EP0000103A1 - Purification of glycoproteins and use of glycoproteins to form antiserum compositions - Google Patents
Purification of glycoproteins and use of glycoproteins to form antiserum compositions Download PDFInfo
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- EP0000103A1 EP0000103A1 EP78300035A EP78300035A EP0000103A1 EP 0000103 A1 EP0000103 A1 EP 0000103A1 EP 78300035 A EP78300035 A EP 78300035A EP 78300035 A EP78300035 A EP 78300035A EP 0000103 A1 EP0000103 A1 EP 0000103A1
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- gel
- antigen
- subunit
- hcg
- human
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- 102000003886 Glycoproteins Human genes 0.000 title claims abstract description 24
- 108090000288 Glycoproteins Proteins 0.000 title claims abstract description 24
- 238000000746 purification Methods 0.000 title abstract description 4
- 239000000203 mixture Substances 0.000 title abstract description 3
- 238000000034 method Methods 0.000 claims abstract description 27
- 239000000427 antigen Substances 0.000 claims abstract description 24
- 102000036639 antigens Human genes 0.000 claims abstract description 24
- 108091007433 antigens Proteins 0.000 claims abstract description 24
- 230000003053 immunization Effects 0.000 claims abstract description 17
- 210000000952 spleen Anatomy 0.000 claims abstract description 10
- 229960004407 chorionic gonadotrophin Drugs 0.000 claims abstract description 8
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 7
- 159000000003 magnesium salts Chemical class 0.000 claims abstract description 7
- 239000000872 buffer Substances 0.000 claims description 15
- 241001465754 Metazoa Species 0.000 claims description 14
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 7
- 238000001962 electrophoresis Methods 0.000 claims description 7
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 claims description 7
- FWEOQOXTVHGIFQ-UHFFFAOYSA-N 8-anilinonaphthalene-1-sulfonic acid Chemical compound C=12C(S(=O)(=O)O)=CC=CC2=CC=CC=1NC1=CC=CC=C1 FWEOQOXTVHGIFQ-UHFFFAOYSA-N 0.000 claims description 5
- 238000005520 cutting process Methods 0.000 claims description 5
- 238000001502 gel electrophoresis Methods 0.000 claims description 3
- HKDVEBUNYUKMMN-UHFFFAOYSA-N 2-anilinonaphthalene-1-sulfonic acid Chemical compound C1=CC2=CC=CC=C2C(S(=O)(=O)O)=C1NC1=CC=CC=C1 HKDVEBUNYUKMMN-UHFFFAOYSA-N 0.000 claims description 2
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 claims description 2
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 claims description 2
- 102000003864 Human Follicle Stimulating Hormone Human genes 0.000 claims description 2
- 108010082302 Human Follicle Stimulating Hormone Proteins 0.000 claims description 2
- 102000009151 Luteinizing Hormone Human genes 0.000 claims description 2
- 108010073521 Luteinizing Hormone Proteins 0.000 claims description 2
- 102000011923 Thyrotropin Human genes 0.000 claims description 2
- 108010061174 Thyrotropin Proteins 0.000 claims description 2
- 229940040129 luteinizing hormone Drugs 0.000 claims description 2
- 229960004509 serum gonadotrophin Drugs 0.000 claims description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 238000002649 immunization Methods 0.000 abstract description 13
- 229920002401 polyacrylamide Polymers 0.000 abstract description 2
- 239000000499 gel Substances 0.000 description 32
- 102000004169 proteins and genes Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 229940088597 hormone Drugs 0.000 description 6
- 239000005556 hormone Substances 0.000 description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 5
- 239000004202 carbamide Substances 0.000 description 5
- 239000000975 dye Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
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- 239000007983 Tris buffer Substances 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000010254 subcutaneous injection Methods 0.000 description 3
- 239000007929 subcutaneous injection Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 230000009260 cross reactivity Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000035931 haemagglutination Effects 0.000 description 2
- PSFDQSOCUJVVGF-UHFFFAOYSA-N harman Chemical compound C12=CC=CC=C2NC2=C1C=CN=C2C PSFDQSOCUJVVGF-UHFFFAOYSA-N 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- CBUWTGCATVNMJE-UHFFFAOYSA-N 6,6-dimethylheptanal Chemical compound CC(C)(C)CCCCC=O CBUWTGCATVNMJE-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000002068 Glycopeptides Human genes 0.000 description 1
- 108010015899 Glycopeptides Proteins 0.000 description 1
- 206010021118 Hypotonia Diseases 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- 241000283977 Oryctolagus Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
