Construction of a human Fab phage display library from antibody repertoires of osteosarcoma patients.
Osteosarcoma is the commonest type of primary malignant bone tumor, frequently found in adolescents at sites of rapid bone growth. Despite current management protocols, up to half of the patients succumb to this disease. Moreover, there is no well-characterized molecular marker for diagnosis and prognosis. Since phage display methodology allows the selection of human antibody fragments with potential use in clinical applications, we applied this procedure to construct a recombinant Fab (antigen binding fragment) library from patients with osteosarcoma. We used peripheral blood lymphocyte total RNA from 11 osteosarcoma patients and cloned recombinant Fab representing the micro, gamma and kappa chain antibody repertoires of these individuals. The resulting library was cloned in the pComb3X vector and attained 1.45 x 10(8) different functional forms. BstO I fingerprinting and DNA sequencing analysis of randomly selected clones revealed the diversity of the library, demonstrating that Fab harbors Vkappa chains from subgroups I to V, biased towards the A27 fragment, as normally reported for the human repertoire. Analysis of the VH repertoire revealed that our library has a slight bias towards the VH4 family, instead of the usually reported VH3. This is the first description of a phage display library from osteosarcoma patients. We believe these human Fab fragments will provide a valuable tool for the study of this neoplasia and could also contribute to improvements in the diagnosis of this disease.
Dantas-Barbosa
Carmela
C
Laboratório de Genética Molecular, Patologia, Rede Sarah de Hospitais de Reabilitação, 70330-150 Brasília, DF, Brazil.
Brígido
Marcelo M
MM
Maranhão
Andréa Q
AQ
D000328:Adult; D001666:Binding Sites, Antibody / Q000235:genetics*; D001859:Bone Neoplasms / Q000175:diagnosis / Q000235:genetics*; D002648:Child; D005260:Female; D005819:Genetic Markers / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D008214:Lymphocytes / Q000737:chemistry; D008297:Male; D012516:Osteosarcoma / Q000175:diagnosis / Q000235:genetics*; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D012334:RNA, Neoplasm / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
D005819:Genetic Markers; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D012334:RNA, Neoplasm