DE10028725B4 - Primary structure of HNA-2a (formerly NB1) antigen - Google Patents
Primary structure of HNA-2a (formerly NB1) antigen Download PDFInfo
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- DE10028725B4 DE10028725B4 DE2000128725 DE10028725A DE10028725B4 DE 10028725 B4 DE10028725 B4 DE 10028725B4 DE 2000128725 DE2000128725 DE 2000128725 DE 10028725 A DE10028725 A DE 10028725A DE 10028725 B4 DE10028725 B4 DE 10028725B4
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- hna
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- acid sequence
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- nucleotide sequence
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- 102100024209 CD177 antigen Human genes 0.000 title claims abstract description 35
- 101000980845 Homo sapiens CD177 antigen Proteins 0.000 title claims abstract description 35
- 239000000427 antigen Substances 0.000 title claims abstract description 24
- 102000036639 antigens Human genes 0.000 title claims abstract description 24
- 108091007433 antigens Proteins 0.000 title claims abstract description 24
- 239000002773 nucleotide Substances 0.000 claims abstract description 11
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 11
- 229940039227 diagnostic agent Drugs 0.000 claims abstract 2
- 239000000032 diagnostic agent Substances 0.000 claims abstract 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 8
- 210000003714 granulocyte Anatomy 0.000 description 11
- 238000001514 detection method Methods 0.000 description 5
- 108090000288 Glycoproteins Proteins 0.000 description 4
- 102000003886 Glycoproteins Human genes 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 238000003149 assay kit Methods 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 208000004235 neutropenia Diseases 0.000 description 3
- 150000007523 nucleic acids Chemical group 0.000 description 3
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 206010055128 Autoimmune neutropenia Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 206010069351 acute lung injury Diseases 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003460 anti-nuclear Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000013321 baculovirus-insect cell expression system Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
Classifications
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- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70539—MHC-molecules, e.g. HLA-molecules
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Nucleotidsequenz umfassend eine Nucleotidsequenz SEQ ID NO 1, die ein HNA-2a-Antigen codiert, zur Verwendung als Diagnostikum, welches mit Alloantikörpern reagiert, die HNA-2a spezifisch sind.A nucleotide sequence comprising a nucleotide sequence of SEQ ID NO 1 encoding a HNA-2a antigen for use as a diagnostic agent which reacts with alloantibodies specific to HNA-2a.
Description
Es ist bekannt, daß das HNA-2a (NB1)-Merkmal zur Bildung von Allo- und Autoantikörpern führen kann. Diese Antikörper können zu Erkrankungen wie der neonatalen Immunneutropenie, der transfusionsassoziierten akuten Lungeninsuffizienz, der Immun neutropenie nach Knochenmarktransplantation, der Medikamentinduzierten Immunneutropenie sowie der Autoimmunneutropenie führen. Man weiß, daß es sich bei dem HNA-2a (NB1)-Antigen um ein auf einer Subpopulation der Granulozyten exprimiertes Glykoprotein handelt. Die dem Glykoprotein zugrundeliegende Nukleotid- und Aminosäuresequenz ist bisher nicht bekannt gewesen, weshalb die fragilen und nicht lagerbaren Granulozyten für die Antigen und Antikörper-Diagnostik benutzt werden müssen. Hierzu sind die Granulozyten zunächst arbeits- und zeitaufwendig aus dem Blut von Patienten (Antigennachweis) bzw. ausgesuchter, gesunder Spender (Antikörpernachweis) zu isolieren. Erst dann können die notwendigen Untersuchungen für die Diagnostik der oben angeführten Erkrankungen durchgeführt werden. Dies hatte zur Folge, daß diese Untersuchungen nur von wenigen Speziallaboratorien durchgeführt werden, was wiederum Postversand notwendig macht, bzw. der Patient wegen der Instabilität der Granulozyten zur Blutentnahme zum Speziallabor reisen muß. It is known that the HNA-2a (NB1) feature can lead to the formation of allo- and autoantibodies. These antibodies can lead to diseases such as neonatal immune neutropenia, transfusion-associated acute lung injury, immune neutropenia after bone marrow transplantation, drug-induced immune neutropenia and autoimmune neutropenia. It is known that the HNA-2a (NB1) antigen is a glycoprotein expressed on a subpopulation of the granulocytes. The nucleotide and amino acid sequence underlying the glycoprotein has not hitherto been known, which is why the fragile and non-storable granulocytes must be used for antigen and antibody diagnostics. For this purpose, the granulocytes are laborious and time consuming to isolate from the blood of patients (antigen detection) or selected, healthy donor (antibody detection). Only then can the necessary examinations for the diagnosis of the above-mentioned diseases be carried out. This had the consequence that these examinations are carried out by only a few specialized laboratories, which in turn makes mailing necessary, or the patient must travel to the special laboratory for blood collection because of the instability of the granulocytes.