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- 238000001949 anaesthesia Methods 0.000 description 1
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- 230000000890 antigenic effect Effects 0.000 description 1
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- 230000003115 biocidal effect Effects 0.000 description 1
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- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 150000001720 carbohydrates Chemical group 0.000 description 1
- 238000012754 cardiac puncture Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229940090343 human menopausal gonadotrophin Drugs 0.000 description 1
- -1 hydroxymethylene diamine Chemical class 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000005499 meniscus Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000036640 muscle relaxation Effects 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- DIHKMUNUGQVFES-UHFFFAOYSA-N n,n,n',n'-tetraethylethane-1,2-diamine Chemical compound CCN(CC)CCN(CC)CC DIHKMUNUGQVFES-UHFFFAOYSA-N 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 229940105631 nembutal Drugs 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000003393 splenic effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229960001005 tuberculin Drugs 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0006—Contraceptive vaccins; Vaccines against sex hormones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- This invention relates to the purification of glycoproteins and to immunization with glycoproteins.
- hCG Human chorionic gonadotrophin
- hCG and subunit are present to some extent as contaminants in these preparations. These antigenic contaminants will thus also produce antibodies when injected along with the primary antigen in immunization procedures. It is therefore desirable to further purify ⁇ -hCG and other glycoproteins prior to antibody production in order to produce a highly specific antiserum with low cross reactivity to interfering substances. Such an antiserum would make possible the design of more sensitive agglutination reactions with antigen sensitized carriers.
- the present invention is directed to provide purified forms of the beta subunit of hCG and other glycoproteins (or subunits or subfragments thereof). Accordingly the present invention provides a method for purifying a glycoprotein antigen or a subunit or subfragment thereof by gel electrophoresis characterised in that the antigen or subunit or subfragment is subjected to polyacrylamide gel electrophoresis in a tube containing an upper gel and a lower gel suspended in an electrophoretic apparatus having upper and lower buffer chambers wherein both the lower gel and the upper chamber buffer contain a fluorescent probe comprising the magnesium salt of 8-anilino-1-naphthalene-sulfonic acid, terminating the electrophoresis and cutting out the fluorescent boundaries corresponding to the antigen or subunit or subfragment.
- glycoprotein antigens and their subunits or subfragments may be purified by the method of this invention.
- glycoproteins besides hCG, would illustratively include human luteinizing hormone, human follicle stimulating hormone, human thyroid stimulating hormone, human menopausal gonadotrophin, pregnant mare's serum gonadotrophin, and carcino embryonic antigen.
- the preferred glycoprotein is the beta subunit of human chorionic gonadotrophin and thus.
- the invention provides a method for purifying the beta subunit of hCG which comprises subjecting the ⁇ -hCG subunit to polyacrylamide gel electrophoresis in a tube containing an upper gel and a lower gel suspended in an electrophoretic apparatus having upper and whose buffer chambers wherein both the lower gel and the upper chamber buffer.contain a fluorescent probe comprising the magnesium salt of 8-anilino-1-naphthalene-sulfonic acid, terminating the electrophoresis and cutting out the fluorescent boundaries corresponding to the ⁇ -hCG subunit located at the most anodic portion of the gel.
- the present invention also concerns to a method for purifying the beta subunit of human chorionic gonadotrophin which comprises subjecting the ⁇ -hCG subunit to polyacrylamide gel electrophoresis in a tube containing an upper gel and a lower gel suspended in an electrophoretic apparatus having an upper tank buffer and a lower tank buffer wherein the upper tank buffer contains a fluorescent probe consisting of the magnesium salt of B - anilino-1-naphthalene-sulfonic acid and a tracking dye, and, after terminating the electrophoretic run, cutting out the fluorescent boundaries corresponding to the ⁇ -hCG subunit.
- the invention also comprises the ⁇ -hCG produced by the above method.
