DD230882B1

DD230882B1 - METHOD FOR THE PRODUCTION OF MONOCLONAL ANTIBODIES FOR A MONOCYTE ANTIGEN

Info

Publication number: DD230882B1
Application number: DD25465183A
Authority: DD
Inventors: Helmut Fiebig; Ingrid Behn; Hartmut Kupper; Herwart Ambrosius
Original assignee: Univ Leipzig
Priority date: 1983-09-08
Filing date: 1983-09-08
Publication date: 1987-06-17
Also published as: DD230882A1

Description

Zellen mittels FITC-markiertem Ziege-anti-Mausimmunglobulin-Serum bestimmt. Mit der Methode der indirekten Immunfluoreszenz wird ausgeschlossen, daß u.U. Hybridome ausgewählt werden, deren Antikörper mit B-Lymphozyten, T-Lymphozyten und Erythrozyten reagieren.Cells determined by FITC-labeled goat anti-mouse immunoglobulin serum. With the method of indirect immunofluorescence is ruled out that u.U. Hybridomas are selected whose antibodies react with B lymphocytes, T lymphocytes and erythrocytes.

Das durch diese Selektionen erhaltene Hybridom H-BL-M/G wird rekloniert und ein Subklon wird selektioniert, die Antikörper, die zur Bestimmung humaner Monozyten, Granulozyten und Nullzellen geeignet sind, produziert. Diese überraschende und vorteilhafte Eigenschaft eröffnet sehr günstige diagnostische Möglichkeiten.The hybridoma H-BL-M / G obtained by these selections is recloned and a subclone is selected which produces antibodies suitable for the determination of human monocytes, granulocytes and null cells. This surprising and advantageous property opens up very favorable diagnostic possibilities.

Die Herstellung des Antikörpers erfolgt erfindungsgemäß dergestalt, daß antikörperproduzierende Hybridomzellen H-BL-M/G in einem Kulturmedium mitSerumzuskatzin Massenkultur aufgezogen werden. Nach entsprechender Kultivierung in CO₂-haitiger Atmosphäre wird das nunmehr antikörperenthaltende Kulturmedium mit 50% gesättigtem (NH₄J₂SO₄ versetzt und eine Gammaglobulinfraktion erhalten. Weitere Reinigung des Antikörpers ist nach an sich bekannten Verfahren wie Gelfiltration, lonenaustauschchromatographieo.dgl. möglich. Der derart erzeugte monoklonaIe Antikörper BL-M/G ist potentiell in der Lage, Komplement zu binden und die Zielzellen zytotoxisch zu zerstören.The preparation of the antibody is carried out according to the invention in such a way that antibody-producing hybridoma cells H-BL-M / G are cultivated in a culture medium with serum accumulation in mass culture. After appropriate cultivation in an atmosphere containing CO ₂ , the culture medium now containing the antibody is mixed with 50% saturated (NH ₄ J ₂ SO ₄₎ and a gamma globulin fraction is obtained Further purification of the antibody is possible by conventional methods such as gel filtration, ion exchange chromatography or the like. The monoclonal antibody BL-M / G thus produced is potentially capable of binding complement and cytotoxically destroying the target cells.

Es ist zweckmäßig, als Kulturmedium MEM Eagle, Dulbeccos MEM, RPMI o. dgl. anzuwenden. Besonders günstig ist die Verwendung von RPMI-1640, das in einer bevorzugten Ausführungsform der Erfindung mit 1OmM HEPES, 5 · 10"⁵M Mercaptoethanol, lOOmg/l Gentamysin versetzt ist. Als Serumzusatz dient fetales Käiberserum oder Pferdenormalserum mit einem Anteil am Kulturmedium von 5 bis 20%. Vorteilhaft ist es, mit einem Serumanteil von 10% zu arbeiten. Die Temperatur des Kulturmediums liegt zwischen 35⁰C und 38°C. Vorteilhaft ist es, bei 37°C zu arbeiten.It is expedient to use MEM Eagle, Dulbecco's MEM, RPMI or the like as the culture medium. It is particularly favorable to use RPMI-1640 in a preferred embodiment of the invention, with 1OmM HEPES, 5 x 10 ^"5 M mercaptoethanol, lOOmg / l Gentamysin is added. Fetal Käiberserum or normal horse serum is used as the addition of serum to a share of the culture medium of 5 to work with a serum content of 10% to 20% is advantageous.. the temperature of the culture medium is between 35 ⁰ C and 38 ° C. it is advantageous to operate at 37 ° C.

