Recombinant rabbit Fab with binding activity to type-1 plasminogen activator inhibitor derived from a phage-display library against human alpha-granules.
The display of panels of antibody (Ab) fragments on the surface of filamentous bacteriophage offers a way of making Ab with defined binding specificities. Because rabbit Ab are routinely utilized as immunologic probes in a variety of biological techniques, the aim of this study was to design and utilize primers for the amplification of mRNAs encoding rabbit kappa light and gamma heavy chains for the construction of an Ab library from this species. Using the polymerase chain reaction, a diverse Ab library with a repertoire of 2 x 10(7) clones was derived from the spleen and bone marrow of a rabbit that had been immunized with purified human platelet alpha-granules. From this library, specific clones were isolated after three rounds of affinity selection with binding activity to type-1 plasminogen activator inhibitor, a trace protein contained in platelet alpha-granules. These data indicate that recombinant phage-displayed Ab libraries obtained after immunization with complex biological antigens can be employed for the isolation of rabbit monoclonal Fab against specific antigens contained in the biological sample.
Lang
I M
IM
Department of Vascular Biology, Scripps Research Institute, La Jolla, CA 92037, USA.
Barbas
C F
CF
Schleef
R R
RR
D000595:Amino Acid Sequence; D000818:Animals; D017104:Bacteriophage M13; D001483:Base Sequence; D001792:Blood Platelets / Q000276:immunology; D003001:Cloning, Molecular; D017931:DNA Primers; D015723:Gene Library; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D017395:Plasminogen Activator Inhibitor 1 / Q000276:immunology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D017395:Plasminogen Activator Inhibitor 1; D011993:Recombinant Fusion Proteins