Das Problem der Erfindung lag daher in der Aufklärung der kodierenden Nukleotidsequenz und der hieraus ableitbaren Aminosäuresequenz, um auf die Verwendung von humanen Granulozyten verzichten zu können. The problem of the invention was therefore to elucidate the coding nucleotide sequence and the amino acid sequence derivable therefrom in order to be able to dispense with the use of human granulocytes.
Zur Lösung dieses Problems wurden zunächst Granulozyten aus dem Blut HNA-2a (NB1) tragender Individuen separiert und anschließend lysiert. Mittels Immunaffinitätschromatographie wurde das Antigen aus dem Granulozytenlysat isoliert. Nach Auftrennung in der SDS-Polyacrylamid- Gelelektropherese wurde das isolierte Antigen mittels Immunoblot unter Verwendung von mehreren Referenzantikörpern als HNA-2a (NB1)-Antigen bestätigt. Die N-terminale Aminosäuresequenz wurde durch Edman-Abbau ermittelt und Primer zur Aufklärung der cDNA-Sequenz konstruiert. Nach Isolierung von m-RNA aus Granulozyten HNA-2a (NB1)-positiver Individuen wurde die cDNA-Sequenz mittels RACE-PCR ("rapid amplification of cDNA ends polymerase chain reaction) ermittelt. Die aus der cDNA-Sequenz abgeleitete Aminosäuresequenz enthielt Aminosäuresequenzen von 3 Trypsinspaltprodukten des immunchromatographisch isolierten Antigens. Die gefundene Nukleinsäuresequenz wurde in COS- und CHO-Säugerzellen zur Expression gebracht. Das exprimierte Glykoprotein (Antigen) wurde von monoklonalen und humanen Antikörpern sowohl in der Durchflußzytometrie als auch in immunchemischen Methoden wie dem Immunoblot und der Immunpräzipitation erkannt. Somit ist nach derzeit gültigen wissenschaftlichen Maßstäben nachgewiesen, daß die von uns aufgeklärte Nukleinsäuresequenz für das bekannte HNA-2a-(NB1)-Antigen codiert. Nukleinsäuresequenz-Datenbanksuchen zeigten, daß der Anfangsteil der Nukleotidsequenz bereits im Rahmen des Human Genome Projekts sequenziert worden waren, ohne jedoch die Bedeutung der Nukleotidsequenz als kodierende Sequenz für das HNA-2a (NB1)-Antigen zu erkennen, was sich auch in den Protein-Datenbanksuchen bestätigte. Bei dem HNA-2a (NB1)-Antigen handelte es sich somit um ein Glykoprotein der Granulozytenmembran mit bislang unbekannter Primärstruktur. To solve this problem, granulocytes were first separated from the blood HNA-2a (NB1) carrying individuals and then lysed. The antigen was isolated from the granulocyte lysate by immunoaffinity chromatography. After separation in SDS-polyacrylamide gel electropheresis, the isolated antigen was confirmed by immunoblot using several reference antibodies as HNA-2a (NB1) antigen. The N-terminal amino acid sequence was determined by Edman degradation and primers designed to elucidate the cDNA sequence. After isolation of mRNA from granulocytes of HNA-2a (NB1) -positive individuals, the cDNA sequence was determined by means of rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) .The amino acid sequence derived from the cDNA sequence contained amino acid sequences of The truncated nucleic acid sequence was expressed in COS and CHO mammalian cells The expressed glycoprotein (antigen) was recognized by both monoclonal and human antibodies in both flow cytometry and immunochemical techniques such as immunoblotting and immunoprecipitation Thus, according to current scientific standards, the nucleic acid sequence we have elucidated has been shown to encode the known HNA-2a (NB1) antigen, Nucleic acid sequence database searches showed that the initial part of the nucleotide sequence had already been sequenced in the human genome project , oh However, the importance of the nucleotide sequence as a coding sequence for the HNA-2a (NB1) antigen to recognize, which was also confirmed in the protein database searches. The HNA-2a (NB1) antigen was thus a granulocyte membrane glycoprotein of previously unknown primary structure.
Mit Hilfe der gewonnenen Nukleotidsequenz lassen sich einfache und sichere molekularbiologische Methoden, z. B. Polymerase-Ketten-Reaktionen, zur Antigenbestimmung entwickeln, die nur geringe Mengen lagerbare DNA und nicht fragile Granulozyten als Ausgangsmaterial erfordern. Die DNA-basierten Techniken erlauben im Gegensatz zu intakten Granulozyten auch einen Versand der Patientenblutproben. Weiterhin kann mit Hilfe der Rekombinationstechnologie das HNA-2a (NB1)-Antigen in großer Menge und ohne Verunreinigungen durch andere humane Glykoproteine hergestellt und in Antikörpernachweisverfahen, z. B. ELISA, eingesetzt werden. With the help of the obtained nucleotide sequence can be simple and safe molecular biology methods, eg. As polymerase chain reactions, to develop antigens that require only small amounts of storable DNA and not fragile granulocytes as starting material. The DNA-based techniques, unlike intact granulocytes, also allow patient blood samples to be shipped. Furthermore, by means of recombinant technology, the HNA-2a (NB1) antigen can be produced in large quantity and without contamination by other human glycoproteins, and can be detected in antibodies, e.g. B. ELISA used.