Der CO₂-Gehalt der Atmosphäre über dem Kulturmedium beträgt günstig 2 bis 10%, insbesondere 5%. Vorteilhaft ist eine hohe Luftfeuchtigkeit, die bis zur Sättigung der Atmosphäre reichen kann.The CO ₂ content of the atmosphere above the culture medium is favorably 2 to 10%, in particular 5%. The advantage is a high humidity, which can reach saturation of the atmosphere.

Die Kultivierung des Hybridoms erfolgt über einen üblichen Zeitraum von mehreren Tagen. Zweckmäßig ist eine Kultivierung von 3 bis 4 Tagen. Danach ist ein teilweiser Mediumwechsel notwendig. Dabei ist erneut die optimale Zelldichte einzustellen.The cultivation of the hybridoma takes place over a customary period of several days. It is advisable to cultivate for 3 to 4 days. Thereafter, a partial medium change is necessary. Here, the optimal cell density is again set.

Der monoklonal Antikörper weist das in Tabelle 1 dokumentierte Reaktionsmuster mit humanen Biutzellen auf. Die Bindung wurde mittels indirekter Radiobindungstechnik (RBT) und indikreter Immunfluoreszenz (UF) bestimmt.The monoclonal antibody has the human biocell reaction pattern documented in Table 1. Binding was determined by indirect radio-binding technique (RBT) and inductive immunofluorescence (UF).

In einer zweiten Ausführungsform der Erfindung wird das Hybridom H-BL-M/G in histokompatible A χ BALBZo-F₁-MaUSe i. p.In a second embodiment of the invention, the hybridoma H-BL-M / G is transformed into histocompatible A χ BALBZo-F ₁ mice ip

injiziert. Es ist vorteilhaft, wenn die eingesetzten Mäuse mit einem Tumorstimulator, wie Paraffinum subliquidum. Pristan o.dgl., vorbehandelt worden sind. Nach der Aszitesbildung, die durch Anschwellen sichtbar wird, wird die den monoklonalen Antikörper BL-M/G enthaltende Aszitesflüssigkeit entnommen, z. B. durch Punktion. Aus der Aszitesflüssigkeit werden die zellulären Bestandteile abgetrennt, z.B. durch Zentrifugieren. Der zelluläre Anteil besteht zum überwiegenden Teil ausZellen des Hybridoms H-BL-M/G. Er wird zweckmäßigerweise mit physiologischer Kochsalzlösung gewaschen und kann erneut injiziert werden. Der klare Überstand, der den monoklonalen Antikörper BL-M/G enthält, wird entweder direkt bzw. in geeigneter Verdünnung eingesetzt oder weiterer Reinigung unterzogen. Zur weiteren Reinigung kommen Verfahren wie DEAE-lonenaustauschchromatographie, Protein-A-Adsorption, Affinitätschromatographie o. dgl. in Frage, die den Isotyp des BL-M/G spezifisch anreichern, oder Verfahren der Immunadsorption, die den Antikörper BL-M/G antigenspezifisch isolieren. Die Aszitesflüssigkeit enthält bei erfindungsgemäßer Verfahrensdurchführung 5 bis 10 mg BL-M/G pro ml Flüssigkeit. Anreicherung führt zu einem Produkt mit einem Antikörpergehalt von ca. 20mg/mI.injected. It is advantageous if the mice used with a tumor stimulator, such as Paraffinum subliquidum. Pristan or the like., Have been pretreated. After ascites formation, which becomes visible by swelling, the ascites fluid containing the monoclonal antibody BL-M / G is removed, e.g. B. by puncture. From the ascites fluid, the cellular components are separated, e.g. by centrifuging. The cellular part consists predominantly of cells of the hybridoma H-BL-M / G. It is conveniently washed with physiological saline and can be re-injected. The clear supernatant containing the monoclonal antibody BL-M / G is either used directly or in a suitable dilution or subjected to further purification. For further purification, methods such as DEAE ion exchange chromatography, protein A adsorption, affinity chromatography or the like, which specifically enrich the isotype of the BL-M / G, or methods of immunoadsorption, the antibody BL-M / G antigen-specific isolate. The ascitic fluid contains 5 to 10 mg of BL-M / G per ml of liquid in the process according to the invention. Enrichment leads to a product with an antibody content of about 20 mg / ml.

