RB066 and RB067 recognize human CDKN2A-derived peptides by ELISA

The recombinant antibodies RB066 and RB067 detect by ELISA two synthetic peptides from the human CDKN2A protein.


Introduction
CDKN2A (UniProt #P42771) is a protein involved in cell cycle regulation through interaction with cyclin-dependent kinases 4 and 6 (Foulkes et al., 1997).This paper reports the evaluation of 5 recombinant antibodies (RB066, RB067, RB068, RB069 and RB070) for the detection of human CDKN2A-derived peptides by ELISA.RB066 exhibited specific recognition of a CDNK2A Nterminal peptide, while RB067 bound to a C-terminal peptide, both in a concentration-dependent manner.
Antigen: All the antibodies were tested by ELISA against the biotinylated peptide antigens they were selected for.RB066 was assayed against an N-biotinylated peptide derived from residues 2-18 of the CDKN2A protein (peptide 1, sequence: EPAAGSSMEPSADWLAT).RB067, RB068, RB069, and RB070 were evaluated against an N-biotinylated peptide derived from residues 137-156 of the CDKN2A protein (peptide 2, sequence: TRGSNHARIDAAEGPSDIPD).An irrelevant Nbiotinylated peptide from the human NDC80 protein (UniProt #O14777) was used as a negative control.
Protocol: MaxiSorp 96-well plates (Nunc #44-2404-21) were coated with 50 μL of streptavidin per well at a concentration of 1 μg/mL.The coating was performed overnight at 4°C.The rest of the procedure was carried out at room temperature.As a saturation agent, 50 μL of PBS-BSA 3% (w/v) was added, followed by 20 minutes of incubation.The sample was then rinsed three times with 100 μL of washing buffer (PBS + 0.5% (w/v) BSA + 0.05% (w/v) Tween20).Biotinylated peptides were then added at a concentration of 0,2 nmol/mL in washing buffer for 30 minutes.After another 3 times rinsing, 50µl of serially diluted RB066-RB070 primary antibodies were added according to specifications described in figure 1.After three further washes, a secondary antibody (goat anti-rabbit IgG coupled to horseradish peroxidase, Sigma-Aldrich #A8275, dilution 1:1000, 50 μl per well) was incubated for 30 minutes.Finally, the wells were washed 5 times before exposure to 50 μl of tetramethylbenzidine (TMB) HRP substrate (Sigma #T5569).The reaction was stopped by the addition of 25 µl of 2 M H2SO4 and the absorbance (OD) was measured at 450 nm.

Results
As illustrated in Figure 1, antibodies RB066 and RB067 exhibited concentration-dependent binding to CDKN2A peptides 1 and 2, respectively.No binding was observed to the peptide derived from the human NDC80 protein (negative control).Despite being selected against this antigen, RB068, RB069, and RB070 did not recognize peptide 2 derived from the CDKN2A protein.
Although RB066 and RB067 antibodies specifically recognize CDKN2A peptides by ELISA, their ability to bind the full-length protein should be determined in future experiments.
Fig. 1.RB066 and RB067 bound to CDNK2A peptide 1 and peptide 2, respectively, but not to the negative control peptide NDC80 (shown only for RB066; the other background curves were superimposed).RB068, RB069 and RB070 did not bind to CDKN2A peptide 2.