Abstract
Antigen Used for Immunization
A 12-mer peptide of PEVGDEEALRPL from human nestin corresponding to amino acids 681-692 (NM_006617) was selected as immunogen. A cysteine residue was added to the C-terminal end of the peptide to facilitate conjugation to the carrier protein keyhole limpet hemocyanin (KLH). KLH-conjugated peptide was used for generating mouse monoclonal antibody.(1)
Method of Immunization
Female BALB/c mice were used for immunization with peptide-KLH conjugate. The first immunization was performed using Freund's complete adjuvant. Incomplete Freund's adjuvant was used for the second, third, fourth, and fifth immunizations. Finally, 3 days before cell fusion, KLH-peptide (without adjuvant) was injected intravenously. Mice with higher titers of specific antibody were selected for fusion.(2,3)
Parental Cell Line Used for Fusion
Mouse myeloma SP2/0-Ag14 cells.
Selection and Cloning Procedure
Splenocytes of the immunized mouse were removed under sterile conditions. SP2/0-Ag14 cell line was mixed with the splenocytes at a ratio of 1:10 (1 SP2/0 to 10 splenocytes). The cell mixture was washed twice with RPMI medium. Pre-warmed (37°C) 50% PEG 1500 (Sigma-Aldrich, St. Louis, MO) was added to the cell pellet slowly by mixing at the same time. The cells were washed twice and selective HAT medium (Sigma-Aldrich) was added to the pellet, and the cells were seeded into 96-well plates. Supernatants from growing wells were separated and ELISA assay was performed. Positive clones were subcloned four times by limiting dilution method.(2,3)
Heavy and Light Chains of Immunoglobulin
Mouse IgG1 kappa light chain.
Specificity
NES-4G10 showed a very high reactivity with immunogenic peptide in ELISA assay. Further characterization of the antibody showed that it was able to detect nestin protein in human cell lines (PaCa2, U373), a mouse cell line (4T1), a monkey cell line (Cos-7), and bovine sertoli cells in Western blotting and immunocytochemistry tests. Bovine sertoli cells showed good reaction in flow cytometry test under permabilaztion condition. Nestin expression by the above cells was confirmed by RT-PCR reaction using specific primers. The NES-4G10 antibody detected nestin from species other than humans. This might imply recognition of an interspecies common epitope.
Specific Antigen Identified and Cross-reactivity
Nestin protein in human, mouse … monkey?