Zum Herstellen einer stabilen Aufbewahrungsform, die auch ais Handelspräparat geeignet ist, wird die Immunglobulinfraktion der zellfreien Aszitesflüssigkeit präzipitiert, z. B. mit 50% gesättigter (NH₄)₂SO₄-Lösung und in gepufferter physiologischer Kochsalzlösung aufgenommen. Dialysieren gegen NH₄HCGyLösung führt zu einem lyophilisierbaren Produkt, das lyophilisiert bei +4⁰C haltbar ist.To produce a stable storage form which is also suitable as a commercial preparation, the immunoglobulin fraction of the cell-free ascites fluid is precipitated, e.g. B. with 50% saturated (NH ₄ ) ₂ SO ₄ solution and taken up in buffered saline. Dialyzing against NH ₄ HCGy solution results in a lyophilizable product which is lyophilized at + 4 ⁰ C preserved.

Table 1

Bindungsmuster des monoklonalen Antikörpers BL-M/G mit verschiedenen BlutzellenBinding pattern of monoclonal antibody BL-M / G with different blood cells

Testzellen MarkierteZellen (%) Rl¹'Test Cells Labeled cells (%) Rl ¹ '

PMZ²¹ PMZ ²¹ <5<5 Monozytenmonocytes >90> 90 Granulozytengranulocytes >80> 80 T-LymphozytenT lymphocytes <2<2 B-LymphozytenB lymphocytes <2<2 O-ZellenO cells >70> 70 Erythrozytenerythrocytes 00

1) Rl bedeutet die relative Fluoreszenzintensität der Membrananfärbung1) Rl means the relative fluorescence intensity of membrane staining

2) Periphere mononukleäre Zellen2) Peripheral mononuclear cells

1. Ausführungsbeispiel:1st embodiment:

Auf flüssigem Stickstoff gelagerte Hybridzellen (H-BL-M/G) werden aufgetaut und zweimal mit Kulturmedium gewaschen. Die Zelldichte wird auf 1-5 χ 10⁵ Zeilen pro ml Kulturmedium eingestellt und in geeigneten Gewerbekulturflaschen kultiviert. Das Kulturmedium besteht aus RPMI-1640, welches mit Natriumhydrogenkarbonat (2g/l), Mercaptoethanol (5 x 10"⁵M), Gentamycin (100mg/l) und N-2-Hydroxyethylpiperazin-N'-ethansulfonsäure (1OmM) ergänzt wird. Dieses Medium wird mitHybrid nitrogen cells (H-BL-M / G) are thawed and washed twice with culture medium. The cell density is adjusted to 1-5 χ 10 ⁵ lines per ml culture medium and cultured in appropriate commercial culture bottles. The culture medium consists of RPMI-1640 supplemented with sodium bicarbonate (2g / l), mercaptoethanol (5 x 10 ^"5 M), gentamycin (100 mg / l) and N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (1OmM) is added. This medium comes with

a) fetalem Kälberserum odera) fetal calf serum or

b) Pferdenormalserumb) Horse Normal Serum

supplementiert. Die möglichen Anteile sind 5 bis 20%. Als geeignet hat sich 10% Serumanteil erwiesen.supplemented. The possible shares are 5 to 20%. 10% serum content has proven to be suitable.

