Korean J Pediatr Hematol Oncol.  2001 Oct;8(2):331-343.

Anti-CD1a Single-Chain Fv Synthesis Using Phage Display and Flow Cytometry Screening

• Oh SH

Abstract

PURPOSE: CD1a antigen is observed in diseases such as Langerhans cell histiocytosis, T cell acute lymphoblastic leukemia, acute myelogenous leukemia and CD1a presenting cells would be associated with transplantation disorders. In this study, anti-CD1a single-chain Fv (scFv) was made and its DNA sequence was obtained to use as useful implement and informations for the diagnosis, therapy, and study of the diseases mentioned above.
METHODS
The cDNA of anti-CD1a scFv was constructed with multiple steps of PCR from NA1/34.HLK and it was inserted into pCANTAB 5 E phagemid. The anti-CD1a scFvs were expressed on transformed DH5alpha Eschericia coli and secreted into cultured media. Thymocytes were used as CD1a antigen presenting cells. ScFvs were tested with flow cytometry. DNA sequence was obtained with PCR generated DNA sequencing method.
RESULTS
There were 7 clones that secreted scFvs binding thymocytes. When using thymocytes after being incubated with anti-CD1a antibody, there was only one clone (D-24) scFv of which binding reaction was significantly inhibited. DNA sequence of scFv of D-24 was obtained.
CONCLUSION
The obtained scFv of D-24 should be anti-CD1a scFv because of its binding thymocytes and being inhibited by anti-CD1a antibody. It needs further purification and confirmative test such as immunoprecipitation. The obtained DNA sequence would be informative to construct various and humanized antibodies for CD1a antigen.

Keyword

Phage display; Single-Chain Fv (scFv); Flow cytometry; CD1a

MeSH Terms

Antibodies, Monoclonal, Humanized
Antigen-Presenting Cells
Bacteriophages*
Base Sequence
Clone Cells
Diagnosis
DNA, Complementary
Flow Cytometry*
Histiocytosis, Langerhans-Cell
Immunoprecipitation
Leukemia, Myeloid, Acute
Mass Screening*
Polymerase Chain Reaction
Precursor Cell Lymphoblastic Leukemia-Lymphoma
Sequence Analysis, DNA
Single-Chain Antibodies*
Thymocytes
Antibodies, Monoclonal, Humanized
DNA, Complementary
Single-Chain Antibodies