DD286876A5 - METHOD FOR DETERMINING A LOCAL HUMAN MONOCYTE SURFACE ANTIGEN - Google Patents
METHOD FOR DETERMINING A LOCAL HUMAN MONOCYTE SURFACE ANTIGEN Download PDFInfo
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- DD286876A5 DD286876A5 DD33165089A DD33165089A DD286876A5 DD 286876 A5 DD286876 A5 DD 286876A5 DD 33165089 A DD33165089 A DD 33165089A DD 33165089 A DD33165089 A DD 33165089A DD 286876 A5 DD286876 A5 DD 286876A5
- Authority
- DD
- German Democratic Republic
- Prior art keywords
- monoclonal antibody
- romo
- surface antigen
- antibody
- enzyme immunoassay
- Prior art date
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- 239000000427 antigen Substances 0.000 title claims abstract description 14
- 102000036639 antigens Human genes 0.000 title claims abstract description 14
- 108091007433 antigens Proteins 0.000 title claims abstract description 14
- 210000001616 monocyte Anatomy 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 title claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims abstract description 7
- 210000001124 body fluid Anatomy 0.000 claims abstract description 7
- 239000010839 body fluid Substances 0.000 claims abstract description 7
- 238000003018 immunoassay Methods 0.000 claims abstract description 7
- 238000011895 specific detection Methods 0.000 claims description 3
- 102000004506 Blood Proteins Human genes 0.000 claims description 2
- 108010017384 Blood Proteins Proteins 0.000 claims description 2
- 238000011891 EIA kit Methods 0.000 claims description 2
- 239000007790 solid phase Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 7
- 102000003886 Glycoproteins Human genes 0.000 abstract 1
- 108090000288 Glycoproteins Proteins 0.000 abstract 1
- 238000000159 protein binding assay Methods 0.000 abstract 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 2
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- RPNUMPOLZDHAAY-UHFFFAOYSA-N Diethylenetriamine Chemical compound NCCNCCN RPNUMPOLZDHAAY-UHFFFAOYSA-N 0.000 description 1
- 101710181478 Envelope glycoprotein GP350 Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
Die Erfindung betrifft eine Bestimmungsmethode zum schnellen und zuverlaessigen Nachweis eines loeslichen humanen Monozytenoberflaechenantigens, vorzugsweise des Glykoproteins CD 14, in Koerperfluessigkeiten. Die quantitative Bestimmung des CD 14 erfolgt unter Verwendung des CD 14-spezifischen monoklonalen Antikoerpers RoMo 1, der mit einem Enzym direkt markiert ist. Es kommt ein Zwei-Seiten-Bindungstest (Enzymimmunoassay) zur Anwendung, in dem als Faengerantikoerper polyklonale Anti-CD 14-Antiseren oder andere CD 14-spezifische monoklonale Antikoerper eingesetzt werden, wenn diese eine andere Epitop-Spezifitaet als RoMo 1 besitzen. Anwendungsgebiet sind die Immunologie und Immundiagnostik.{Enzymimmunoassay; loesliches Monozytantigen, human; monoklonaler Antikoerper, markiert; Faengerantikoerper; Immundiagnostik}The invention relates to a method of determination for the rapid and reliable detection of a soluble human monocyte surface antigen, preferably the glycoprotein CD 14, in body fluids. The quantitative determination of the CD 14 is carried out using the CD 14-specific monoclonal antibody RoMo 1, which is directly labeled with an enzyme. A two-site binding assay (enzyme immunoassay) is used in which polyclonal anti-CD 14 antisera or other CD 14-specific monoclonal antibodies are used as the parent antibody if they have a different epitope specificity than RoMo 1. Applications include immunology and immunodiagnostics. {Enzyme immunoassay; soluble monocyte antigen, human; monoclonal antibody, labeled; Faengerantikoerper; Immunodiagnostics}
Description
Die Erfindung betrifft eine Methodo zur schnellen und zuverlässigen Bestimmung eines löslichen Monozytenoberflächenantigens in menschlichen Körperflüssigkeiten und findet bei Immuntests in der Immunologie und Immundiagnostik Anwendung.The invention relates to a method for the rapid and reliable determination of a soluble monocyte surface antigen in human body fluids and is used in immunoassays in immunology and immunodiagnostics.