Die günstigsten Kulturbedingungen werden erreicht, wenn die Zellen bei +37⁰C in einer wassergesättigten 5%igen CO₂-Atmosphäre bebrütet werden.The best culture conditions are achieved when the cells at +37 ⁰ C in a water-saturated 5% CO ₂ atmosphere are incubated strength.

Nach 3 bis 4 Tagen werden die Zellkulturüberstände steril entnommen. Die z.T. an der Gefäßwand anhaftenden Zellen können erneut mit frischem Medium versorgt und bebrütet werden. Dabei ist jedoch auf die optimale Zelldichte zu achten. Die im Kulturüberstand enthaltenden Hybridzellen werden durch Zentrifugation abgetrennt. Sie können zur Aussaat in neue Kulturgefäße verwendet werden.After 3 to 4 days, the cell culture supernatants are removed under sterile conditions. The z.T. Cells adhering to the vessel wall can be re-supplied with fresh medium and incubated. However, it is important to pay attention to the optimal cell density. The hybrid cells in the culture supernatant are separated by centrifugation. They can be used for sowing in new culture vessels.

Die zellfreien Kulturüberstände enthalten den monoklonalen Antikörper BL-IWG. Die Konzentration des Antikörpers BL-M/G ist ausreichend fürdie üblichen diagnostischen Nachweisverfahren für Monozyten, Granulozyten undNullzellen(wiez.B. indirekte Immunfluoreszenz, enzymimmunologische Nachweise, Radiobindungstechniken).The cell-free culture supernatants contain the monoclonal antibody BL-IWG. The concentration of the antibody BL-M / G is sufficient for the usual diagnostic detection methods for monocytes, granulocytes and null cells (such as indirect immunofluorescence, enzyme immunological detection, radio linkage techniques).

Gegebenenfalls kann der Titer mit einer geeigneten Technik (z. B. indirekte Immunfluoreszenz) bestimmt werden. Hochtitrige Kulturüberstände gestatten eine Verdünnung bis zur gewünschten Arbeitskonzentration. Werden höhere Konzentrationen des Antikörpers benötigt, so werden die Kulturüberstände mit 50% gesättigtem Ammoniumsulfat gefällt. Die so erhaltene Gammaglobulinfraktion kann durch weitere bekannte Trenntechniken (lonenaustauschchromatographie, Gelfiltration usw.) weitergereinigt werden.Optionally, the titer may be determined by a suitable technique (eg, indirect immunofluorescence). High titre culture supernatants allow dilution to the desired working concentration. If higher concentrations of the antibody are required, the culture supernatants are precipitated with 50% saturated ammonium sulfate. The gamma globulin fraction thus obtained can be further purified by further known separation techniques (ion exchange chromatography, gel filtration, etc.).

Werden hochgereinigte monoklonale Antikörper benötigt, so können diese durch Immunadsorption an trägerfixierte Anti-Mausimmunglobuiinantikörper von den aus Kälberserum bzw. Pferdeserum stammenden Begleitproteinen abgetrennt werden und durch schonend wirkende Desorbentien (3M KSCN oder Glycin/HCI-Puffer pH2,8) von diesem Immunadsorbens wieder isoliert werden.If highly purified monoclonal antibodies are required, they can be separated from the calf serum or horse serum-derived accompanying proteins by immunoadsorption on carrier-fixed anti-mouse immunoglobin antibodies and isolated again from this immunoadsorbent by gently acting desorbents (3M KSCN or glycine / HCl buffer pH2.8) become.

2. Ausführungsbeispiel:2nd embodiment:

Eingesetzt werden Zellen des Hybridoms H-BL-M/G, die gegebenenfalls in vitro vorkultiviert werden. Nach Waschen in serumfreier physiologischer Kochsalzlösung werden 10⁵ bis 5 10⁷ lebende Zellen histokompatiblen Mäusen intraperitorieal injiziert. Als histokompatible Mäuse werden F^-Hybride der Stämme A und BALB/c eingesetzt, die mit eirem Tumorstimulator, hiermit0,5mlParaffinumsubliquidum,i.p. 2 Wochen vor der Hybridom injektion behandelt worden sind.Cells of the hybridoma H-BL-M / G, which are optionally pre-cultured in vitro, are used. After washing in serum-free physiological saline, 10 ⁵ to 5 10 ⁷ live cells are intraperitoneally injected into histocompatible mice. The histocompatible mice used are F 1 hybrids of strains A and BALB / c, which have been treated with a tumor stimulator, herewith 0.5 ml paraffin fluid, ip 2 weeks before the hybridoma injection.

Nach dem Einsetzen der Aszitesbildung werden die Mäuse punktiert. Die abgenommenen Aszitesflüssigkeiten, die den monoklonalen Antikörper BL-M/G enthalten, werden durch Zentrifugieren in zelluläre Bestandteile und in Aszitesflüssigkeit getrennt.After the onset of ascites formation, the mice are punctured. The collected ascites fluids containing the monoclonal antibody BL-M / G are separated by centrifugation into cellular components and ascites fluid.

Der zelluläre Anteil besteht überwiegend aus Zellen des Hybridoms H-BL-M/G. Diese Hybridomzellen werden mit physiologischer Kochsalzlösung gewaschen und wie oben beschrieben eingesetzt, indem sie histokompatiblen Mäusen i. p.The cellular part consists predominantly of cells of the hybridoma H-BL-M / G. These hybridoma cells are washed with physiological saline and used as described above by injecting histocompatible mice i. p.

injiziert werden. Solche Passagen sind wiederholt möglich.be injected. Such passages are possible repeatedly.

Die Immunglobulinfraktion der zellfreien Aszitesflüssigkeit wird mit 50% gesättigtem (NHa)₂SO präzipitiert und in gepufferter physiologischer Kochsalzlösung aufgenommen. Diese Lösung wird gegen 0,5%ige NH₄HCO₃-Lösung dislysiert. Anschließend erfolgt die Lyophilisierung. Der Antikörper BL-M/G ist lyophilisiert bei +4⁰C ohne Verlust der Bindungseigenschaften haltbar.The immunoglobulin fraction of the cell-free ascitic fluid is precipitated with 50% saturated (NH ₂ ) ₂ SO and taken up in buffered saline. This solution is disiluted against 0.5% NH ₄ HCO ₃ solution. Subsequently, the lyophilization takes place. The antibody BL-M / G is lyophilized at +4 ⁰ C without loss of binding properties.

Der Antikörpertiter wird durch Herstellen einer Verdünnungsreihe und Austestung mittels indirekter Immunfluoreszenz unter Verwendung frischer humaner Blutmonozyten und einem geeigneten FITC-markierten Anti-Mausimmunglobulinserum bestimmt. Als Titer wird diejenige größte Verdünnung angegeben, bei der noch deutlich alle Monozyten markiert werden.Antibody titer is determined by preparing a dilution series and testing by indirect immunofluorescence using fresh human blood monocytes and a suitable FITC-labeled anti-mouse immunoglobulin serum. The titer is given as the largest dilution at which clearly all monocytes are still marked.

3. Ausführungsbeispiel:3rd embodiment

Zellen des Hybridoms H-BL-M/G werden wie in Beispiel 1 kultiviert und vermehrt. Nach Erreichen einer hinreichenden Zeilanzahl werden die Hybridomzellen geerntet und mehrmals mit serumfreiem Zellzuchtmedium gewaschen.Cells of the hybridoma H-BL-M / G are cultured and propagated as in Example 1. After reaching a sufficient number of cells, the hybridoma cells are harvested and washed several times with serum-free cell culture medium.

