Selection of vimentin-specific antibodies from the HuCAL phage display library by subtractive panning on formalin-fixed, paraffin-embedded tissue.
We describe the direct isolation of specific antibodies on formalin-fixed, paraffin-embedded (FFPE) tissue. The technique involves subtractive selection of a large and highly diverse combinatorial human antibody phage library (HuCAL) on lymphocyte FFPE tissue sections. Tissue sections from normal human tonsil tissue were used to deplete the library of binders to most housekeeping proteins. Mantle-cell lymphoma tissue was used for positive selection and enrichment of mantle cell or tumor-specific antibody phage. We established a high-throughput immunohistochemical method for screening of antibody clones selected from FFPE tissue. One recombinant antibody showed specific staining for interfollicular and mantle cells in FFPE tissue. Immunoprecipitation with this antibody and subsequent mass spectrometric analysis revealed specificity for vimentin.
Jarutat
Tiantom
T
Department of Pathology, University Hospital of Schleswig-Holstein/Campus Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany. jarutat@patho.uni-luebeck.de
Nickels
Cora
C
Frisch
Christian
C
Stellmacher
Florian
F
Hofig
Kai Peter
KP
Knappik
Achim
A
Merz
Hartmut
H
D000911:Antibodies, Monoclonal / Q000276:immunology*; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D020522:Lymphoma, Mantle-Cell / Q000378:metabolism*; D014066:Palatine Tonsil / Q000378:metabolism*; D016612:Paraffin Embedding; D011994:Recombinant Proteins / Q000276:immunology; D014746:Vimentin / Q000276:immunology*
D000911:Antibodies, Monoclonal; D005404:Fixatives; D011994:Recombinant Proteins; D014746:Vimentin; D005557:Formaldehyde