Lek, Tovarna Farmacevtskih In Kemicnih Izdelkov, D.D.
Zavod Republike Slovenije Za Transfuzijo Krvi
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Priority to SI9500249ApriorityCriticalpatent/SI9500249B/en
Priority to DE69626829Tprioritypatent/DE69626829T2/en
Priority to EP96926042Aprioritypatent/EP0846169B1/en
Priority to AT96926042Tprioritypatent/ATE234918T1/en
Priority to PCT/SI1996/000018prioritypatent/WO1997006251A1/en
Priority to AU66345/96Aprioritypatent/AU703091B2/en
Publication of SI9500249ApublicationCriticalpatent/SI9500249A/en
Publication of SI9500249BpublicationCriticalpatent/SI9500249B/en
The submitted invention belongs to the field of pharmaceutical industry and refers to new monoclonal antibodies and their fragments which bind to an accessible surface of human recombinant TNF-Alpha, TNF-Alpha analogues and TNF-Alpha soluble receptors. The invention also refers to stable cell lines of hybridomas which are capable of producing such monoclonal antibodies, to the procedure for their preparation and to pharmaceutical preparations which contain them. Further, the invention refers to the method for a quantitative determination of a human TNF-Alpha, free and complex with soluble TNF-Alpha receptors p55 and p75 as well as putty which is used in this procedure. It was not possible to simultaneously determine free TNF-Alpha macromolecules and their soluble receptors as well as their complexes by means of monoclonal antibodies known so far and the reagents developed from them. This invention also refers to TNF-Alpha analogue TNF-Alpha His 107/108, Cys95/148 which is used in the complex with TNF-Alpha receptors as an antigen for the immunization of mice.
Claims (2)
Λ PATENTNI ZAHTEVKIΛ PATENT CLAIMS4, Monoklonska protitelesa in njihovi fragmenti, ki so zmožni vezati dostopno površino humanega TNF-α ali TNF-α analogov ali prostih topnih TNF-α receptorjev p55 ali p75 ter njihovih kompleksov, označeni s tem, da jih pridobimo z gojenjem stabilne celične linije hibridomov ECACC 95071217. Z. Monokion8ka protitelesa in njihovi fragmenti, ki so zmožni vezati se ali na dostopno površino humanega TNF-α ali TNF-α analogov ali prostih topnih TNF-α receptoijev p55 ali p75 ter njihovih kompleksov, označeni s tem, da jih pridobimo z gojenjem stabilne celične linije hibridomov ECACC 95071218. 3 Stabilna celična linija hibridomov ECACC 95071217. L). Stabilna celična linija hibridomov ECACC 95071218. 5\ Postopek za pripravo stabilne celične linije hibridomov po zahtevkih 6 do V označen s tem, da obsega: - imunizacijo miši z zmesjo kompleksa TNF-α analoga Cys95His107His108Cys148 in topnih nativnih ali rekombinantnih TNF-a receptorjev p55 in/aii p75, - fuzijo izolirani mišjih vraničnih celic s celično linijo mielomskih celic, in - kloniranje hibridomov, ki proizvajajo monoklonska protitelesa. G, Postopek po zahtevku ξ, označen s tem, da se za imunizacijo uporabi kompleks TNF-α analoga Cys95His107His108Cys148 in topnega nativnega ali rekombinantnega TNF-α receptorja p55. 'f. Postopek po zahtevku £T, označen s tem, da se za imunizacijo uporabi kompleks TNF-α analoga Cys95His107His108Cys148 in topnega nativnega ali rekombinantnega TNF-α receptorja p75. $ \ Postopek po zahtevku 5, označen s tem, da se za imunizacijo uporabi kompleks TNF-α analoga Cys95His107His108Cys148 in obeh topnih nativnih ali rekombinahtnih receptoijev p55 In p75. 9 Metoda za kvantitativno določanje TNF-α receptorja p55, prostega in kompleksiranega s TNF-α, v bioloških vzorcih z uporabo monoklonskih protiteles in njihovih fragmentov po zahtevku /I * 40 Metoda za kvantitativno določanje TNF-α receptorjev p75, prostih ali kompleksiranih s TNF-α, v bioloških vzorcih z uporabo monoklonskih protiteles po zahtevku %.