Generation and characterization of anti-alpha-enolase single-chain antibodies in chicken.
It was previously reported that up-regulation of alpha-enolase protein was detected in 65% of patients with non-small cell lung cancers (NSCLC). Moreover, a high titer of anti-alpha-enolase antibodies was developed in a smaller proportion (7.4%) of these patients than in non-tumor-associated patients and healthy subjects. In the present study, we characterized polyclonal and single-chain variable fragment (scFv) anti-alpha-enolase antibodies from immunized chickens. The E. coli-derived recombinant alpha-enolase protein was purified to its high homogenicity as verified by SDS-PAGE. After the 4th immunization, a high titer of specific polyclonal anti-alpha-enolase antibodies was elicited in immunized chickens and specifically recognized the purified human alpha-enolase antigen as determined by Western blot and ELISA. The expressed heavy and light chain variable genes (VH and VL) were isolated from spleen B cells and amplified to construct phage antibody libraries containing scFv molecules. After four rounds of panning selection, the scFv antibodies of randomly chosen clones were expressed and their binding specificity to alpha-enolase protein was verified using competitive ELISA, flow cytometry and immunofluorescence staining. Nucleotide sequence analysis from 10 alpha-enolase binding clones showed that 3 (30%) clones used identical heavy and light genes for scFv antibody expression, as represented by EnL5. Notably, amino acid changes in complementarity-determining regions (CDRs) were more frequently observed than those in framework regions (FRs) in all clones, indicating a strong affinity selection through mutations. All together, it is believed that these polyclonal and scFv IgY antibodies may be helpful in the development of molecular diagnostic and therapeutic agents for lung cancers in the future.
Leu
Sy-Jye
SJ
Department of Microbiology and Immunology, School of Medicine, Taipei Medical University, Taipei 110, Taiwan.
Lee
Yu-Ching
YC
Shih
Neng-Yao
NY
Huang
I-Jen
IJ
Liu
Ko-Jiunn
KJ
Lu
Hsu-Feng
HF
Huang
Shih-Yi
SY
Yang
Yi-Yuan
YY
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D007114:Immunization; D007136:Immunoglobulins / Q000096:biosynthesis / Q000276:immunology*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D019151:Peptide Library; D010751:Phosphopyruvate Hydratase / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
C016512:IgY; D007136:Immunoglobulins; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D010751:Phosphopyruvate Hydratase