wherein the quantitative determination of the coupling protein is selected from one or more of the following methods: high performance liquid chromatography, ultraviolet spectrophotometry, Coomassie brilliant blue binding, Folin-phenol reagent, biuret, BCA.
3. The method for determining the coupling rate of the GnRH ligand dimer artificial antigen as claimed in claim 1, wherein the quantitative determination of the coupling protein, the residual GnRH ligand dimer and the free GnRH ligand single chain is performed by high performance liquid chromatography, the chromatographic column is a C4 or C8 column, and mobile phase gradient elution is performed, wherein the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is 0.05-0.1% (v/v) trifluoroacetic acid aqueous solution, the mobile phase B is acetonitrile, and the detection wavelength is 210-280 nm.
4. The method of claim 3, wherein the quantitative determination of the conjugated protein is performed by high performance liquid chromatography, the column is C4, and the gradient elution is performed by using a mobile phase, wherein the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is 0.05-0.1% (v/v) trifluoroacetic acid in water, the mobile phase B is acetonitrile, and the detection wavelength is 210 nm.
5. The method of claim 3, wherein the detection wavelength is 280nm in the HPLC assay for the residual GnRH ligand dimer and the free GnRH ligand single chain.
6. The method for determining the coupling ratio of a GnRH paralog dimer artificial antigen as claimed in claim 1, wherein the step of subjecting the coupling protein to a denaturing reduction treatment comprises: adding a protein reducing agent and a protein denaturant into the coupled protein sample solution, wherein the reaction temperature is 20-50 ℃, and the reaction time is 0.5-2 h.
7. The method of determining the coupling ratio of a GnRH ligand dimer artificial antigen as claimed in claim 6, wherein the protein denaturant is selected from one or two of the following: urea, guanidine hydrochloride; the protein reducing agent is selected from one or more of the following: dithiothreitol, beta-mercaptoethanol, tris (2-carboxyethyl) phosphine, and dithioerythritol.
8. The method for determining the coupling rate of a GnRH ligand dimer artificial antigen as claimed in claim 1, wherein the GnRH ligand dimer is obtained by connecting GnRH ligand monomers through intermolecular disulfide bonds.
9. The method of determining the coupling rate of a GnRH paralog dimer artificial antigen as claimed in claim 1, wherein the carrier protein is selected from one or more of the following: bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, thyroglobulin, diphtheria toxoid, and tetanus toxoid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2019111406168 | 2019-11-20 | ||
| CN201911140616 | 2019-11-20 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110954706A CN110954706A (en) | 2020-04-03 |
| CN110954706B true CN110954706B (en) | 2020-10-02 |
Family
ID=69981659
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911285131.8A Active CN110954706B (en) | 2019-11-20 | 2019-12-13 | Method for measuring coupling rate of GnRH paralog dimer artificial antigen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110954706B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4608251A (en) * | 1984-11-09 | 1986-08-26 | Pitman-Moore, Inc. | LHRH analogues useful in stimulating anti-LHRH antibodies and vaccines containing such analogues |
| WO2001012216A1 (en) * | 1999-08-13 | 2001-02-22 | Proteinix Company | Heat shock fusion-based vaccine system |
| CN1508153A (en) * | 2002-12-18 | 2004-06-30 | 章孝荣 | Gonadotropin-releasing hormone juxtaposition dimer-egg albumin preparing method |
| CN103554265A (en) * | 2013-11-11 | 2014-02-05 | 宁波市三生药业有限公司 | Solid-phase synthesis method for gonadotropin-releasing hormone (GnRH) castration vaccine |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060121051A1 (en) * | 1998-02-19 | 2006-06-08 | Kenten John H | Heat shock fusion-based vaccine system |
| EP1564554A1 (en) * | 2004-02-12 | 2005-08-17 | Pepscan Systems B.V. | Method for the detection of early B cell populations in vaccine development |
-
2019
