[The epitope analysis of beta Netrin and preparation and characterization of its antibodies].
OBJECTIVE
To prepare and characterize anti-human beta-Netrin antibodies.


METHODS
B cell dominant epitopes of human beta-Netrin C-terminal 114 amino acid sequences were predicated by the GoldKey software. One of the epitopes was synthesized and coupled with bovine serum album (BSA) by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The BALB/c mice were immunized with the coupled protein. The splenocytes of immunized mice were fused with Sp2/0 cells by routine method and the hybridomas were selected in HAT medium. The hybridoma cells secreting specific antibody were detected by ELISA and cloned by limiting dilution. The titer specificity, and Ig subclass of anti-beta-Netrin mAbs were characterized by ELISA, Western blot and immunocytochemical staining. In addition, New Zealand rabbits were immunized with the coupled protein to prepare polyclonal antibody against beta-Netrin. The specificity of the antiserum was verified by Western blot.


RESULTS
A 16-mer peptide NH2-FRGKRTLYPES-WTDRG-COOH was the dominant epitope of the B cells. Synthesized peptide coupled with BSA was used as the immunogen to immunize BALB/c mice. Three hybridoma cell lines that stably secrete specific mAbs were obtained. The result of immunocytochemical staining showed that prepared mAb specifically recognize the antigen in the neuronal cells. The polyclonal antibody against beta-Netrin had high specificity. Western blot analysis showed that the antiserum bind with the prokaryotically expressed beta-Netrin specifically.


CONCLUSIONS
Using the synthesized peptides as hapten, we have prepared epitope-specific mAbs and pAb against beta-Netrin successfully.
Yu
Li-hong
LH
Department of Neurobiology, Beijing Institute of Radiation Medicine, Beijing 100850, China. yyulihongg@hotmail.com
Gao
Yan
Y
Wang
Li-hong
LH
Han
Hong-yan
HY
Sun
Zhi-xian
ZX
Zhang
Cheng-gang
CG
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000276:immunology*; D000918:Antibody Specificity; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D005260:Female; D005786:Gene Expression Regulation; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D000075385:Netrins; D009474:Neurons / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D017434:Protein Structure, Tertiary; D051381:Rats
D000906:Antibodies; D000939:Epitopes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D000075385:Netrins; D010455:Peptides; C415837:beta-netrin