RESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Partículas de Ribonucleoproteínas em Forma de Abóbada/imunologia , Sequência de Bases , Western Blotting , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Dados de Sequência Molecular , Biblioteca de Peptídeos , Testes de PrecipitinaRESUMO
The Mr 110,000 lung resistance-related protein (LRP), also termed the major vault protein (MVP), constitutes >70% of subcellular ribonucleoprotein particles called vaults. Overexpression of LRP/MVP and vaults has been linked directly to MDR in cancer cells. Clinically, LRP/MVP expression can be of value to predict response to chemotherapy and prognosis. Monoclonal antibodies (MAbs) against LRP/MVP have played a critical role in determining the relevance of this protein in clinical drug resistance. We compared the applicability of the previously described MAbs LRP-56, LMR-5, LRP, 1027, 1032, and newly isolated MAbs MVP-9, MVP-16, MVP-18, and MVP-37 for the immunodetection of LRP/MVP by immunoblotting analysis and by immunocyto- and histochemistry. The availability of a broader panel of reagents for the specific and sensitive immunodetection of LRP/MVP should greatly facilitate biological and clinical studies of vault-related MDR.