Sowohl die DNA-basierten Antigentests als auch die Antikörpernachweisverfahren unter Verwendung rekombinanter Antigene lassen sich in Form von fertig konfektionierten Testkits als einfache diagnostische Verfahren für Laboratorien vermarkten. Die Aufklärung der HNA-2a (NB1)-Primärstruktur führt somit zu einer entscheidenden Vereinfachung seiner Antigen- und Antikörper-Diagnostik. Both the DNA-based antigen tests and the antibody detection methods using recombinant antigens can be marketed in the form of ready-made test kits as simple diagnostic procedures for laboratories. The elucidation of the HNA-2a (NB1) primary structure thus leads to a significant simplification of its antigen and antibody diagnostics.
Die Ausführung der Testkits kann zum Beispiel analog zu den bereits im Handel befindlichen PCR-SSP-Testkits für die molekulare HLA-Antigenbestimmung erfolgen, wobei die Nukleotidsequenzen für die HLA-Primer durch HNA-2a (NB1)-Antigenspezifische Primersequenzen zu ersetzen wären. The execution of the test kits can be carried out, for example, analogously to the already commercially available PCR-SSP test kits for molecular HLA antigen determination, wherein the nucleotide sequences for the HLA primers would have to be replaced by HNA-2a (NB1) antigen-specific primer sequences.
Der Antikörpernachweistest könnte entsprechend einem ebenfalls bereits auf dem Markt befindlichen Testkit zum Nachweis antinukleärer Antikörper unter Verwendung rekombinat hergestellter Antigene konfiguriert werden. Hierzu könnte die rekombinate Herstellung des HNA-2a (NB1)-Antigens in größerem Stile ebenfalls im Baculovirus-Insektenzellen-Expressionssystem erfolgen. The antibody detection assay could be configured in accordance with an already on the market assay kit for detection of antinuclear antibodies using recombinantly produced antigens. For this purpose, the recombinant production of HNA-2a (NB1) antigen in a larger scale could also be carried out in the baculovirus insect cell expression system.
Zum Sequenzprotokoll wird auf die Offenlegungsschrift
Claims (6)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2000128725 DE10028725B4 (en) | 2000-06-09 | 2000-06-09 | Primary structure of HNA-2a (formerly NB1) antigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2000128725 DE10028725B4 (en) | 2000-06-09 | 2000-06-09 | Primary structure of HNA-2a (formerly NB1) antigen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| DE10028725A1 DE10028725A1 (en) | 2001-12-13 |
| DE10028725B4 true DE10028725B4 (en) | 2013-03-21 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DE2000128725 Expired - Lifetime DE10028725B4 (en) | 2000-06-09 | 2000-06-09 | Primary structure of HNA-2a (formerly NB1) antigen |
Country Status (1)
| Country | Link |
|---|---|
| DE (1) | DE10028725B4 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0223655D0 (en) | 2002-10-10 | 2002-11-20 | Mabtech Ab | Method of diagnosis |
| DE102008045696A1 (en) | 2008-09-04 | 2010-03-11 | Drk Blutspendedienst West Ggmbh | Granulocyte HNA-3a / b antigen |
-
2000
- 2000-06-09 DE DE2000128725 patent/DE10028725B4/en not_active Expired - Lifetime
Non-Patent Citations (1)
| Title |
|---|
| SKUBITZ, K.M. u.a.: Neutrophil-specific antigen NB1 is anchored via a glycosyl-phosphatidylinositol linkage. J. Leukoc. Biol. (1991) 49 (2) 163-71 * |
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| Publication number | Publication date |
|---|---|
| DE10028725A1 (en) | 2001-12-13 |
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| Date | Code | Title | Description |
|---|---|---|---|
| 8122 | Nonbinding interest in granting licences declared | ||
| 8127 | New person/name/address of the applicant |
Owner name: MAYER JUN., NORBERT, DR., 61462 KOENIGSTEIN, DE Owner name: FARKAS, FERENC, BUDAPEST, HU |
|
| 8127 | New person/name/address of the applicant |
Owner name: BUX, JUERGEN, DR., 58093 HAGEN, DE |
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| 8110 | Request for examination paragraph 44 | ||
| R018 | Grant decision by examination section/examining division | ||
| R020 | Patent grant now final |
Effective date: 20130622 |
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| R082 | Change of representative |
Representative=s name: ADVOTEC. PATENT- UND RECHTSANWAELTE, DE Representative=s name: ETL WABLAT & KOLLEGEN PATENT- UND RECHTSANWALT, DE |
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| R082 | Change of representative |
Representative=s name: ADVOTEC. PATENT- UND RECHTSANWAELTE, DE |
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| R081 | Change of applicant/patentee |
Owner name: BAG HEALTH CARE GMBH, DE Free format text: FORMER OWNER: BUX, JUERGEN, DR., 58093 HAGEN, DE |
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| R071 | Expiry of right |