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 PDF

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Publication number
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
Authority
EP
European Patent Office
Prior art keywords
gel
antigen
subunit
hcg
human
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.)
Withdrawn
Application number
EP78300035A
Other languages
German (de)
English (en)
French (fr)
Inventor
John Krupey
Ewald Frank Welchner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wyeth LLC
Original Assignee
American Home Products Corp
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by American Home Products Corp filed Critical American Home Products Corp
Publication of EP0000103A1 publication Critical patent/EP0000103A1/en
Withdrawn legal-status Critical Current

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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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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Endocrinology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Reproductive Health (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Virology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
EP78300035A 1977-06-14 1978-06-12 Purification of glycoproteins and use of glycoproteins to form antiserum compositions Withdrawn EP0000103A1 (en)

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)

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US (1) US4123343A (it)
EP (1) EP0000103A1 (it)
JP (1) JPS548718A (it)
CA (1) CA1114772A (it)
GB (2) GB1589108A (it)
IT (1) IT1096734B (it)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
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 (es) * 1981-04-13 1983-08-01 Hoechst Co American Un metodo de determinar la presencia de un antigeno en un medio liquido sospechoso de contenerlo.
JP2804038B2 (ja) * 1988-02-24 1998-09-24 株式会社日立製作所 塩基配列決定方法
JPH0752194B2 (ja) * 1988-03-05 1995-06-05 常廣 北川 固体抗原に対する抗体を用いるイムノアッセイ
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)

* Cited by examiner, † Cited by third party
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

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
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
US4123343A (en) 1978-10-31
IT7824571A0 (it) 1978-06-14
GB1589108A (en) 1981-05-07
CA1114772A (en) 1981-12-22
JPS548718A (en) 1979-01-23
GB1587776A (en) 1981-04-08
IT1096734B (it) 1985-08-26

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