Phage-displayed Fab fragments against anti-human interleukin-2 receptor alpha. Detection of antigen-bound phages with anti-cpIII monoclonal antibodies.
The genes encoding the VHCH1 and VLCL parts of the mouse anti-human IL-2R alpha antibody 7G7B6 were amplified by PCR and the corresponding antibody fragments displayed on the surface of filamentous phages. The expression of Fab fragments was analysed by immunoblotting using HRP-labelled goat anti-mouse Ig antisera. By traditional hybridoma technology, splenocytes from Balb/c mice, immunized with native phage particles, were fused with P3X63-Ag8.653 myeloma cells in order to yield monoclonal antibodies against filamentous phage proteins. The obtained monoclonal antibody IF8 (mu/kappa) recognized the minor coat protein III as a 65-70 kDa protein band by immunoblotting, whereas the monoclonal antibody IVC8 (mu/kappa), in addition to cpIII, recognized a protein with an approximate molecular weight of 38-43 kDa. Both antibodies were employed to determine the binding specificity of the phage-displayed anti-human IL-2R alpha Fab fragments in an ELISA using recombinant baculovirus-expressed human IL-2R alpha proteins as antigens.
Raivio
E
E
Abo Akademi University, Department of Biochemistry and Pharmacy, Finland.
Kronqvist
M
M
Brodin
T N
TN
Persson
M A
MA
Lindqvist
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry*; D000941:Antigens / Q000378:metabolism*; D002213:Capsid / Q000276:immunology*; D036022:Capsid Proteins; D003090:Coliphages / Q000276:immunology* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D015375:Receptors, Interleukin-2 / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000941:Antigens; D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D007140:Immunoglobulin Fab Fragments; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D014760:Viral Fusion Proteins