WO1991008488A1 - Peptide (alpha, at) inhibiteur de trypsine, immunoanalyse, anticorps monoclonal et hybridome - Google Patents
Peptide (alpha, at) inhibiteur de trypsine, immunoanalyse, anticorps monoclonal et hybridome Download PDFInfo
- Publication number
- WO1991008488A1 WO1991008488A1 PCT/SE1990/000768 SE9000768W WO9108488A1 WO 1991008488 A1 WO1991008488 A1 WO 1991008488A1 SE 9000768 W SE9000768 W SE 9000768W WO 9108488 A1 WO9108488 A1 WO 9108488A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lys
- antitrypsin
- monoclonal antibodies
- amino acid
- thr
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8121—Serpins
- C07K14/8125—Alpha-1-antitrypsin
Definitions
- the present invention relates to a decapeptide cor- responding to the amino acids 338-347 of Lys 342- ⁇ .-anti- trypsin (Z-protein), and the use of this decapeptide coupled to an inert carrier, in a method for obtaining a hybridoma cell line producing monoclonal antibodies speci- fically binding to only one epitope of Lys 342- ⁇ .-anti- trypsin comprising Lys 342.
- the invention further encom- passes monoclonal antibodies specifically binding to said epitope and ,a hybridoma cell line producing said mono ⁇ clonal antibodies.
- the invention is directed to a method for in vitro diagnosis of a. -antitrypsin defi ⁇ ciency in man, using said monoclonal antibodies, and an immunoassay kit for said diagnosis.
- ⁇ .-glycoprotein was isolated and desig ⁇ nated a.-antitrypsin ( ⁇ .AT) after its ability to bind tryp ⁇ sin had been observed (Schultze, H.E., Heide, K., and Haupt, H., .-Antitrypsin aus Humanserum. Klin. ochenschr. 40, 427-429 (1962)).
- ⁇ .-antitrypsin As the name . -antitrypsin indicates, it is the main component in the . -electrophoretic band of human plas aproteins and acts as an inhibitor of proteo- lytic enzymes. It is now known that ⁇ .-antitrypsin is one of the major extracellular serine protease inhibitors in man.
- CC-AT is the most important extra ⁇ cellular protease inhibitor whose main function is to in ⁇ activate elastase liberated from leucocytes and macro- phages. Therefore, ⁇ .-antitrypsin has also been referred to as ⁇ .-protease inhibitor (cc.PI) which better indicates its function (Johnson, D.R., Pannell, R.N., and Travies, J. , The molecular stoichiometry of trypsin inhibition by human ⁇ .-proteinase inhibitor. Biochem. Biophys. Res. Commun. 57, 584-589 (1974)).
- cc.PI ⁇ .-protease inhibitor
- ⁇ ..-antitrypsin has a polypeptide chain of 394 amino acid residues, and three carbohydrate side chains, giving a molecular weight of 51 kDa (Carrel, R.W., Jeppsson, J-0 and Laurell C-B. Structure and variation of human .-antitryp ⁇ sin. Nature (1982) 298, 329).
- oc.AT exhibits a considerable genetic variation with more than 60 variants (Jeppsson, J-0 and Franzen, B. Typing of Genetic variants of ⁇ .-antitryp- sin by Electrofocusing. Clin Chemistry (1982) 28, 219).
- variants or, in other words, alleles are designated by letters according to their electrophoretic mobility, primarily in starch gel electrophoresis and later according to their appearance in isoelectric focusing.
- Pi prote inhibitor
- Fagerhol M.K. Ser. Haemat. 1 , Suppl. 1, 153-161 (1968).
- the PI* M-allele is the most common and corresponds to a normal amount of c.AT in plasma. Of clinical interest are three alleles giving lower concentrations of ⁇ .AT in plasma.
- Variants of particular interest are the common Z- and S-mutants giving low concentrations of ⁇ ---antitrypsin in plasma; the product of the Z-allele gives concentra- tions corresponding to 15% of the concentration of the normal M-allele, while the product of the S-allele gives concentrations of 60% of the normal allele.
- Mixed hetero- zygotes, such as MZ, MS and SZ give proportionally chang ⁇ ed plasma levels.
- the genotypes giving such a deficiency of ⁇ . -antitrypsin in plasma which is an evident health hazard, are PiZZ (15% of normal concentration), PiSZ (35% of normal concentration) and combinations with an uncommon 0 (-)gene, such as PiZ- (8% of normal concentration).
- the difference in charge between M-, S- and Z-pro- teins depends on amin ⁇ acid substitutions. Glutamic acid in position 342 of the M-protein is replaced by lysine in the Z-protein, and glutamic acid in position 264 of the M-protein is replaced by valine in the S-protein.
- Deficiency in, or rather low concentration of, the plasmaprotein .-antitrypsin is correlated with the pre ⁇ sence of lung emphysema (Eriksson, S. Acta Med. Scand. 175, 197-205 (1964)) and certain liver diseases (Sharp. H.L. Hosp. Pract. 6. 83-96 (1971)).
- augmentation therapy with isolated human a.-antitrypsin might be offered to prevent chronic de ⁇ struction of the lung (Hubbard R.C. et al. Biochemical efficacy and safety of monthly augmentation therapy for ⁇ 1 -antitrypsin deficiency. JAMA (1988) 260, 1259).
- Diagnosing .-antitrypsin deficiency has hitherto been done by quantitative determination of cc.-antitrypsin in plasma, supplemented with electrofocusing at low le ⁇ vels. This is a relatively laborious and complicated tech- nique. Therefore, there is a need for a simpler and faster technique allowing routine analysis of blood samples with an aim to locate at an early stage individuals having a.
- Wallmark A. and co-workers produced an antibody against cc..-antitrypsin isolated from the liver of a PiZZ indivi ⁇ dual and used said antibody in ELISA for the detection of PiZ gene carriers (Wallmark A., et al. Monoclonal antibody specific for the mutant PiZ cc.-antitrypsin and its appli- cation in an ELISA procedure for identification of PiZ gene carriers. Proc.Natl.Acad.Sci. USA, Vol. 81, pp 5690-5693 (1984)).
- the present invention is based on a decapeptide cor ⁇ responding to amino acids 338-347 in the amino acid se ⁇ quence of the Z-protein. With the aid of this decapeptide, it has been possible to produce monoclonal antibodies spe- cifically binding to only one epitope of Lys 342 - .-anti- trypsin comprising Lys 342. Said monoclonal antibodies have then been used in a method of in vitro diagnosis of a. - antitrypsin deficiency, that is the presence in the blood of Lys 342- ⁇ .-antitrypsin, in man, and distinguishing between a heterozygote and a homozygote PiZ gene carrier.
- One aspect of the invention is directed to a decapep ⁇ tide having the amino acid sequence
- X 1 and X2 each represent an optional coupling-faci ⁇ litating amino acid residue and Y represents -NH 9 or -OH.
- the peptide according to the invention can be produced by suitable known methods for synthesising peptides; for instance, an amino acid can be coupled to the next one to form a peptide bond, either in solution or by so-called solid phase technique.
- the peptide according to the invention can be synthesised either in amide form or as a free acid, which is indicated by Y representing -NH- or -OH.
- Another aspect of the invention is directed to mono- clonal antibodies specifically binding to only one epitope of Lys 342 -a. -antitrypsin comprising Lys342.
- the expression "Lys 342 - . -antitrypsin” is equivalent to the expression
- the mono ⁇ clonal antibodies should specifically bind to only one epitope of the Z-protein, which epitope comprises the amino acid residue -Lys- which is in position 342 in the Z-protein and which is considered to be the determining amino acid residue distinguishing the Z-protein from the most commonly existing M-protein.
- Yet another aspect of the invention is directed to a hybridoma cell line producing monoclonal antibodies speci-
- a further aspect of the invention is directed to a method for producing a hybridoma cell line producing mo ⁇ noclonal antibodies specifically binding to only one epitope of Lys 342-cc. -antitrypsin comprising Lys342. This method consists in that spleen cells derived from a mouse previously immunised with a decapeptide having the amino acid sequence
- X 1 and X2 each represent an optional coupling-faci ⁇ litating amino acid residue and Y represents - H 2 or -OH, coupled to an inert carrier, are fused with myeloma cells, followed by selection of the hybridoma cells producing said monoclonal antibodies.
- Spleen cells from other mam ⁇ mals or poultry might be used, but in practice use is made of spleen cells from mouse for producing hybridoma cell lines.
- the decapeptide coupled to an inert carrier and used for immunising a mouse can be coupled to any inert carrier usable for immunisation purposes.
- the decapeptide may be coupled to an inert protein, such as bovine serum albumin or hemo- cyanin, or plastic particles.
- an inert protein such as bovine serum albumin or hemo- cyanin, or plastic particles.
- the fusion of the spleen cells with myeloma cells can be effected in conventional manner, and a known myeloma cell line can be used.
- the selection of the hybridoma cell lines producing the desired monoclonal antibodies can be effected in known manner.
- a further aspect of the invention is directed to a method for in vitro diagnosis of cc, -antitrypsin deficien-
- 342 cy that is the presence in the blood of Lys -cc -anti ⁇ trypsin, in man, and distinguishing between a heterozygote and a homozygote PiZ gene carrier.