# 4/j.Kit za določanje topnega TNF-α receptorja p55, prostega ali kompleksiranega z TNF-α v bioloških vzorcih, označen s tem, da vsebuje monoklonska protitelesa in njihove fragmente po zahtevki) A· /£,Kit za določanje topnega TNF-α receptorja p75, prostega ali kompleksiranega z TNF-α v bioloških vzorcih, označen s tem, da vsebuje monoklonska protitelesa in njihove fragmente po zahtevku /3-TNF-a analog Cys95His107His108Cys148. .Postopek za pripravo TNF-α analogov po zahtevku označen s tem, da obsega: - ekspresijo v rekombinantnem sevu E. coli in - enostopenjsko čiščenje s keiatno kromatografijo. 4$, Uporaba TNF-α analogov po zahtevku /3 za izolacijo TNF-α topnih receptoijev p55 in p75 iz humanega urina.4, Monoclonal antibodies and fragments thereof that are able to bind the accessible surface of human TNF-α or TNF-α analogs or free soluble TNF-α p55 or p75-TNF-α receptors and their complexes, characterized in that they are obtained by cultivating a stable cell line of hybridoma ECACC 95071217. Z. Monoclonal antibodies and fragments thereof that are able to bind to or access the surface of human TNF-α or TNF-α analogs or free soluble TNF-α receptors p55 or p75 and their complexes, characterized in that they are obtained by cultivating a stable cell line of hybridomas ECACC 95071218. 3 Stable hybridoma cell line ECACC 95071217. L). A stable cell line of hybridoma ECACC 95071218. A method for the preparation of a stable hybridoma cell line according to claims 6 to V comprising: - immunizing mice with a mixture of the TNF-α complex of the Cys95His107His108Cys148 analogue and soluble native or recombinant TNF-a receptor p55 and / aii p75, - fusion of isolated mouse spleen cells with a cell line of myeloma cells, and - cloning of hybridomas that produce monoclonal antibodies. G, Method according to claim ξ, characterized in that the TNF-α complex of the Cys95His107His108Cys148 analogue and the soluble native or recombinant TNF-α receptor p55 are used for immunization. 'f. A method according to claim E, characterized in that the TNF-α complex of the Cys95His107His108Cys148 analogue and the soluble native or recombinant TNF-α receptor p75 are used for immunization. A method according to claim 5, characterized in that the TNF-α complex of the Cys95His107His108Cys148 complex and the two soluble native or recombinant receptors p55 and p75 are used for immunization. Method for the quantification of TNF-α TNF-α TNF-α receptor p55 free and complexed in biological samples using monoclonal antibodies and fragments thereof according to claim I * 40 Method for the quantitative determination of TNF-α peptide receptors p75 free or complexed with TNF -α in biological samples using monoclonal antibodies according to Claim 4, for the determination of a soluble TNF-α receptor p55 free or complexed with TNF-α in biological samples, characterized in that it contains monoclonal antibodies and their fragments according to claims) A · / £, Kit for the determination of soluble TNF-α receptor p75, free or complexed with TNF-α in biological samples, characterized in that it contains monoclonal antibodies and fragments thereof according to claim 3-TNF-a analog Cys95His107His108Cys148. . Procedure for the preparation of TNF-α analogs according to the claim, characterized in that it comprises: - expression in a recombinant strain E. coli, and - single-step purification by kayat chromatography. 4 $, Use of TNF-α analogs according to claim 3 for the isolation of TNF-α soluble p55 and p75 human urine.
SI9500249A1995-08-041995-08-04Monoclonal antibodies versus soluble tnf-alpha receptors p55 and p75 and tnf-alpha and its analogues
SI9500249B
(en)
The development of a quantitative RIA for basic fibroblast growth factor using polyclonal antibodies against the 157 amino acid form of human bFGF: the identification of bFGF in adherent elicited murine peritoneal macrophages
Antigenic relationships of fractionated western diamondback rattlesnake (Crotalus atrox) hemorrhagic toxins and other rattlesnake venoms as indicated by monoclonal antibodies