INTRODUCTIONSince undetectable BCR-ABL mRNA transcription does not always indicate eradication of the Ph+ CML clone and since transcriptionally silent Ph+ CML cells exist, quantitation by genomic PCR of bcr-abl genes can be clinically useful. Furthermore, hotspot mutations in the Abelson tyrosine kinase (ABLK) domain of the bcr-abl gene translocation in Philadelphia chromosome-positive (Ph+) chronic myeloid leukaemia (CML) cells confer resistance on the specific kinase blocking agent, STI571.

MATERIALS AND METHODSGenomic DNA from K562, CESS and patient CML cells were amplified using rapid cycle quantitative real-time polymerase chain reaction for the gene regions spanning the mutation hotspots. In assays for ABLK exons 4 or 6, exonic or intronic PCR primers were used.

RESULTSWe show that separation of cycle threshold (CT) values for log-fold amplicon quantification was 2.9 cycles for ABLK exon 4, and 3.8 cycles for exon 6 with rapid amplification times. K562 CML cells were found to have a approximately 2 log-fold ABLK gene amplification. In contrast, patient CML cells had CT differences of 2.2 for both exon, suggesting that there was no significant ABLK gene amplification. DNA sequencing confirmed that neither K562 nor patient CML cells contained ABLK hotspot mutations. Messenger RNA transcription analysis permitted the assessment of BCR-ABL transcription, which was qualitatively correlated to genomic amplification.

CONCLUSIONSThis novel Q-PCR assay was found to have high fidelity and legitimacy, and potentially useful for monitoring minimal residual disease, transcriptionally silent Ph+ CML cells, and bcr-abl gene amplification.

Chronic Disease ; Drug Resistance ; genetics ; Fusion Proteins, bcr-abl ; genetics ; Gene Amplification ; Genes, abl ; genetics ; Hematologic Neoplasms ; genetics ; Humans ; Leukemia, Myeloid ; genetics ; Mutation ; Protein-Tyrosine Kinases ; genetics ; RNA, Messenger ; Reverse Transcriptase Polymerase Chain Reaction

INTRODUCTIONMultiple myeloma (MM), a malignancy of plasma cells, accounts for 10% of all haematological malignancies and is currently incurable. Although it can be treated, the disease tends to relapse after several years and becomes increasingly resistant to conventional therapy. Investigations into using humoral therapy for MM are now underway with a view that novel therapeutic agents may provide a more targeted therapy for MM.

MATERIALS AND METHODSHere, phage display, a faster and more efficient method compared to classical hybridoma fusion technology, was used as a proof-of-concept to isolate several single-chain Fragment variables (scFv) against Ku86.

RESULTSAnti-Ku86 polyclonal scFvs biopanning was successful where third round scFvs (A(450)~1.1) showed a 1/3 increase in binding as compared to the fi rst round scFvs (A(450)~0.4) with 100 microg/mL of antigen (purified human Ku86). Subsequent selection and verification of monoclonal antibodies using third round biopanning revealed 4 good affinity binding clones ranging from A(450)~0.1 to A450~0.15 on 12.5 microg/mL of antigen as compared to low binders (A(450)~0.07) and these antibodies bind to Ku86 in a specific and dose-dependent manner. Comparative studies were also performed with commercially available murine antibodies and results suggest that 2 of the clones may bind close to the following epitopes aa506-541 and aa1-374.

CONCLUSIONSThese studies using phage display provide an alternative and viable method to screen for antibodies quickly and results show that good affinity antibodies against Ku86 have been successfully isolated and they can be used for further studies on MM and form the basis for further development as anti-cancer therapeutic agents.

Antibodies, Monoclonal ; isolation & purification ; Antibody Affinity ; Cell Line ; DNA Helicases ; immunology ; Humans ; Immunoglobulin Idiotypes ; immunology ; isolation & purification ; Immunoglobulin Variable Region ; isolation & purification ; Ku Autoantigen ; Multiple Myeloma ; immunology ; Peptide Library ; Recombinant Proteins