- Human chorionic gonadotrophin a glycoprotein hormone may be obtained by extraction from the urine of pregnant women in the first trimester of pregnancy. Follouing a crude purification, the two subunits may be further purified and separated through a combination of ion exchange chromatography and sieve chromatography. In the last steps the alpha subunit is disassociated from the beta subunit in a 10M urea solution. The hCG/urea solution may then be sieve chromatographed twice on a urea impregnated column. The purified subunits may be eluted from the'column, the urea removed and the products lyophilized.
- the ⁇ -hCG obtained from the above described chromatographic procedures may then be purified by polyacrylamide gel electrophoresis according to the invention prior to immunization.
- the lower gel which is the separating gel contains about 5.4% polymerized acrylamide and about 0.5 mg percent of the magnesium salt of 8-anilino-1-naphthalene-sulfonic acid (referred to hereinafter as ASN). It will be understood by those skilled in the art that the amount of free acrylamide and ANS may vary with the individual glycoprotein hormone which is electrophoresed.
- the subunits isolated from the hormone complex reveal 3 components for hCG-a and 5 components for hCG- ⁇ .
- hCG- ⁇ was contaminated by extraneous substances which had the slowest anodic mobility.
- hCG and its a subunit and other glycoprotein hormones were also present in the hCG- ⁇ preparation.
- this invention also includes the method of producing an antiserum to a glycoprotein antigen, such as ⁇ -hCG, which comprises immunizing the host animal in the spleen and isolating the antiserum from the host animal.
- a glycoprotein antigen such as ⁇ -hCG
- the antigen utilized for the immunization is the highly purified ⁇ -hCG obtained from the polyacrylamide gel electrophesis and the spleen immunization is also followed by subcutaneous injection of the antigen.
- the antiserums of the present invention are useful immunological reagents.
- the ⁇ -hCG antiserum produced by utilizing the highly purified ⁇ -hCG obtained from polyacrylamide gel electrophoresis is highly specific and possesses very low cross reactivity to interfering antigens.
- an immunologic reagent preferably in lyophilized form in haemagglutination test method, more reliable results are obtained and more sensitive tests can be designed as described in our co-pending European Application No 78300034.2 filed concurrently herewith under Representative's reference AHP-6522 (corresponding to U.S.Serial Numbar B06563 filed 14th June, 1977).
- BIS is N,N'-methylene bis acrylamide
- TRIS is tris (hydroxymethylene diamine)
- TEMED is N,N,N',N'-tetra- ethylene diamine.
- the lower gel was prepared by, pouring a fresh solution containing 2.5ml of A, 5ml of C, 2.5ml of distilled deionized water with an equal volume of ammonium persulfate solution (0.14g/100ml) and 0.1ml of ANS (1 mg/ml solution of the Mg salt of 1-anilino-8-naphthalene sulfonic acid) into a gel tube having 0.6cm inner diameter and 9.5cm in length, the bottom end being temporarily sealed. Water was then layered on'top of the solution to eliminate any'meniscus. The gel formed after polymerizing for 1 ⁇ 2 hour and excess unpolymerized acrylamide on top was removed by washing with a solution containing 1 part B: 2 parts E: 6 parts water, and the bottom seal was then removed.
- An upper spacer gel was then prepared by layering or top of the lower gel a fresh solution containing 1 ml of B, 2 ml of D, 1 ml of E and 4 ml of F. Water was then layered over the upper gel solution and the gel was then formed by photopolymerization with fluorescent light for hour and removing excess water.
- the lower gel contains about 5.4% of unpolymerized acrylamide and 0.5 mg percent of ANS and the upper gel contains about 2.5% unpolymerized acrylamide.
- the gel tubes were marked at a distance of 5.5 cm from the top of the lower gel. Then 200 ⁇ g of the protein ( ⁇ -hCG, ⁇ -hCG, etc.) was layered on the upper spacer gel, the protein solution contains 2 mg of protein per ml of 0.15 M phosphate buffered saline at pH 7.4 containing 10% dextrose.
- Tank buffer (0.6g TRIS, 2.88g glycine, 5mg ANS in water q.s. to 1 litre and pH 8.3) was then layered over the sample.
