Nucl Med Mol Imaging.  2009 Oct;43(5):487-494.

Production and Evaluation of Immunoreactivity of Poly Lysine-Tagged Single Chain Fragment Variable (ScFv) Lym-1 Antibody for Direct Conjugation to Fluorescence Dye

• Jung JH
• Choi TH
• Woo KS
• Chung WS
• Kang JH
• Jeong SY
• Choi CW
• Lim SM
• Cheon GJ

Abstract

PURPOSE
Small size of recombinant scFv antibody has many advantages such as rapid blood clearances and improved targeting antibodies to tumor region. On the other hand owing to small size, number of amino group is insufficient in conjugation with chelator and fluorescence labeling. This study is to introduce poly lysine tag to the C-terminal end of scFv lym-1 sequence for fluorescence chelator conjugation.
MATERIALS AND METHODS
Poly lysine scFv lym-1 gene, cloned into pET-22b (+) vector, was expressed in E. coli BL21 (DE3) strain. Antibody purification was performed with Ni-NTA column and then size exclusion column chromatography. Expression and purification levels of poly lysine tagged scFv lym-1 antibody were confirmed by western blot analysis. I-124, I-125, I-131 and Tc-99m were used for radiolabeling of purified poly lysine scFv lym-1. Flow cytometry analysis of FITC conjugated poly lysine scFv lym-1 was performed for confirmation of immunoreactivity of human Burkitt`s lymphoma cells.
RESULTS
Poly lysine scFv lym-1 antibody was purified through two steps and identified as molecular weight of 48 KDa. Radiolabeling yields of I-124, I-125, I-131 and Tc-99m into poly lysine scFv lym-1 were >99%, >99%, >95% and >99%, respectively. Flow cytometry analysis of poly lysine scFv and scFv lym-1 was showed similar immunoreactivity to human Burkitt`s lymphoma cells.
CONCLUSION
Poly lysine tag was useful for the sufficient number of amino groups to scFv lym-1 antibody for chelator conjugation with minimizing loss of immunoreactivity.

Keyword

Anti-B cell lymphoma antibody (Lym-1); single chain fragment variable (scFv); poly lysine tag

MeSH Terms

Antibodies
Blotting, Western
Chromatography
Clone Cells
Flow Cytometry
Fluorescein-5-isothiocyanate
Fluorescence
Hand
Humans
Lymphoma
Lysine
Molecular Weight
Sprains and Strains
Antibodies
Fluorescein-5-isothiocyanate
Lysine