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Soluble thymic stromal lymphopoietin receptors are absent in murine sera – detection with anti‐mTSLPR monoclonal antibodies
Author(s) -
ROSENQVIST LINDA,
ANDERSSON ANNEMARIE,
OHLS SARA,
BOSTRÖM MARINA,
NORDSTRÖM TOMMY,
LINDQVIST CHRISTER
Publication year - 2005
Publication title -
apmis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.909
H-Index - 88
eISSN - 1600-0463
pISSN - 0903-4641
DOI - 10.1111/j.1600-0463.2005.apm_113503.x
Subject(s) - monoclonal antibody , microbiology and biotechnology , sf9 , hybridoma technology , antiserum , recombinant dna , antibody , biology , splenocyte , thymic stromal lymphopoietin , virology , transfection , receptor , cell culture , immunology , biochemistry , spodoptera , genetics , gene
Two monoclonal antibodies, termed nnIE11 and nnIG11, were generated against the murine thymic stromal lymphopoietin receptor, mTSLPR, using traditional hybridoma technology. The antibody‐producing hybridoma clones were obtained by fusing P3X63‐Ag8.653 myeloma cells with splenocytes from Balb/c mice immunized with anti‐FLAG M2 affinity‐purified FLAG‐tagged mTSLPR from pSVL‐mTSLPR‐FLAG‐transfected COS cells and Ni‐NTA‐purified his‐tagged mTSLPR from recombinant FastBacHisB‐mδ1 baculovirus‐infected Sf9 cells. Several monoclonal anti‐mTSLPR‐specific hybridoma clones were obtained and two of these clones are further characterized here. The generated antibodies could in an immunoblotting identify baculovirus‐expressed mTSLPR proteins with a molecular weight corresponding to 50 kDa. Both immunoblotting and ELISA with recombinant mouse TSLPR/Fc chimera as antigen, having only the N‐terminal domain of mTSLPR present, indicated that the generated monoclonal antibodies identify the C‐terminus of mTSLPR. Although sandwich ELISAs performed with a goat anti‐mTSLPR antiserum as capture antibody and nnIE11 as indicator antibody were able to detect mTSLPR in the range of 5 ng/ml, no souble mTSLPR could be observed in serum samples from CBA/H, Balb/c and C57Bl/6 mice.