- the tubes to be electrophoresed were placed in an electrophoretic apparatus having upper and lower chambers for receiving said tank buffer.
- the acrylamide tablets from the ANS stained gels, twelve in number, containing approximately a total of 2.4 mg hCG- ⁇ were suspended in 4 ml of phosphate buffer 0.15M, pH 7.4 and homogenized for 30 seconds. An equal volume of Freund's adjuvant (complete) was then added and the mixture emulsified. One ml of this emulsion contained approximately 300 micrograms of hCG- ⁇ .
- a left subcostal incision was then made exposing the spleen.
- the organ was gently grasped with two surgical sterile gauze compresses presoaked in saline at 37°C.
- the antigen was injected into the spleen using a sterile 1 ml tuberculin syringe with a No. 20 gauge needle. No more than 0.1 ml was injected per location. The total volume injected varied from 0.5-1.0 ml depending on the size of the spleen.
- the incision was closed using 00 silk sutures.
- the remaining antigen was injected subcutaneously in preshaven areas around the neck. The animal was then given a one millilitre injection, intraperitoneal with a suitable antibiotic.
- the animals were bled via the central ear artery using a No. 20 hypodermic needle and the antibody titre determined by microtitre haemagglutination. Each animal then received multiple subcutaneous injections of a total concentration of 300 ⁇ g/ml of hCG- ⁇ in Freund's adjuvant. This process of subcutaneous immunization and testing of serum extended over a period of 64 days with the immuno- gen being administered over 10-14 day intervals. Finally, the animals were exsanguinated by cardiac puncture and the antiserum tested for specificity.
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Endocrinology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Reproductive Health (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
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Abstract
A method is disclosed for the purification of glycoproteins, particularly the beta subunit of human chorionic gonadotrophin, by polyacrylamide gel electrohoresis using the magnesium salt of8-anilino-1- naphthalene-sulfonic acid as a fluorescent probe. Also disclosed is an antiserum com- position produced by using spleen Immunization of glycoprotein antigens.
Description
- This invention relates to the purification of glycoproteins and to immunization with glycoproteins.
- Human chorionic gonadotrophin (hereinafter referred to as hCG) is composed of two subunits; the α subunit is largely identical to the a subunit of TSH, FSH and LH; the B subunit also possesses areas which are homologous with these glycoprotein hormones as well as a 30 amino acid sequence that is not homologous. Many preparations of the β subunit of hCG obtained by chromatography of urea dissociated hCG exhibit microheterogeneity, Donini et al, Acta Endocr., 73: 133-145, 1973, i.e., they contain carbohydrate chains of different lengths and complexity. Besides other glycoprotein hormones or their metabolites, it is also possible that undisassociated hCG and subunit are present to some extent as contaminants in these preparations. These antigenic contaminants will thus also produce antibodies when injected along with the primary antigen in immunization procedures. It is therefore desirable to further purify β-hCG and other glycoproteins prior to antibody production in order to produce a highly specific antiserum with low cross reactivity to interfering substances. Such an antiserum would make possible the design of more sensitive agglutination reactions with antigen sensitized carriers.
- It was thought that gel electrophoresis would provide a suitable approach to obtain the desired, purified antigen. Such a procedure for the separation of human serum proteins is described by Davis in Ann. N.Y. Ac. Sci., 121:404-427 (1964). But, the staining procedure for localizing the separated protein requires prior fixation of the protein in acid which causes complete denaturation of the protein. Hartman et al in Anal. Biochem., 30: 391-394 (1969) attempted to provide a method of staining the electrophoresed protein with minimal denaturation by utilizing anilinonaphthalene sulfonate to stain the gels once they are removed from their tubes following electrophoresis. It has been found, however, that this latter procedure is unsatisfactory with hCG and no staining is observed. The further suggestion by .Harman to utilize HCl to intensify the stain is also counterproductive causing further denaturation of the protein.
- The present invention, therefore, is directed to provide purified forms of the beta subunit of hCG and other glycoproteins (or subunits or subfragments thereof). Accordingly the present invention provides a method for purifying a glycoprotein antigen or a subunit or subfragment thereof by gel electrophoresis characterised in that the antigen or subunit or subfragment is subjected to polyacrylamide gel electrophoresis in a tube containing an upper gel and a lower gel suspended in an electrophoretic apparatus having upper and lower buffer chambers wherein both the lower gel and the upper chamber buffer contain a fluorescent probe comprising the magnesium salt of 8-anilino-1-naphthalene-sulfonic acid, terminating the electrophoresis and cutting out the fluorescent boundaries corresponding to the antigen or subunit or subfragment.
- It is to be understood, besides hCG, that other glycoprotein antigens and their subunits or subfragments may be purified by the method of this invention. These glycoproteins, besides hCG, would illustratively include human luteinizing hormone, human follicle stimulating hormone, human thyroid stimulating hormone, human menopausal gonadotrophin, pregnant mare's serum gonadotrophin, and carcino embryonic antigen. However the preferred glycoprotein is the beta subunit of human chorionic gonadotrophin and thus. in a preferred aspect the invention provides a method for purifying the beta subunit of hCG which comprises subjecting the β-hCG subunit to polyacrylamide gel electrophoresis in a tube containing an upper gel and a lower gel suspended in an electrophoretic apparatus having upper and louer buffer chambers wherein both the lower gel and the upper chamber buffer.contain a fluorescent probe comprising the magnesium salt of 8-anilino-1-naphthalene-sulfonic acid, terminating the electrophoresis and cutting out the fluorescent boundaries corresponding to the β-hCG subunit located at the most anodic portion of the gel.
- The present invention also concerns to a method for purifying the beta subunit of human chorionic gonadotrophin which comprises subjecting the β-hCG subunit to polyacrylamide gel electrophoresis in a tube containing an upper gel and a lower gel suspended in an electrophoretic apparatus having an upper tank buffer and a lower tank buffer wherein the upper tank buffer contains a fluorescent probe consisting of the magnesium salt of B-anilino-1-naphthalene-sulfonic acid and a tracking dye, and, after terminating the electrophoretic run, cutting out the fluorescent boundaries corresponding to the β-hCG subunit. The invention also comprises the β-hCG produced by the above method.
- Human chorionic gonadotrophin, a glycoprotein hormone may be obtained by extraction from the urine of pregnant women in the first trimester of pregnancy. Follouing a crude purification, the two subunits may be further purified and separated through a combination of ion exchange chromatography and sieve chromatography. In the last steps the alpha subunit is disassociated from the beta subunit in a 10M urea solution. The hCG/urea solution may then be sieve chromatographed twice on a urea impregnated column. The purified subunits may be eluted from the'column, the urea removed and the products lyophilized.
- The β-hCG obtained from the above described chromatographic procedures may then be purified by polyacrylamide gel electrophoresis according to the invention prior to immunization.
- Preferably, the lower gel which is the separating gel contains about 5.4% polymerized acrylamide and about 0.5 mg percent of the magnesium salt of 8-anilino-1-naphthalene-sulfonic acid (referred to hereinafter as ASN). It will be understood by those skilled in the art that the amount of free acrylamide and ANS may vary with the individual glycoprotein hormone which is electrophoresed.
- The subunits isolated from the hormone complex reveal 3 components for hCG-a and 5 components for hCG-β.
- Besides these glycopeptides it was also found that hCG-β was contaminated by extraneous substances which had the slowest anodic mobility. In addition one cannot exclude the possibility that hCG and its a subunit and other glycoprotein hormones were also present in the hCG-β preparation.
- In order to circumvent the problem of contamination in hCG-β only material which had an electrophoretic mobility remote from that of other substances was excised from gels and used for immunization.
- While an antiserum to glycoproteins such as the highly purified β-hCG may be produced by many standard methods of immunization it is preferred according to this invention to produce the antiserum by immunizing the host animal in the spleen. Thus, this invention also includes the method of producing an antiserum to a glycoprotein antigen, such as β-hCG, which comprises immunizing the host animal in the spleen and isolating the antiserum from the host animal. Preferably the antigen utilized for the immunization is the highly purified β-hCG obtained from the polyacrylamide gel electrophesis and the spleen immunization is also followed by subcutaneous injection of the antigen.