Anschließend werden die Hybridomzellen in einer Dichte von 2-5 · TO⁶ Zellen pro 1 ml in einem serumfreien Zellzuchtmedium wie z. B. RPMI-1640, das mit NaHCO₃ (2g/l), Mercaptoethanol (5 · 10""⁵M), Gentamycin (lOOmg/l) und N-2-Hydroxyethylpiperazin-N'-ethansulfonsäure (1OmM) ergänzt wird, kultiviert. Die günstigsten Kulturbedingungen werden erreicht, wenn die Zellen bei 37⁰C in einer 5%igen CO₂-Atmosphäre bebrütet werden. Die Inkubation dauert 12-24 Stunden. Der zellfreie Kulturüberstand enthält den monoklonalen Antikörper des jeweiligen Hybridoms H-BL-M/G ohne Serumproteine, die sonst als Mediumzusatz verwendet werden. Der monoklonale Antikörper kann durch DEAE-Ionenaustauschchromatographie oder Protein-A-Adsorption o.dgl. weiter konzentriert werden, wenn das gewünscht wird.Subsequently, the hybridoma cells at a density of 2-5 · TO ⁶ cells per 1 ml in a serum-free cell culture medium such. B. RPMI-1640 supplemented with NaHCO ₃ (2g / l), mercaptoethanol, and N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (1OmM) is added (5 x 10 "" ⁵ M), gentamycin (lOOmg / l), cultured. The most favorable culture conditions are achieved when the cells are incubated at 37 ^° C. in a 5% CO ₂ atmosphere. The incubation takes 12-24 hours. The cell-free culture supernatant contains the monoclonal antibody of the respective hybridoma H-BL-M / G without serum proteins, which are otherwise used as a medium supplement. The monoclonal antibody may be prepared by DEAE ion exchange chromatography or protein A adsorption or the like. be further concentrated if desired.

Mit den erfindungsgemäß hergestellten Antikörpern BL-M/G gelingt es besser und genauer als bisher, Monozyten, Granulozyten und Nullzellen zu bestimmen. Ein weiterer wesentlicher Vorteil der geschilderten Verfahrensweise ist die Möglichkeit, das Hybridom H-BL-M/G über längere Zeit aufzubewahren und auch nach Ruhezeiten wieder zur Anti körper produktion einzusetzen. In diesem Sinne wird durch die zur Verfügung gestellte Lehre der Stand der Technik wesentlich bereichert. Das Hybridom H-BL-M/G ist an der Sektion Biowissenschaften der Karl-Marx-Universität Leipzig zugänglich. Der monoklonale Antikörper BL-M/G ist im Zentralinstitut für Molekularbiologie der Akademie der Wissenschaften der DDR unter der Nummer DDR 0044 registriert.With the antibodies BL-M / G according to the invention it is better and more accurate than before to determine monocytes, granulocytes and null cells. Another major advantage of the described procedure is the ability to store the hybridoma H-BL-M / G for a long time and to use after rest periods again for anti-body production. In this sense, the teaching provided by the prior art significantly enriched. The hybridoma H-BL-M / G is available at the Life Sciences Section of Karl Marx University Leipzig. The monoclonal antibody BL-M / G is registered in the Central Institute of Molecular Biology of the Academy of Sciences of the GDR under the number DDR 0044.

Claims

Invention claim:

1. A process for preparing monoclonal antibodies for a monocyte antigen, characterized in that female Α mice are immunized with human monocytes, lymphocytes are separated from the spleens of mice treated in this way, these are hybridized with mouse B myeloma cells, hybridoma cells of the type H-BL-M / G selected, the selected hybridoma cells are cultured or injected into mice, the ascites fluid removed and the antibody BL-M / G is obtained.

2. The method according to item 1, characterized in that the hybridoma H-BL-M / G is recloned and that a Subkion is selected, which produces antibodies suitable for the determination of monocytes, granulocytes and null cells.

3. The method according to item 1, characterized in that 6 to 8 weeks Aite female A mice are used for immunization.

4. The method according to item 1, characterized in that are used as Myelomzeüen those of the line P3-X-63 Ag 8.653.

5. The method according to item 1 to 4, characterized in that the immunization of the mice by a series of injections with human monocytes he ^f o! Gt.

6. The method according to item 1 to 5, characterized in that the last immunization tion 4 Ta ge before isolation of the dia hybridization determined B lymphocytes is made.

7. The method according to item 1 to 6, characterized in that aas the ratio of myeloma cells and B-lymphoblasts, which are intended for fusion, about 1: 1.

