Selection and characterisation of a phage-displayed human antibody (Fab) reactive to the lung resistance-related major vault protein.
The major vault protein is the main component on multimeric vault particles, that are likely to play an essential role in normal cell physiology and to be associated with multidrug resistance of tumour cells. In order to unravel the function of vaults and their putative contribution to multidrug resistance, specific antibodies are invaluable tools. Until now, only conventional major vault protein-reactive murine monoclonal antibodies have been generated, that are most suitable for immunohistochemical analyses. The phage display method allows for selection of human antibody fragments with potential use in clinical applications. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human Fab fragments recognising major vault protein we used a large non-immunized human Fab fragment phage library. Phages displaying major vault protein-reactive Fabs were obtained through several rounds of selection on major vault protein-coated immunotubes and subsequent amplification in TG1 E coli bacteria. Eventually, one major vault protein-reactive clone was selected and further examined. The anti-major vault protein Fab was found suitable for immunohistochemical and Western blot analysis of tumour cell lines and human tissues. BIAcore analysis showed that the binding affinity of the major vault protein-reactive clone almost equalled that of the murine anti-major vault protein Mabs. The cDNA sequence of this human Fab may be exploited to generate an intrabody for major vault protein-knock out studies. Thus, this human Fab fragment should provide a valuable tool in elucidating the contribution(s) of major vault protein/vaults to normal physiology and cellular drug resistance mechanisms.
Scheffer
G L
GL
Department of Pathology, Free University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
Reurs
A W
AW
Jutten
B
B
Beiboer
S H W
SH
van Amerongen
R
R
Schoester
M
M
Wiemer
E A C
EA
Hoogenboom
H R
HR
Scheper
R J
RJ
D018528:ATP-Binding Cassette Transporters / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D019151:Peptide Library; D011233:Precipitin Tests; D020394:Vault Ribonucleoprotein Particles / Q000276:immunology*; D000099187:Major Vault Protein
D018528:ATP-Binding Cassette Transporters; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein