ABCD_RB066, ABCD_RB067, ABCD_RB068, ABCD_RB072 and ABCD_RB073 antibodies recognize the full length CDKN2A protein by ELISA

Abstract

The recombinant antibodies ABCD_RB066, ABCD_RB067, ABCD_RB068, ABCD_RB072 and ABCD_RB073 detect by ELISA the full-length human protein CDKN2A.

Introduction

The cyclin-dependent kinase inhibitor 2A protein (CDKN2A, UniProt #P42771) is a tumor suppressor protein. By binding to CDK4 or CDK6, CDKN2Ainhibits the formation of the CDK4/6-cyclin D complex, thus inducing a G1 phase cell cycle arrest (Zhao et al., 2016). Inactivation of the CDKN2A gene is associated with the development of various cancers, such as melanoma (Kreuger et al., 2023). Previous work reported that ABCD_RB066 binds an N-terminal peptide of CDKN2A, while RB067 binds a C-terminal peptide (Almeida et al., 2024). This study reports the ability of antibodies ABCD_RB066, ABCD_RB067, ABCD_RB068, ABCD_RB072 and ABCD_RB073 to bind the full-length human protein CDKN2A by ELISA.

Materials & Methods

Antibodies: ABCD_RB066 (RB066), ABCD_RB067 (RB067), ABCD_RB068 (RB068), ABCD_RB072 (RB072) and ABCD_RB073 (RB073) (ABCD nomenclature, http://web.expasy.org/abcd/) were generated at the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/). Briefly, a semi-synthetic human phage display library (Novimmune) was screened against the antigens indicated in table 1. After three rounds of panning, selected phage vectors were isolated using a plasmid preparation kit (Qiagen), and the single-chain variable fragment (scFv) inserts were subcloned into custom-made expression vectors and sequenced. The selected antibodies were expressed as mini-antibodies, consisting of the antigen-binding scFv domain fused to rabbit IgG Fc region. Synthesized scFv sequences (GeneArt, Invitrogen) corresponding to the sequences of the heavy and light variable regions were joined by a peptide linker (GGGGS)₃. HEK293 suspension cells growing in HEK TF medium (Xell #861-0001, Sartorius), supplemented with 0.1% Pluronic F68 (Sigma #P1300), were transiently transfected with the vector coding for the scFv-Fc of each antibody. Supernatants containing each antibody at a concentration ranging from 60 to 95 mg/L were collected after 4 days.

Table 1. Antigens used for antibody selection (GST fusion proteins)

Name	Selection antigens
RB066	1-MEPAAGSSMEPSADWLAT-18
RB067	137-TRGSNHARIDAAEGPSDIPD-157
RB068	137-TRGSNHARIDAAEGPSDIPD-157
RB072	Full length CDKN2A
RB073	Full length CDKN2A

Antigen: E. coli BL21(DE3) bacteria harboring a plasmid encoding GST-CDKN2A (full-length) or GST proteins were inoculated into 3 mL LB medium containing 100 µg/mL ampicillin and 10μg/ml chloramphenicol and grown overnight at 37 °C. Half of this pre-culture was used to inoculate 350 mL of fresh TYH medium with ampicillin and 20% of sterile glucose and incubated at 37 °C until OD600 reached ~0.5. Protein expression and biotinylation was induced with 1.5 mM IPTG and 5 mM Biotin for 4 h at 37 °C. Induced cells were harvested by centrifugation (5,000 × g, 15 min, 4 °C) and resuspended in lysis buffer (PBS + 1% Triton X-100; protease inhibitor cocktail). Lysis was performed by bead beating on ice, and the lysate was clarified by centrifugation (20,000 × g, 20 min, 4 °C). The supernatant was then applied to glutathione-coupled sepharose beads (GE Healthcare Life Sciences) equilibrated in PBS buffer and incubated for 30 min at room temperature under rotation. After washing with 20 column volumes, the GST-tagged protein was eluted with 10 mM reduced glutathione in 50 mM Tris-HCl pH 8.0. Eluted fractions were analysed and quantified on SDS-PAGE gel using calibrated BSA.

ELISA: The whole procedure was carried out at room temperature. Antigens were immobilized on streptavidin-coated ELISA plates (10 µM, Pierce #15124) for 30 min. Each well was rinsed three times with 100 μl of washing buffer (PBS + 0.5% (w/v) BSA + 0.05% (w/v) Tween20), then incubated for 30 minutes with 50 µl of antibody-containing supernatant diluted in washing buffer (Fig. 1). After rinsing 3 times (100 µl washing buffer), wells were incubated with horseradish peroxidase-coupled goat anti-rabbit IgG (Sigma #A8275, dilution 1:1000, 50 μl per well) for 20 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 2M H₂SO₄. The absorbance (OD) was measured at 450 nm, and the absorbance at 570 nm was subtracted.

Results & Discussion

As shown in Fig. 1, all five antibodies tested (RB066, RB067, RB068, RB072, and RB073) exhibited concentration-dependent binding to full-length CDKN2A. No signal was detected with GST alone, confirming the specificity of CDKN2A recognition.

Figure 1. Antibodies RB066, RB067, RB068, RB072, and RB073 were tested at serial dilutions against immobilized GST–CDKN2A full-length protein. Curves represent the mean OD (450 nm) obtained from two independent experiments. Binding of RB073 to GST alone was used as a negative control; all other GST control curves were superimposed.

RB066, which was generated against a N-terminal peptide, displayed a moderate signal only at low dilution (66 µg/mL) that rapidly decreased upon further dilution. Although RB066 was previously shown to recognize its cognate peptide in an ELISA setting, its signal against the full-length protein was weak, which may suggest limited accessibility of the N-terminal epitope or a low apparent affinity in this context. RB067 and RB068 were generated against a C-terminal peptide. RB067 was previously shown to recognize its immunizing peptide (Almeida et al,. 2024) and results show here that it also recognizes the full-length CDKN2A protein by ELISA in a dilution-dependent manner. It may indicate that the C-terminal epitope is accessible under the assay conditions used. In contrast, previous study shows that RB068 did not recognize its immunizing peptide by ELISA (Almeida et al., 2024) but our results showed clear binding to the full-length CDKN2A protein, highlighting a discrepancy between peptide- and protein-based ELISA results for this antibody. RB072 and RB073 were generated using the full-length protein as immunogen; therefore, the epitopes they recognize remain unknown.

Conflict of interest

Camille Mary is an associate-editor of the journal Antibody Reports.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.