- This method involves subjecting a blood sample from a human to an immunoassay using monoclonal antibodies specifically binding to only one epitope of Lys 342-cc. -antitrypsin comprising Lys342 as diagnostic antibody.
- any suitable immuno- assay using a diagnostic antibody can be used for the purposes of the invention, especially suitable methods being radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA) and Time-Resolved-Fluorescence.
- the analysed presence of antigen-antibody complexes confirms the pre- sence of Lys 342-cc.-antitrypsin in the blood sample, that is cc-antitrypsin deficiency in the human from whom the blood sample was taken and analysed and the amount of said complexes revealing if said human is a homozygote or heterozygote PiZ gene carrier. If no antigen-antibody com- plex is detected by the method according to the invention, this means that the blood sample does not contain any Z-protein.
- the analysed presence of antigen-antibody com ⁇ plexes also indicates whether the blood sample contains homozygous protease inhibitor ZZ or heterozygous protease inhibitor comprising only one Z when the concentration is compared with references.
- the currently most preferred immunoassay techniques are enzyme linked immunosorbent assay and Time-Resolved-Fluorescence.
- a final aspect of the invention is directed to an im ⁇ munoassay kit for diagnosing ⁇ .-antitrypsin deficiency in man, which kit comprises a container holding monoclonal antibodies specifically binding to only one epitope of
- Lys 342-c -antitrypsin comprising Lys342, in an inert medium.
- the inert medium may be e.g. a phosphate buffer or a physiological saline solution.
- the immunoassay kit may optionally contain further reagents, diluents etc. in accordance with the immunoassay method to be used. Preparation of peptide coupled to inert carrier A synthetic peptide comprising 11 amino acids was ob ⁇ tained from Peninsula Laboratories, San Fransisco, Cali ⁇ fornia:
- hemocyanin H-Leu-Thr-Ile-Asp-Lys-Lys-Gly-Thr-Gly-Ala-Cys-OH
- This peptide was coupled C-terminally to an inert carrier, viz. hemocyanin.
- the hemocyanin Sigma
- MBS maleimidobenzoyl-n-hydroxy succinic acid ester
- Activated hemocyanin was gel-fil- tered on a G-25 column in 10 mM sodium phosphate, pH 7.0.
- the protein fraction from the gel filtration was mixed with the peptide under agitation for 3 h at room tempera ⁇ ture and was thereafter dialysed against 50 mM Tris-HCl, 0.15 M NaCl, pH 7.5. The resulting coupling product was used for immunisation.
- the fusion was carried out in polyethylene glycol/dimethyl sulphoxide/water in proportions 45:7:48.
- the fused cells
- 5x10 cells of a master clone were injected intrape ⁇ ritoneally in Balb/c mice after an injection of 0.5 ml Pristine (2,6,10,14-tetramethylpentadecane. After 8 days, all ascites fluid was aspirated, and the resulting immu- noglobulins were precipitated with 50% ammonium sulphate. The precipitate was dissolved in water and dialysed against 50 mM Tris-HCl, pH 7.4, and purified on a Mono-Q (Pharmacia) column equilibrated with 20 mM Tris-HCl, pH 8.0. Immunoglobulin fractions, which had been detected with agarose electrophoresis, were pooled and frozen at -70°C. Detection with ELISA technique
- Monoclonal antibodies according to the invention were labelled in known manner with the earth metal Europium giv ⁇ ing a strong fluorescence of short duration. (See e.g. Hemmila, I. et al, Europium as a Label in Time-Resolved Immunofluorometric Assays, Analytical Biochemistry 137, 335-343 (1984)). Wells in a microtiter plate were incubated at pH 9.6 with a polyclonal antibody against x.-antitrypsin derived from rabbit (Dako No. A 012). To avoid unspecific adsorp ⁇ tion, the remaining surfaces were blocked with 0.1% Tween 20 in phosphate buffer, pH 8.0.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
Décapeptide de la séquence d'acides aminés H-X1-Leu-Thr-Ile-Asp-Lys-Lys-Gly-Thr-Gly-Ala-X2-Y, dans laquelle X1 et X2 représentent chacun un reste d'acides aminés facilitant un couplage facultatif, et Y représente NH¿2? ou OH. Sont également décrits des anticorps monoclonaux se liant spécifiquement à uniquement un épitope de Lys?342¿-α¿1?-antitrypsine, par exemple une protéine Z, comprenant Lys?342¿; une lignée cellulaire d'hybridomes produisant lesdits anticorps monoclonaux; ainsi qu'un procédé de préparation de la lignée cellulaire d'hybridomes. Sont églament décrits un procédé de diagnostic in vitro de carence en α¿1?-antitrypsine, par exemple la présence dans le sang de Lys?342¿-α¿1?-antitrypsine, chez l'homme, ainsi qu'un kit d'immunoanalyse permettant ledit diagnostic.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE8904007-5 | 1989-11-28 | ||