8. The method according to item 1 to 7, characterized in that in the step of separating the unhybridized myeloma cells from the hybrid lines, the culture medium used contains spleen cells of non-immunized A χ BALBZc-F ₁ hybrid mice.

9. The method according to item 1, characterized in that the testing of the supernatants of the growing hybrid cell-containing cultures on their reaction with human monocytes, granulocytes, null lines by indirect immunofluorescence using FITC-labeled goat anti-Mausimmunglobuiinserum.

10. The method according to item 1, characterized in that the examination of the supernatants of the growing hybrid cell-containing cultures on the non-reacting ren with B and T lymphocytes by means of indirect immunofluorescence.

11. The method according to item 1, characterized in that antibody-producing hybridoma cells H-BL-M / G are grown in a culture medium in mass culture, wherein the culture medium, a serum is added, after appropriate cultivation in CO ₂ -containing atmosphere the now antibody-containing culture medium 50% saturated (NHj) ₂ SO ₁ J is added and a gamma globulin fraction is obtained, from which the monoclonal antibody is isolated after further workup in a manner known per se.

12. The method according to item 1 and 11, characterized in that the culture medium MEMN Eagle, Dulbecco's MEM, RPMI or the like. Is used.

13. The method according to item 1 and 11 to 12, characterized in that is used as the culture medium RPM 1-1640.

14. The method according to item 1 and 11 to 13, characterized in that the culture medium 1OmM HEPES, 5 10 " ⁵ M mercaptoethanol, 100mg / l gentamycin be added.

15. The method according to item 1 and 11 to 14, characterized in that are used as serum fetal calf serum or normal horse serum.

16. The method according to item 15 .., characterized in that the proportion of fetal calf serum in the culture medium is about 10%, that of the horse normal serum is also about 10%.

17. The method according to item 1 and 11 to 16, characterized in that the cultivation temperature 35 ³ C to 38 ⁰ C.

18. The method according to item 1 and 11 to 17, characterized in that is cultivated at 37'C.

19. The method according to item 1 and 11 to 18, characterized in that the CO ₂ -Geha t of the atmosphere over the culture medium is 2 to 10%.

20. The method according to item 1 and 11 to 19, characterized in that the CO ₂ content over the culture medium is 5%.

21. The method according to item 1 and 11 to 20, characterized in that above the culture medium high humidity prevails, which may extend to saturation of the Aimosphäre with water vapor.

22. The method according to item 1 and 11 to 21, characterized in that 3 to 4 days is cultivated.

23. The method according to item 1, characterized in that the hybridoma H-BL-M / G is injected ip in histocompatible A χ BALB / c Frmäuse, after the ascites the ascites is removed, the cellular components are separated, the clear supernatant containing the monoclonal antibody BL-M / G, either used directly or undergoing purification and enrichment operations, or by precipitating the immunoglobulin fraction of the cell-free ascitic fluid, e.g. B. with 50% saturated (NH ₄ I ₂ SCVLOSUHg, receiving the precipitate in buffered saline, dialyzed against dilute NH ₄ HCO3 solution and subsequent lyophilization in a permanent storage form is transferred.

24. The method according to item 1 and 23, characterized in that the separated cellular components, which consist mainly of cells of the hybridoma H-BL-M / G, washed with saline and can be injected again histocompatible mice.

25. The method according to item 1 and 23, characterized in that the mice used with a tumor stimulator such as Paraffinumsubliquidum, Pristano.dgl. pretreated.

26. Method according to Puniet 1,23 and 25, characterized in that a corresponding tumor stimulator is given 3 days to 6 weeks before hybridinjection.

27. The method according to item 1, characterized in that the lyophilized antibody BL-M / G is stored at +4 ⁰ C.

Field of application of the invention

The invention relates to a method for producing monoclonal antibodies which react with human monocytes, granulocytes and null cells.

