RESUMO
Recently, because of highly advanced protein engineering technology, beyond the chimeric antibody, highly humanized and fully human antibody development is becoming crucial in the medical field. In the last decade, investigational approaches using clinical samples for fully human antibody production have been performed, but there are still problems with efficiency and accuracy, which should be solved. In the present study, based on novel IgG antibody-measuring ELISA and antibody gene copy number-quantitative PCR, a human single B cell RT-PCR-mediated IgG monoclonal antibody (mAb) gene cloning method was established, and CMVpp65-specific human mAbs were successfully identified. Quantitative PCR for the human IgG mRNA copy number per cell demonstrated that the detection range was 10-250copies/cell. CMVpp65(+)surfaceIgG(+) B cells were collected from melanoma patients who showed high titers of serum anti-CMVpp65 IgG antibody. RT-PCR was successful in 64% (IGH) and 84% (ß-actin) of 88 single B cells. Finally, both IGH and IGL gene amplifications in the same cell were successful in 21 single cells, and 18 IgG antibody genes specific for CMVpp65 antigen were cloned. Four of 13 recombinant human single-chain fragment variable (scFv) antibodies showed strong responses to full-length CMVpp65 protein. These results suggested that the current fully human mAb production procedure through antibody-titer screening by ELISA, single B cell RT-PCR-based antibody gene cloning, and the making of scFv recombinant antibody is an efficient method of therapeutic antibody development.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Citomegalovirus/imunologia , Imunoglobulina G/imunologia , Melanoma , Fosfoproteínas/imunologia , Anticorpos de Cadeia Única/imunologia , Proteínas da Matriz Viral/imunologia , Anticorpos Monoclonais/genética , Anticorpos Antivirais/genética , Clonagem Molecular , Citomegalovirus/genética , Humanos , Fosfoproteínas/genética , Anticorpos de Cadeia Única/genética , Proteínas da Matriz Viral/genéticaRESUMO
BACKGROUND: Melanoma is an intractable cancer with a poor prognosis and increasing prevalence worldwide. Specific biomarkers for early diagnosis have yet to be found. MATERIALS AND METHODS: Serum samples from melanoma patients and healthy volunteers were utilized for identifying melanoma marker proteins using a serological proteome approach. Specifically, G361 cell protein spots separated by 2-dimensional gel electrophoresis and transferred to a membrane were incubated with patient sera, and positive spots that reacted with more than 5 serum samples were identified using time of flight mass spectrometry. RESULTS: Only patient sera showed many spots reacted in G361 gels. A total of 13 positive spots were detected and 5 proteins were identified: eukaryotic elongation factor2 (EEF2), enolase1 (ENO1), aldolase A (ALDOA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heterogeneous nuclear ribonucleoproteins (HNRNP) A2B1. The mRNAs of four proteins (EEF2, ENO1, ALDOA and HNRNPA2B1) were highly expressed in G361 cells compared with melanocytes. EEF2, ENO1 and ALDOA mRNAs were also frequently expressed in other melanoma cell lines. CONCLUSION: The autoantibody-based proteomic approach was effective for investigating melanoma biomarkers. This study might contribute to the development of a diagnostic device for the early detection of cancer.