- The most widely used immunization procedures for the induction of antibodies to proteins and other macromolecules employ foot pad-innoculations. This method has a major disadvantage in that the foot pad occasionally becomes infected and necrotic. Furthermore, the disease may become disseminated and prove fatal to the animal and a costly antigen may be lost. This has not been observed in the case of animals immunized via the splenic route.
- The potential use of spleen immunization followed by subcutaneous injection is reflected in the consistently high antibody titres produced to hCG-β in a short period of time.
- The antiserums of the present invention are useful immunological reagents. For example, the β-hCG antiserum produced by utilizing the highly purified β-hCG obtained from polyacrylamide gel electrophoresis is highly specific and possesses very low cross reactivity to interfering antigens. Thus, when used as an immunologic reagent preferably in lyophilized form in haemagglutination test method, more reliable results are obtained and more sensitive tests can be designed as described in our co-pending European Application No 78300034.2 filed concurrently herewith under Representative's reference AHP-6522 (corresponding to U.S.Serial Numbar B06563 filed 14th June, 1977).
- The invention may be further illustrated by reference to the following examples.
-
- The lower gel was prepared by, pouring a fresh solution containing 2.5ml of A, 5ml of C, 2.5ml of distilled deionized water with an equal volume of ammonium persulfate solution (0.14g/100ml) and 0.1ml of ANS (1 mg/ml solution of the Mg salt of 1-anilino-8-naphthalene sulfonic acid) into a gel tube having 0.6cm inner diameter and 9.5cm in length, the bottom end being temporarily sealed. Water was then layered on'top of the solution to eliminate any'meniscus. The gel formed after polymerizing for ½ hour and excess unpolymerized acrylamide on top was removed by washing with a solution containing 1 part B: 2 parts E: 6 parts water, and the bottom seal was then removed.
- An upper spacer gel was then prepared by layering or top of the lower gel a fresh solution containing 1 ml of B, 2 ml of D, 1 ml of E and 4 ml of F. Water was then layered over the upper gel solution and the gel was then formed by photopolymerization with fluorescent light for hour and removing excess water. The lower gel contains about 5.4% of unpolymerized acrylamide and 0.5 mg percent of ANS and the upper gel contains about 2.5% unpolymerized acrylamide.
- The gel tubes were marked at a distance of 5.5 cm from the top of the lower gel. Then 200µg of the protein (β-hCG, α-hCG, etc.) was layered on the upper spacer gel, the protein solution contains 2 mg of protein per ml of 0.15 M phosphate buffered saline at pH 7.4 containing 10% dextrose. Tank buffer (0.6g TRIS, 2.88g glycine, 5mg ANS in water q.s. to 1 litre and pH 8.3) was then layered over the sample. The tubes to be electrophoresed were placed in an electrophoretic apparatus having upper and lower chambers for receiving said tank buffer. To 400 ml of tank buffer in the upper chamber was added 1ml of 0.001% Bromophenol Blue as a tracking dye. The cathode was contacted to the top of the tube and the anode to the bottom. Electrophcresis was conducted at 4°C and a current of 2 milliamps per tube was applied till the. tracking dye reached the interface of the two gels and then a current of 3 milliamps per tube until the tracking dye reached the 5.5cm mark as described, a total of about 90 minutes. The tubes were removed from the apparatus and the gels were reamed out of the tubes. The portions of the gel-, which under long wave UV light corresponded for example, to β-hCG were excised immediately in the form of discs or tablets and frozen to avoid possible diffusion of the protein.
- For first establishing the location of the β-hCG after conducting the electrophoresis, samples of alpha and beta hCG were electrophoresed as described. The glycoproteins on the gels were then immediately fixed by staining with Amido Schwartz also known as Buffalo Black (0.2g/100ml of 7% acetic acid), fdr 90 minutes, then destained electrophoretically in 7% acetic acid. It was determined that the portions of β-hCG to be excised were in the far anodic regions of the gel.
- The acrylamide tablets from the ANS stained gels, twelve in number, containing approximately a total of 2.4 mg hCG-β were suspended in 4 ml of phosphate buffer 0.15M, pH 7.4 and homogenized for 30 seconds. An equal volume of Freund's adjuvant (complete) was then added and the mixture emulsified. One ml of this emulsion contained approximately 300 micrograms of hCG-β.