Characteristic of the known technical solutions

Monocytes and macrophages play an important role in various pathogenic immune reactions. Analysis of the function of these cells and their diagnostic use are linked to the availability of antibodies to specific monocyte / macrophage antigens. There has been no lack of attempts to generate anti-macrophage sera by xeno or alloimmunization followed by tissue absorption (UNANUE, ER, Nature 218, [1968], 36; STINNETTJ.D., et al., J. Immunol. [1976], 273; BREARD, J. et al., Clin Immunol Immunopathol 15, [1980], 438). However, these antisera can only be produced to a limited extent, are not reproducible and therefore can not be standardized. One solution to these problems could be the monoclonal antibodies secreted by hybridomas. It is already known to produce monoclonal antibodies. For example, KÖHLER and MILSTEIN (Nature, 256, [1975] 495) have generated a hybrid cell line by fusion of anti-body-forming cells with a permanently growing plasmocytoma line, which permanently grows and stably secretes antibodies. This principle solution has led to various hybridoma lines that produce antibodies of different splice specificity have been produced. Thus, nonionic rat anti-mouse macrophage antibodies have also been described (SPRINGER, T.A. et al., Eur. J. Immuno! 9, [1979], 301). These antibodies also cross-react with antigens present on human monocytes and granulocytes (AULT, K.A. and T.A. SPRINGER, J. Immunol., 126, [1980], 359). Monoclonal antibodies reactive with human monocytes have also been described (Nunez, G. et al., Scand, J. Immunol., 18, [1982], 515). Since only two monoclonal monoclonal antibodies are currently known and available, it seems to make sense to enrich the state of the art with another, easily accessible and inexpensive monoclonal antibody.

Object of the invention

The object of the invention is to provide a method for producing a monoclonal antibody of the BL-M / G type, which in an easily controllable manner allows the cost-effective production of larger amounts of this specific antibody for a cell surface antigen of human monocytes, granulocytes and null cells. In this case, consistent quality should be ensured, the antibody should be stable and storable and suitable for use in simple and exact detection tests for human monocytes, granulocytes and null cells, with cross-reactions with cell surface antigens of other lines should not occur.

Explanation of the essence of the invention

The invention has for its object to provide a production process for monoclonal antibodies that react specifically with human monocytes, granulocytes and null cells, in the course of which first a hybridoma is to be generated, which is easy to handle and its cultivation is technically and economically advantageous , The cultivation of the hybridoma should lead to monoclonal antibodies which have the abovementioned properties and can be used in clinical diagnostics.

The object is achieved in that first mononuclear cells are isolated from the peripheral blood of healthy donors by the known methods of density gradient centrifugation. By incubating these cells in plastic petri dishes, the ad parating η cells are isolated. After detachment of these cells, e.g. with a rubber scraper, these are used according to the invention for Immunisie tion of mice. Preferably, 6 to 3 week old female Α mice are used. The immunization is carried out by repeated application of the antigen. From the spleens of such hyperimmune mice, the lymphocytes are separated in a conventional manner. The B lymphocytes of this cell mixture are fused or hygridised with mouse B myeloma cells. The necessary mouse B myeloma cells P3-X-63 Ag 8.653 (Keearney et al., J. Immunol. 123, [1979] 1548) are cultured in RPMI-1640 with 10% fetal calf serum (FCS). In a culture medium containing hypoxanthine, aminopterin and thymidine (LITTLEFIELD, Science 145, [1964], 709), the hybrids formed are separated from the unfused myeloma cells and, according to the invention, a cell clone is selected which secretes antibodies of the desired kind. The selection of the hybridoma is carried out in such a way that the cells are divided immediately after the fusioin into a plurality of separate 200-дІ-KiИигеп. The supernatants of cultures containing the mature hybrid cells are screened for the presence of antibodies which react with cell surface antigens of monocytes, granulocytes and null cells. For this purpose, all culture supernatants which react with adherent cells (monocytes), with granulocytes, which are obtained by methods known per se by density gradient centrifugation from peripheral blood of healthy donors tested. The supernatants are tested by indirect immunofluorescence, it being necessary to check in the phase contrast method that they are of the respective type. Only those cultures are used which react with the majority of monocytes and granulocytes. The culture supernatants are then incubated with the adherent monocytes and with the granulocytes and the proportion of the labeled