SE8904007A SE8904007D0 (sv) | 1989-11-28 | 1989-11-28 | Monoklona antikroppar och diagnostiseringsmetod |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1991008488A1 true WO1991008488A1 (fr) | 1991-06-13 |
Family
ID=20377617
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/SE1990/000768 WO1991008488A1 (fr) | 1989-11-28 | 1990-11-23 | Peptide (alpha, at) inhibiteur de trypsine, immunoanalyse, anticorps monoclonal et hybridome |
Country Status (5)
Country | Link |
---|---|
AU (1) | AU7896891A (fr) |
IE (1) | IE904273A1 (fr) |
IL (1) | IL96369A0 (fr) |
SE (1) | SE8904007D0 (fr) |
WO (1) | WO1991008488A1 (fr) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006019906A1 (fr) * | 2004-07-14 | 2006-02-23 | The Regents Of The University Of California | Biomarqueur pour detecter de maniere precoce un cancer des ovaires |
US7670792B2 (en) | 2004-07-14 | 2010-03-02 | The Regents Of The University Of California | Biomarkers for early detection of ovarian cancer |
US9241976B2 (en) | 2011-08-29 | 2016-01-26 | The Regents Of The University Of California | Use of HDL-related molecules to treat and prevent proinflammatory conditions |
US9487575B2 (en) | 2004-07-14 | 2016-11-08 | The Regents Of The University Of California | Compositions and methods for treatment of gynecologic cancers |
US9488655B2 (en) | 2004-07-14 | 2016-11-08 | The Regents Of The University Of California | Biomarkers for detection of early- and late-stage endometrial cancer |
KR101753447B1 (ko) * | 2010-09-24 | 2017-07-03 | 그리폴스 테라퓨틱스 인코포레이티드 | 면역크로마토그래피 장치, 방법 및 키트 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989005308A1 (fr) * | 1987-12-03 | 1989-06-15 | Gesellschaft Für Biotechnologische Forschung Mbh ( | Anticorps monoclonaux et lignees de cellules d'hybridomes utilisees pour les obtenir |
-
1989
- 1989-11-28 SE SE8904007A patent/SE8904007D0/xx unknown
-
1990
- 1990-11-16 IL IL96369A patent/IL96369A0/xx unknown
- 1990-11-23 WO PCT/SE1990/000768 patent/WO1991008488A1/fr unknown
- 1990-11-23 AU AU78968/91A patent/AU7896891A/en not_active Abandoned
- 1990-11-27 IE IE427390A patent/IE904273A1/en unknown
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989005308A1 (fr) * | 1987-12-03 | 1989-06-15 | Gesellschaft Für Biotechnologische Forschung Mbh ( | Anticorps monoclonaux et lignees de cellules d'hybridomes utilisees pour les obtenir |
Non-Patent Citations (4)
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006019906A1 (fr) * | 2004-07-14 | 2006-02-23 | The Regents Of The University Of California | Biomarqueur pour detecter de maniere precoce un cancer des ovaires |
US7670792B2 (en) | 2004-07-14 | 2010-03-02 | The Regents Of The University Of California | Biomarkers for early detection of ovarian cancer |
US8323915B2 (en) | 2004-07-14 | 2012-12-04 | The Regents Of The University Of California | Biomarkers for early detection of ovarian cancer |
US9110083B2 (en) | 2004-07-14 | 2015-08-18 | The Regents Of The University Of California | Biomarkers for early detection of ovarian cancer |
US9487575B2 (en) | 2004-07-14 | 2016-11-08 | The Regents Of The University Of California | Compositions and methods for treatment of gynecologic cancers |
US9488655B2 (en) | 2004-07-14 | 2016-11-08 | The Regents Of The University Of California | Biomarkers for detection of early- and late-stage endometrial cancer |
KR101753447B1 (ko) * | 2010-09-24 | 2017-07-03 | 그리폴스 테라퓨틱스 인코포레이티드 | 면역크로마토그래피 장치, 방법 및 키트 |
US9241976B2 (en) | 2011-08-29 | 2016-01-26 | The Regents Of The University Of California | Use of HDL-related molecules to treat and prevent proinflammatory conditions |
US10208103B2 (en) | 2011-08-29 | 2019-02-19 | The Regents Of The University Of California | Use of HDL-related molecules to treat and prevent proinflammatory conditions |
Also Published As
Publication number | Publication date |
---|---|
SE8904007D0 (sv) | 1989-11-28 |
IE904273A1 (en) | 1991-06-05 |
AU7896891A (en) | 1991-06-26 |
IL96369A0 (en) | 1991-08-16 |
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