- White male New Zealand rabbits (2.5-3.0 kg) were fasted 16 hours before surgery. The animals were anaesthetized by slow intravenous injection of Nembutal, 60 mg/kg of body weight. Anaesthesia yes further continued by ether inhalation until good muscle relaxation was insured.
- A left subcostal incision was then made exposing the spleen. The organ was gently grasped with two surgical sterile gauze compresses presoaked in saline at 37°C. The antigen was injected into the spleen using a sterile 1 ml tuberculin syringe with a No. 20 gauge needle. No more than 0.1 ml was injected per location. The total volume injected varied from 0.5-1.0 ml depending on the size of the spleen. The incision was closed using 00 silk sutures. The remaining antigen was injected subcutaneously in preshaven areas around the neck. The animal was then given a one millilitre injection, intraperitoneal with a suitable antibiotic.
- Thirteen days following primary immunization with hCG-β, the animals were bled via the central ear artery using a No. 20 hypodermic needle and the antibody titre determined by microtitre haemagglutination. Each animal then received multiple subcutaneous injections of a total concentration of 300 µg/ml of hCG-β in Freund's adjuvant. This process of subcutaneous immunization and testing of serum extended over a period of 64 days with the immuno- gen being administered over 10-14 day intervals. Finally, the animals were exsanguinated by cardiac puncture and the antiserum tested for specificity.
Claims (10)
1. A method for purifying a glycoprotein antigen or a subunit or subfragment thereof by gel electrophoresis characterised in that the antigen or subunit or subfragment is subjected to polyacrylamide gel electrophoresis in a tube containing an upper gel and a lower gel suspended in an electrophpretic apparatus having upper and lower buffer chambers wherein both the lower gel and the upper chamber buffer contain a fluorescent probe comprising the magnesium salt of β-anilino-1-naphthalene-sulfonic acid, terminating the electrophoresis and cutting out the fluorescent boundaries corresponding to the antigen or subunit or subfragment.
2. A method as claimed in Claim 1 wherein the glycoprotein antigen is human chorionic gonadotrophin, human luteinizing hormone, human follicle stimulating hormone, human thyroid stimulating hormone, human menopausl gonadotrophin, pregnant mare's serum gonadotrophin or carcino embryonic antigen.
3. A method as claimed in Claim 1 for purifying the beta subunit of human chorionic gonadotrophin which comprises subjecting the β-hCG subunit to polyacrylamide gel electrophoresis in a tube containing an.upper gel and a lower gel suspended in an electrophoretic apparatus having upper and lower buffer chambers wherein both the lower gel and the upper chamber buffer contain a fluorescent probe comprising the magnesium salt of 8-anilino-1-naphthalene-sulfonic acid terminating the electrophoresis, and cutting out the fluorescent boundaries corresponding to.the β-hCG subunit located at the most anodic portion of the gel.
4. A method as claimed in Claim 3 wherein the louer gel contains about 5.4% polymerized acrylamide.
5. A method as claimed in Claim 3 or 4 wherein the concentration of the fluorescent probe in both the lower gel and upper chamber buffer is about 0.5 milligram per cent.
6. A method for producing an antiserum of a glycoprotein antigen which comprises immunizing a host animal to the antigen in the spleen and isolating the antiserum from the host animal.
7. A method as claimed in Claim 6 in which the antigen is injected subcutaneously after immunizing the host animal in the spleen and before isolating the antiserum.
8. A method as claimed in Claim 7 wherein the antigen is the glycoprotein or a subunit or subfragment thereof whenever obtained by the method claimed in Claim 1 or 2.
9. A method as claimed in Claim 7 wherein the antigen is the beta subunit of human chorionic gonadotrophin whenever obtained by the method claimed in any one of Claims 3 to 5.
