The AJ513 antibody recognizes the Dictyostelium p25 marker by immunofluorescence

Abstract

The AJ513 antibody, derived from the H72 hybridoma, detects by immunofluorescence the p25-labelled compartments from Dictyostelium discoideum.

Introduction

The H72 monoclonal antibody recognizes the (unidentified) p25 antigen of D. discoideum, used as a marker of cell surface and recycling endosomes (Ravanel et al., 2001; Charette et al., 2006). Here we describe the ability of the AJ513 antibody, a single chain fragment (scFv) derived from the H72 hybridoma, to label p25 compartments by immunofluorescence.

Materials & Methods

Antibodies: ABCD_AJ513 antibody (ABCD nomenclature, http://web.expasy.org/abcd/) was produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) 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 joined by a peptide linker (GGGGS)₃. The sequencing of the H72 hybridoma was performed by the Geneva Antibody Facility. HEK293 suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco #12338) were transiently transfected with the vector coding for the scFv-Fc. Supernatants (~50 mg/L) were collected after 5 days.

Antigen: 5x10⁵ D. discoideum DH1 cells, sedimented on a 22x22 mm glass coverslip (Menzel-Gläser) for 90 minutes at room temperature in HL5 medium, were used.

Protocol: Cells were fixed with HL5 + 4% paraformaldehyde (w/v) (Applichem, #A3013) for 30 min, and blocked with PBS + 40 mM ammonium chloride (NH₄Cl) (Applichem, #A3661) for 5 min. Cells were then permeabilized in methanol at -20 ^oC for 2 min, washed once (5 min) with PBS, and once (15 min) with PBS + 0.2% (w/v) BSA (PBS-BSA). Cells were then incubated for 30 min with the original mouse hybridoma H72 supernatant (dilution 1:3 in PBS-BSA) and with the reformatted scFv antibody (dilution 1:10 in PBS-BSA). After 3 washes (5, 5, 15 min) with PBS-BSA, cells were incubated for 30 min with secondary goat anti-mouse IgG conjugated to AlexaFluor-488 (hybridoma) and goat anti-rabbit IgG conjugated to AlexaFluor-647 (scFv) (1:300, Molecular Probes #A11029 and #A21245, respectively). After 3 washes (5, 5, 15 min) with PBS-BSA and one wash (5 min) with PBS, coverslips were mounted on slides (Menzel-Gläser, 76x26 mm) with Möwiol (Hoechst) + 2.5% (w/v) DABCO (Fluka, #33480). Pictures were taken using a Zeiss LSM700 confocal microscope, with a 63x Neofluar oil immersion objective.

Results

In agreement with the original descriptions of the H72 hybridoma (Ravanel et al., 2001; Charette et al., 2006), the AJ513 antibody labels the cell surface and dot‐like structures in the center of the cell, characterized as recycling endosomes (Fig. 1). The staining with both antibodies appears almost indistinguishable (Fig. 1).

Figure 1. The H72 hybridoma and the AJ513 antibody label the cell surface and endosomal compartments in Dictyostelium cells. A double fluorescence staining with both antibodies was performed. No labelling was seen when the primary antibodies were omitted (No Ab). Scale bar: 10 µm.

Conflict of interest

Pierre Cosson and Wanessa Cristina Lima are editors of the Antibody Reports journal.