The AJ521 antibody detects the human CD1b protein by flow cytometry

Abstract

The AJ521 antibody detects the human CD1b protein by flow cytometry.

Introduction

Human CD1b (Uniprot #P29016), a protein displayed at the surface of antigen-presenting cells, is involved in the presentation of lipid antigens to T cells (Porcelli et al., 1992). Here, we describe the ability of the AJ521 antibody, a single chain fragment (scFv) derived from the BCD1b3.1 hybridoma, to successfully detect the CD1b protein by flow cytometry in CD1b-transfected HEK293 cells.

Materials & Methods

Antibodies: ABCD_AJ521 antibody (ABCD nomenclature, http://web.expasy.org/abcd/; Lima et al., 2019) and IgG produced by BCD1b3.1 hybridoma were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/). AJ521 antibody was produced as mini-antibody with the antigen-binding scFv fused to a rabbit IgG Fc. The synthesized scFv sequence (GeneArt, Invitrogen) corresponds to the sequence of the variable regions of the BCD1b3.1 hybridoma (Behar et al., 1995) joined by a peptide linker (GGGGS)₃. The sequencing of the BCD1b3.1 hybridoma was performed by the Geneva Antibody Facility. HEK293 suspension cells (growing in serum-free FreeStyle^TM 293 Expression Medium, Gibco #12338) were transiently transfected with the vector coding for the scFv-Fc. AJ521 supernatant was collected after 4 days. Production of AJ521 was undetectable in this system, indicating a low production yield (<5 mg/L).

Antigen: The BCD1b3.1 hybridoma was originally raised against human CD1⁺ monocytes in BALB/c mice (Behar et al., 1995). HEK293 suspension cells (growing in FreeStyle^TM 293 Expression Medium, Gibco #12338) were transiently transfected 3 days before the experiment with the vector coding for the human CD1b protein fused to its β2 microglobulin subunit (Mercanti et al., 2010).

Protocol: The whole procedure was carried out at 4°C. 500’000 transfected cells were pelleted and washed once with washing buffer (PBS + 0.2% BSA (w/v)). Cells were then incubated for 20 minutes with either the original mouse hybridoma BCD1b3.1 supernatant (dilution 1:2 in PBS-BSA) or with the reformatted scFv antibody AJ521 (undiluted). After two washes in washing buffer, cells were incubated for 20 minutes with either secondary goat anti-mouse or anti-rabbit IgG conjugated to Alexa Fluor 488 (dilution 1:400, Molecular Probes #A11029 and #A11034 respectively). After two washes in washing buffer, cells were resuspended in 500 µL of washing buffer and analyzed with a flow cytometer (BD Accuri^TM C6).

Results

Both the IgG produced by the mouse hybridoma BCD1b3.1 and the reformatted scFv AJ521 detect the CD1b protein at the cell surface of CD1b transfected cells. No signal was detected in mock transfected cells (Fig. 1).

Figure 1. Both the IgG produced by the mouse hybridoma BCD1b3.1 (blue) and the reformatted scFv AJ521 (orange) label HEK293 cells overexpressing the CD1b protein. No signal was detected in mock transfected cells incubated with the AJ521 antibody (red).

Conflict of interest

The authors declare no conflict of interest.