10. A method as claimed in any one of Claims 6 to 9 wherein the host animal is a rabbit.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US05/806,562 US4123343A (en) | 1977-06-14 | 1977-06-14 | Purification of glycoproteins and immunization therewith |
US806562 | 1977-06-14 |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0000103A1 true EP0000103A1 (en) | 1978-12-20 |
Family
ID=25194312
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP78300035A Withdrawn EP0000103A1 (en) | 1977-06-14 | 1978-06-12 | Purification of glycoproteins and use of glycoproteins to form antiserum compositions |
Country Status (6)
Country | Link |
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US (1) | US4123343A (en) |
EP (1) | EP0000103A1 (en) |
JP (1) | JPS548718A (en) |
CA (1) | CA1114772A (en) |
GB (2) | GB1587776A (en) |
IT (1) | IT1096734B (en) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4271069A (en) * | 1978-09-26 | 1981-06-02 | Population Council, Inc. | Beta-hCG preparation and method |
US4321120A (en) * | 1980-03-19 | 1982-03-23 | Nardi Ronald V | Process for detecting proteins specific to hypertension in mammals |
ES8307897A1 (en) * | 1981-04-13 | 1983-08-01 | Hoechst Co American | Single incubation immunochemical assay for creatin phosphokinase MB. |
JP2804038B2 (en) * | 1988-02-24 | 1998-09-24 | 株式会社日立製作所 | Base sequence determination method |
JPH0752194B2 (en) * | 1988-03-05 | 1995-06-05 | 常廣 北川 | Immunoassays using antibodies against solid antigens |
US5225330A (en) * | 1988-08-01 | 1993-07-06 | The United States Of America As Represented By The Department Of Health And Human Services | Diagnostic kit and diagnostic method utilizing carbohydrate receptors |
US6319504B1 (en) | 1996-06-24 | 2001-11-20 | University Of Maryland Biotechnology Institute | Treatment and prevention of HIV infection by administration of derivatives of human chorionic gonadotropin |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2705696A (en) * | 1951-03-30 | 1955-04-05 | American Cyanamid Co | Production of antigens |
US3410839A (en) * | 1964-04-16 | 1968-11-12 | Rand Dev Corp | Immunochromatographic partition of soluble antigens |
US3789116A (en) * | 1970-12-09 | 1974-01-29 | Abbott Lab | Fluorescent labeled antibody reagent |
US3704282A (en) * | 1971-04-09 | 1972-11-28 | Sidney Spector | Catecholamine antigens and antibodies specific therefor |
US3699033A (en) * | 1971-04-26 | 1972-10-17 | Rashid A Zeineh | Electrophoresis and electrofocusing |
US4065445A (en) * | 1971-09-29 | 1977-12-27 | Behringwerke Aktiengesellschaft | Pregnancy-specific β1 -glycoprotein and process for isolating it |
US3795600A (en) * | 1973-03-23 | 1974-03-05 | Instrumentation Specialties Co | Electrophoresis and method apparatus |
US3948743A (en) * | 1975-04-14 | 1976-04-06 | Bio-Rad Laboratories | Method for gel electrophoresis |
US4030995A (en) * | 1976-07-13 | 1977-06-21 | Sigma Chemical Company | Alkaline phosphatase isoenzyme determination |
-
1977
- 1977-06-14 US US05/806,562 patent/US4123343A/en not_active Expired - Lifetime
-
1978
- 1978-05-26 GB GB38637/78A patent/GB1587776A/en not_active Expired
- 1978-05-26 GB GB23299/78A patent/GB1589108A/en not_active Expired
- 1978-06-02 CA CA304,668A patent/CA1114772A/en not_active Expired
- 1978-06-12 EP EP78300035A patent/EP0000103A1/en not_active Withdrawn
- 1978-06-14 JP JP7269578A patent/JPS548718A/en active Pending
- 1978-06-14 IT IT24571/78A patent/IT1096734B/en active
Non-Patent Citations (2)
Title |
---|
ANALYTICAL BIOCHEMISTRY, vol. 43, 564-574 (1971) * |
CHEMICAL ABSTRACTS, vol. 79, 39383h (1973) & Arch. Biochem. Biophys., 155(2), 478-9 (1973) * |
Also Published As
Publication number | Publication date |
---|---|
GB1587776A (en) | 1981-04-08 |
CA1114772A (en) | 1981-12-22 |
GB1589108A (en) | 1981-05-07 |
IT7824571A0 (en) | 1978-06-14 |
IT1096734B (en) | 1985-08-26 |
US4123343A (en) | 1978-10-31 |
JPS548718A (en) | 1979-01-23 |
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