Introduction
Claudins are transmembrane proteins involved in tight junctions between epithelial cells (Tsukita et al., 2019). Here we describe the ability of six recombinant antibodies (RB599, RB600, RB601, RB602, RB603 and RB605) to detect by ELISA a synthetic biotinylated peptide from the mouse Claudin-9 protein (Uniprot #Q9Z0S7).
Materials & Methods
Antibodies: ABCD_RB599, ABCD_RB600, ABCD_RB601, ABCD_RB602, ABCD_RB603 and ABCD_RB605 nanobodies (ABCD nomenclature, http://web.expasy.org/abcd/) were discovered by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/). They were produced as mini-antibodies with the antigen-binding VHH portion fused to a mouse IgG2A Fc. HEK293 suspension cells (growing in HEK TF medium, Xell#861-0001, supplemented with 0.1% Pluronic F68, Sigma #P1300) were transiently transfected with the vector coding for the VHH-Fc of each antibody. Supernatants (see Table 1 for individual yields) were collected after 4 days.
| Name | Yield (mg/L) |
| RB599 | 30 |
| RB600 | 30 |
| RB601 | 20 |
| RB602 | 50 |
| RB603 | 10 |
| RB605 | 30 |
Antigen: The antibodies were raised against an N-biotinylated synthetic peptide corresponding to residues 189 to 217 (HFERPRGPRLGYSIPSRSGASGLDKRDYV) from the Claudin-9 protein (Uniprot #Q9Z0S7). An irrelevant N-biotinylated antimicrobial peptide LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES, UniProt #P49913) was used as a negative control.
Protocol: The whole procedure was carried out at room temperature. Biotinylated peptides (saturating concentration of 10 pmol/well) were immobilized on streptavidin-coated 8-well ELISA plate (Pierce #15120) for 30 min. Each well was rinsed three times with 100 μL of washing buffer (PBS + 0.1% (w/v) BSA + 0.05% (w/v) Tween20), then incubated for 30 minutes with 50 µl of antibody-containing supernatant diluted in washing buffer as indicated (Fig. 1). After rinsing 3 times (100 µl washing buffer), wells were incubated with horseradish peroxidase-coupled goat anti-mouse IgG (Bio-Rad #170-6516, dilution 1:1000, 50 μl per well) for 30 min. After 5 rinses, Tetramethylbenzidine (TMB) substrate (Sigma T5569) was added (50 μl per well). The reaction was stopped by the addition of 25 μl of 2 M H2SO4. The absorbance (OD) was measured at 450 nm.
Results
Antibodies RB599, RB600, RB601, RB602, RB603 and RB605 bound in a concentration-dependent manner to the Claudin-9 peptide (CLD9), but not to the LL-37 peptide (Fig. 1). Although these antibodies recognize specifically the Claudin-9 peptide by ELISA, their ability to bind the full-length protein should be determined in future experiments.
Figure 1. RB599, RB600, RB601, RB602, RB603 and RB605 bound specifically to the Claudin-9 peptide (CLD9), but not to the negative control peptide (LL-37) (shown only for RB603; the other background curves were superimposed), as detected by ELISA.
Conflict of interest
The authors declare no conflict of interest.