- 2019-12-13 CN CN201911285131.8A patent/CN110954706B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4608251A (en) * | 1984-11-09 | 1986-08-26 | Pitman-Moore, Inc. | LHRH analogues useful in stimulating anti-LHRH antibodies and vaccines containing such analogues |
| WO2001012216A1 (en) * | 1999-08-13 | 2001-02-22 | Proteinix Company | Heat shock fusion-based vaccine system |
| CN1508153A (en) * | 2002-12-18 | 2004-06-30 | 章孝荣 | Gonadotropin-releasing hormone juxtaposition dimer-egg albumin preparing method |
| CN103554265A (en) * | 2013-11-11 | 2014-02-05 | 宁波市三生药业有限公司 | Solid-phase synthesis method for gonadotropin-releasing hormone (GnRH) castration vaccine |
Non-Patent Citations (3)
| Title |
|---|
| A Sandwich Enzyme-Linked Immunoabsorbent Assay for Measurement of Gonadotropin-Releasing Hormone-Toxin Conjugates;Wei-Hsiung Yang et al;《American Journal of Reproductive Immunology》;20061231;第55卷;208-216 * |
| Oligomerization of G Protein-Coupled Receptors: Biochemical and Biophysical Methods;A.A. Kaczor et al;《Current Medicinal Chemistry》;20111231;第18卷(第30期);4606-4634 * |
| Structure, enzymatic stability and antitumor activity of sea lamprey GnRH-III and its dimer derivatives;Gabor Mezo et al;《Peptides》;20070104;第28卷;806-820 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110954706A (en) | 2020-04-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Vaughan et al. | Detection and purification of inhibin using antisera generated against synthetic peptide fragments | |
| AU784285B2 (en) | Methods and compositions for prolonging elimination half-times of bioactive compounds | |
| Yi-An et al. | Chemically unambiguous peptide immunogen: preparation, orientation and antigenicity of purified peptide conjugated to the multiple antigen peptide system | |
| Carraway et al. | Isolation, structure and biologic activity of chicken intestinal neurotensin | |
| CN119119238A (en) | Method for preparing GIP/GLP1 dual agonist | |
| MATSUURA et al. | A Human Chorionic Gotiadotropin-Specific Antiserum against Synthetic Peptide Analogs to the Carboxyl-Terminal Peptide of Its β-Subunit | |
| JPH0848636A (en) | Antibody | |
| Gutkowska | Radioimmunoassay for atrial natriuretic factor | |
| Goos et al. | Purification of gonadotropic hormone from the pituitary of the African catfish, Clarias gariepinus (Burchell), and the development of a homologous radioimmunoassay | |
| JP2980334B2 (en) | Peptides and polypeptides derived from rat submandibular gland, corresponding polyclonal and monoclonal antibodies, corresponding hybridomas and the use of these products for diagnostic, detection and therapeutic purposes | |
| JP2863527B2 (en) | Islet amyloid polypeptide | |
| CN110954706B (en) | Method for measuring coupling rate of GnRH paralog dimer artificial antigen | |
| GB2079289A (en) | Process for preparing antibody | |
| CN114989313B (en) | p-tau181 epitope peptide and its use in Alzheimer's disease detection | |
| JPS6089429A (en) | Manufacture of anti-hcg and anti-lh vaccine | |
| JPH08510210A (en) | Polyoxime compounds and their preparation | |
| EP0323769B1 (en) | Peptide structures, immunogens containing them, and their use in the control of fertility | |
| CN105622745A (en) | Derivative of amino acid fragment of angiotensin II, angiotensin II antigen and preparation method and application of angiotensin II antigen | |
| CN1916591A (en) | Ractopamine column, preparation method and usage | |
| Friedel et al. | A neurosecretory protein from Locusta migratoria | |
| CA1338782C (en) | Antibodies for use in determining human glycoalbumin | |
| CN115015545B (en) | Highly sensitive liver disease detection kit based on chitinase 3-like protein 1 | |
| CN105646660A (en) | Human HSP90[alpha] antigen epitope peptide, antigen and antibody, application of antibody and kit | |
| Trenkle et al. | An immunological investigation of porcine pituitary growth hormone | |
| Kinzel et al. | Synthesis of a functionalized high affinity mannose receptor ligand and its application in the construction of peptide‐, polyamide‐and PNA‐conjugates |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 159 Buzheng East Road, Wangchun Industrial Park, Haishu District, Ningbo City, Zhejiang Province, 315000 Patentee after: Ningbo Sansheng Biotechnology Co.,Ltd. Address before: 159 Buzheng East Road, Wangchun Industrial Park, Haishu District, Ningbo City, Zhejiang Province, 315000 Patentee before: NINGBO SANSHENG BIOLOGICAL TECHNOLOGY Co.,Ltd. |