Masp isoforms as inhibitors of complement activation

a technology of complement activation and isoforms, which is applied in the field of new ficolin-associated polypeptides, can solve problems such as increasing the risk of infections

Inactive Publication Date: 2012-11-08
RIGSHOSPITALET +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is some evidence that polymorphisms in the MAS

Method used

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  • Masp isoforms as inhibitors of complement activation
  • Masp isoforms as inhibitors of complement activation
  • Masp isoforms as inhibitors of complement activation

Examples

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example 1

[0316]Detection of Alternative Transcription of the MASP1 Gene

[0317]Methods: In order to detect the three transcript variants of MASP1: MASP1, MASP3 and FAP, specific primers for each variant were design. PCR was set up with a common forward primer in exon 6 (5′-gcacccagagccacagtg-3′) and specific reverse primers: MASP1 in exon 12 (5′-gccttccagtgtgtgggc-3′), MASP3 in exon 11 (5-gccttccagagtgtggtca-3′) and FAP in exon 8a (5′-cgatctggagagcgaactc-3′) (FIG. 1). PCR amplifications were carried out in 20-μl volumes containing: 50 ng liver cDNA (Clontech), 0.25 μM of each primer, 2.5 mM MgCl2, 0.2 mM dNTP, 50 mM KCl, 10 mM Tris.HCl, pH 8.4, and 0.4 units of Platinum Taq DNA polymerase (Invitrogen). The PCR reactions were performed at the following cycling parameters: 10 min 94° C., 30 or 40 cycles (30 sec 94° C., 50 sec 58° C., 90 sec 72° C.), 10 min 72° C. Samples were analysed on 2% agarose gels.

[0318]Results: Alternative transcription of the MASP1 gene was detected in liver cDNA. The MA...

example 2

[0325]Immunoprecipitation.

[0326]Specific immunoprecipitation of MAP-1 from serum was performed with the MAP-1 specific mAb 20C4 (raised against the 17 MAP-1 specific C-terminal peptide) or mAb 8B3, a monoclonal antibody reacting against the common heavy chain of MASP-1 / 3 used as control precipitation antibody. A total of 10 μg of anti MAP-1 or MASP-1 / 3 antibody was allowed to bind to sheep anti mouse or rabbit IgG Dynabeads (M-280, cat. 112.02D / 112.04D, Dynal / Invitrogen). After a washing step the beads were applied to a pool of normal human serum (diluted 1:1 in TBS) and incubated end over end for 1 hour at 4° C. After final washing steps and magnetic separation the beads were boiled in SDS loading buffer and subjected to SDS-PAGE and western blotting probed with antibodies to MAP-1, MBL, and Ficolin-3.

[0327]The same precipitation procedure as described above was performed with mAbs to MBL (Hyb 131-11, Bioporto, Denmark), Ficolin-2 (FCN219) and Ficolin-3 (FCN334). To compensate for ...

example 3

[0341]Determining serum concentration and association properties of the novel MBL / Ficolin associated protein 1 (MAP-1).

[0342]A full-length non-tagged recombinant constructs of MAP-1 was generated and stably expressed in CHO-DG44 cells. Specific monoclonal antibodies against MAP-1 were raised. Also a quantitative ELISA for MAP-1 serum measurements was established and the associations between serum MAP-1 and Ficolin-2, -3 and MBL was examined by ELISA and density gradient fractionation.

[0343]Recombinant Proteins

[0344]Full length constructs of non-tagged human MAP-1 was expressed in CHO-DG44 cells as described elsewhere (Hummelshoj et al., Mol Immunol 44, 401-11, 2007; Larsen et al., J Biol Chem 279, 21302-11, 2004; Ma et al., 2009 J Biol Chem, October 9; 284(41)) with the modifications that PowerCHO1 serum-free medium (Lonza, Vallensbaek / Denmark, www.lonza.com) was used as the expression medium. We used antibody affinity purification to purify rMAP-1 as described previously (Skjoedt e...

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Abstract

The present invention relates to novel ficolin-associated polypeptides, and polypeptides derived from these ficolin-associated polypeptides for the use in the treatment of conditions associated with inflammation, apoptosis, autoimmunity, coagulation, thrombotic or coagulopathic related diseases, as well as the use as biomarkers. The present invention further relates to anti-bodies recognising such novel ficolin-associated polypeptides, and polypeptides derived thereof, nucleic acid molecules encoding such polypeptides, vectors and host cells used in the production of the polypeptides.

Description

FIELD OF THE INVENTION[0001]The present invention relates to novel ficolin-associated polypeptides, and polypeptides derived from these ficolin-associated polypeptides for the use in the treatment of conditions associated with inflammation, apoptosis, autoimmunity, coagulation, thrombotic or coagulopathic related diseases, as well as the use as biomarkers. The present invention further relates to antibodies recognising such novel ficolin-associated polypeptides, and polypeptides derived thereof, nucleic acid molecules encoding such polypeptides, vectors and host cells used in the production of the polypeptides.BACKGROUND OF THE INVENTION[0002]Activation of the complement system (C) is accomplished via three different initiation pathways: The alternative (AP), the classical (CP), or the lectin pathway (LCP). AP activation occurs on foreign surfaces and is caused by a slow, spontaneous hydrolysis of C3 and the activity of the factors properdin, factor B and factor D to form the functi...

Claims

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Application Information

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IPC IPC(8): A61K38/17C07K16/18C12N15/12A61P29/00A61P43/00C12Q1/68A61P37/00C12N5/10C12N1/19C12N5/12C12P21/02G01N33/566C07K14/435A61K39/00
CPCC07K14/472G01N33/6893A61K38/00G01N2333/4716A61P1/00A61P1/02A61P1/04A61P1/16A61P1/18A61P11/00A61P11/02A61P11/06A61P13/00A61P13/08A61P13/12A61P15/00A61P17/00A61P17/02A61P17/06A61P19/02A61P21/04A61P25/00A61P25/28A61P27/02A61P29/00A61P3/04A61P31/04A61P31/10A61P31/12A61P31/16A61P31/18A61P31/20A61P33/00A61P33/04A61P33/06A61P35/00A61P3/06A61P37/00A61P37/02A61P37/06A61P37/08A61P43/00A61P5/14A61P7/02A61P7/06A61P9/00A61P9/10A61P3/10G01N33/536G01N33/54387
Inventor GARRED, PETERGLUE, TINA HUMMELSHOJSKJODT, MIKKEL-OLE
Owner RIGSHOSPITALET
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