Allgemein ist bekannt, daß sich immunologische Verfahren zum Nachweis von Proteinen an und in menschlichen Zellen und Körporflüssigkeiten mittels monoklonaler Antikörper als sehr zuverlässig erweisen. Für den spezifischen Nachweis der Antigene kommen in vielen Anwendungsfällen diese monoklonalen Antikörper bei Enzymimmunoassays zum Einsatz. Für die Bestimmung des Monozytenoberflächenantigeni CD14 sind verschiedene monoklonale Antikörper beschrieben. So werden durch Todd et al. in: Bernard et öl. (ed.), Leukocyte Typing, Springer Verlag, New York, 1984, S.424, der monoklonale Antikörper Leu M 3, durch Bazil et al. in: Eur. J. Immunol. 16,1984, S. 1563, der monoklonal Antikörper MEM18 und durch Goyert et al. in: McMichael et al. (ed.), Leukocyte Typing III, Springer Verlag, New York, 1987, S. 613, der monoklonale Antikörper My 4 für die Bestimmung von Monozyten in Zellsuspensionen genannt, da CD 14 ein monozytenspezifisches Antigen ist, das auf anderen Zellen nicht exprimiert wird. Aus der Literatur ist jedoch nicht zu entnehmen, daß sich dieso monoklonalen Antikörper auch für den Nachweis des löslichen Monozytenoberflächenantigens CD 14 eignen bzw. dessen Nachweis boschrieben wurde.It is generally known that immunological methods for the detection of proteins on and in human cells and body fluids by means of monoclonal antibodies prove to be very reliable. For the specific detection of antigens, these monoclonal antibodies are used in enzyme immunoassays in many applications. Various monoclonal antibodies are described for the determination of monocyte surface antigen CD14. Thus, Todd et al. in: Bernard et oil. (ed.), Leukocyte Typing, Springer Verlag, New York, 1984, p.424, the monoclonal antibody Leu M 3, by Bazil et al. in: Eur. J. Immunol. 16, 1984, p. 1563, the monoclonal antibody MEM18 and by Goyert et al. in: McMichael et al. (ed.), Leukocyte Typing III, Springer Verlag, New York, 1987, p. 613, called the monoclonal antibody My 4 for the determination of monocytes in cell suspensions, since CD 14 is a monocyte-specific antigen that is not expressed on other cells , However, it can not be deduced from the literature that these monoclonal antibodies are also suitable for the detection of the soluble monocyte surface antigen CD 14 or its detection has been described.
Das Ziel der Erfindung Ist ein kostengünstiges und r. »"!Massiges Verfahren zur Bestimmung des löslichen Monozytenoberflächenantigens CD14 in menschlichen Körperflüssigkeiten.The object of the invention is a cost-effective and r. Massive method for the determination of the soluble monocyte surface antigen CD14 in human body fluids.
Die Aufgabe der Erfindung besteht in einem Verfahren zur quantitativen Bestimmung des löslichen menschlichen Monozytenoberflächenantigens CD 14 in Körperflüssigkeiten unter Verwendung eine·» geeigneten monoklonalen Antikörpers, der keine Kreuzreaktivität zu anderen Serumproteinun zeigt. Die Aufgabe wird erfindungsgemäß dadurch gelöst, daß zur Bestimmung des löslichen Monozytenoberflächenantigens CD 14 in Körperflüssigkeiten der monoklonale Antikörper RoMo 1, welcher direkt markiert ist, als spezifischer Nachweisantikörper in einem Zwei-Seiten-Bindungs-Enzymimmunoassay dien», wobei als Fängerantikörpor an der festen Phaso des EIA-Kits polygonale Anti-CD 14-Antiseren oder andere CD 14-spezifischo monoklonale Antikörper, die eine andere Epitop-Spezifität als der monoklonalo RoMo 1 besitzen, verwendet werden. Der monoklonale Antikörper RoMo 1 ist in seiner Herstellung durch das DD-WP 255543 beschrieben. Als Fängerantikörper eignet sich dor CD 1'1-spozifische monoklonale Antikörper MEM 18. Der spezifischo monoklonalo Antikörper RoMo 1 ist vorteilhafterweise mit dem Enzym Peroxidase direkt markiert.The object of the invention is a method for the quantitative determination of the soluble human monocyte surface antigen CD 14 in body fluids using a suitable monoclonal antibody which shows no cross-reactivity with other serum proteins. The object is achieved in that the determination of the soluble Monozytenoberflächenantigens CD 14 in body fluids of the monoclonal antibody RoMo 1, which is directly labeled as a specific detection antibody in a two-side binding enzyme immunoassay dien », wherein as a catcher antibody on the solid Phaso the EIA kit polygonal anti-CD 14 antisera or other CD 14-specific monoclonal antibodies having a different epitope specificity than the monoclonal RoMo 1 can be used. The monoclonal antibody RoMo 1 is described in its preparation by DD-WP 255543. As a catcher antibody is dor CD 1'1-specific monoclonal antibody MEM 18. The specifico monoclonal antibody RoMo 1 is advantageously labeled with the enzyme peroxidase directly.
RoMo 1 be itrt eine hoho Affinität zu oinem Epitop auf CD 14, einem 53kDa-Olykoprotein. Dadurch ist gesichert, dali dio Verwendung dos monoklonalen Antikörpers RoMo 1 stets zu einem eindeutigen Nachweis des humanen Monozytenmembranantigens in loslicher Form führt.RoMo 1 has a high affinity for an epitope on CD 14, a 53kDa oligonucleotide. This ensures that the use of the monoclonal antibody RoMo 1 always leads to a clear detection of the human monocyte membrane antigen in a soluble form.
Dio Erfindung soll nachstehend an einem Ausführungsbeispiel näher erläutort werden. Daboi sind folgende Verfahrensschriuo vorgosehon.Dio invention will be explained in more detail below using an exemplary embodiment. Daboi are vorgosehon following procedures.
1. Feste Phase: Benutzung mit monoklonalem Antikörper MEM18 (10pg/ml) in 0,1 M Carbonatpuffer, pH 9,5, ca. 12-15h bei Raumtemperatur1. Solid phase: Use with monoclonal antibody MEM18 (10 μg / ml) in 0.1 M carbonate buffer, pH 9.5, at room temperature for approx. 12-15 h
danach ..'weimal waschen mit PBS plus 0,1 % Tween 20afterwards .. 'wash with PBS plus 0.1% Tween 20
2. Antigen (CDM)haltige Probe vsrdünnen in PBS plus 0,1% Tween 20,2. Antigen (CDM) -containing sample in PBS plus 0.1% Tween 20,
3. Konjugat: monoklonaler Antikörper, mit POD direkt markiert,3. Conjugate: monoclonal antibody directly labeled with POD,
verdünnen in PBS, 0,1 % Tween 20,5% Kälberserum, Inkubation ca. 2 h bei 4 Grad Celsius, danach zweimal waschen mit PBS plus 0,1 'oTweon 20dilute in PBS, 0.1% Tween 20.5% calf serum, incubate for approx. 2 h at 4 degrees Celsius, then wash twice with PBS plus 0.1% Tweon 20
-2- 286 87b-2- 286 87b
4. Reaktion des gebundenen POD mit chromogenem Substrat o-Phenylendiamin (0,5Mg/ml) in 0,1 M Citratpuffer, pH 5,0,4. Reaction of the bound POD with chromogenic substrate o-phenylenediamine (0.5 μg / ml) in 0.1 M citrate buffer, pH 5.0,
Dadurch ist es möglich, die untere Nachweisgrenze von 20ng/ml zu garantieren. Als Standard wird affinitätschromatographisch gereinigtes Antigen CD 14 verwendet.This makes it possible to guarantee the lower detection limit of 20ng / ml. The standard used is antigen 14 purified by affinity chromatography.
Ansteile des Fängerantikörpers MEM18 sind andere Antikörper, z.B. polyklonale Antiseren gegen das Antigen CD 14 von Kaninchen oder andere monoklonale Antikörper gegen CD 14 einsetzbar.Part of the capture antibody MEM18 are other antibodies, e.g. polyclonal antisera against the antigen CD 14 of rabbits or other monoclonal antibodies against CD 14 can be used.
Anstoile des POD-markierten RoMo 1 ist ein biotinylierter RoMo 1 einsetzbar. Die Nachweisstrecke besteht dabei aus Avidin-POD-Konjugat und gleichem Substrat.Anstoile the POD-labeled RoMo 1 is a biotinylated RoMo 1 can be used. The detection path consists of avidin-POD conjugate and the same substrate.
normal pathologischnormal pathological
Urin - jUrine - j
Liquor - jLiquor - j
Claims (1)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DD33165089A DD286876A5 (en) | 1989-08-10 | 1989-08-10 | METHOD FOR DETERMINING A LOCAL HUMAN MONOCYTE SURFACE ANTIGEN |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DD33165089A DD286876A5 (en) | 1989-08-10 | 1989-08-10 | METHOD FOR DETERMINING A LOCAL HUMAN MONOCYTE SURFACE ANTIGEN |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| DD286876A5 true DD286876A5 (en) | 1991-02-07 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DD33165089A DD286876A5 (en) | 1989-08-10 | 1989-08-10 | METHOD FOR DETERMINING A LOCAL HUMAN MONOCYTE SURFACE ANTIGEN |
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| Country | Link |
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| DD (1) | DD286876A5 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7465547B2 (en) | 2002-11-12 | 2008-12-16 | Mochida Pharmaceutical Co., Ltd. | Methods for detecting human low molecular weight CD14 |
| US7608684B2 (en) | 2002-11-12 | 2009-10-27 | Mochida Pharmaceuticals Co., Ltd. | Soluble CD14 antigen |
-
1989
- 1989-08-10 DD DD33165089A patent/DD286876A5/en not_active IP Right Cessation
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7465547B2 (en) | 2002-11-12 | 2008-12-16 | Mochida Pharmaceutical Co., Ltd. | Methods for detecting human low molecular weight CD14 |
| US7608684B2 (en) | 2002-11-12 | 2009-10-27 | Mochida Pharmaceuticals Co., Ltd. | Soluble CD14 antigen |
| US7901900B2 (en) | 2002-11-12 | 2011-03-08 | Mochida Pharmaceutical Co., Ltd. | Assay kit and antibody for human low molecular weight CD14 |
| US8124722B2 (en) | 2002-11-12 | 2012-02-28 | Mochida Pharmaceutical Co., Ltd. | Soluble CD14 antigen |
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