The AF122 antibody recognizes the AChR subunit in murine muscle endplates by immunofluorescence
The recombinant antibody AF122 binds to the acetylcholine receptor (AChR) a subunit expressed in murine muscle endplates and can be detected by immunofluorescence in paraformaldehyde (PFA)-fixed tibialis anterior cryosections.
The AChR (UniProt #P04756) is a subunit of the ligand-gated nicotinic AChR ion channel expressed at the neuromuscular junction. Binding of its ligand ACh leads to ion influx, to depolarization of the postsynaptic membrane and to subsequent contraction of the muscle. Here we report that the recombinant antibody AF122 detects by immunofluorescence the AChR subunit in cryosections of the murine tibialis anterior muscle.
Materials & Methods
Antibodies: ABCD_AF122 (ABCD nomenclature, http://web.expasy.org/abcd/) targets the subunit of the AChR. It was produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies) as mini-antibody with the antigen-binding scFv fused to a human IgG1 Fc. The synthesized scFv sequence (GeneArt, Invitrogen) corresponds to the sequence of the variable regions of the clone 637 (Graus et al., 1997) joined by a peptide linker (GGGGS)4. HEK293T cells (growing in DMEM GlutaMAXTM (Gibco, #31966) supplemented with 8% Fetal Bovine Serum (Gibco, #10270)) were transiently transfected with the vector coding for the scFv-Fc of the antibody. Supernatants (<5 g/L) were collected after 4 days.
Antigen: The antibody AF122 was originally generated by a phage display selected for reactivity against the human AChR (UniProt #P02708). The phage display Fab library was generated from thymic lymphocytes derived from two Myasthenia gravis patients (Graus et al., 1997). Tibialis anterior was isolated from C57BL/6 mice and rinsed in PBS. The muscle was embedded in OCT (Tissue-Tek, #4583) and cryosectioned at 10 m using a Hyrax C60 cryostat (Zeiss).
Protocol: Cryosections were fixed with 4% (w/v) PFA (Electron Microscopy Sciences, #15710) in 0.1 M phosphate buffer, pH 7.4, for 10 min at room temperature, washed in PBS, and blocked with PBS supplemented with 0.1% Triton X-100 and 5% normal goat serum. Subsequently, sections were incubated with the following primary antibodies overnight at 4°C: Alexa Fluor 647-conjugated -bungarotoxin (1:300, Thermo Fisher Scientific, #B35450), mouse anti-neurofilament 200 (1:300, Sigma, #N0142) and the undiluted human monoclonal antibody AF122. Sections were then washed in PBS and incubated with Alexa Fluor 488-labeled goat anti-human and Alexa Fluor 555-labeled goat anti-mouse secondary antibody overnight at 4°C (1:250 Life, Technologies, #A11013, #A28180). Counterstaining was performed using SlowFade Gold antifade reagent with DAPI (Invitrogen, #S36936). Fluorescence photomicrographs were captured with a SP5 Leica confocal laser scanning microscope (SP5; Leica, Heerbrug, Switzerland) equipped with argon and helium lasers using the 40x objective (oil immersion, NA1.25). Images were processed and merged by Imaris imaging software (Bitplane, Zurich, Switzerland).
The AF122 antibody staining in the neuromuscular endplates of the murine tibialis anterior colocalizes with bungarotoxin (Fig. 1), which binds with high affinity to the AChR subunit (Tzartos et al., 1983). Neuromuscular endplates are identified by the presence of AChR in the proximity of nerve fibers in the muscle. Thus, AF122 recognizes the AChR in murine PFA-fixed muscle endplates.
Conflict of interest
The authors declare no conflict of interest.
Graus YF, De Baets MH, Van Breda Vriesman PJ, Burton DR. Anti-acetylcholine receptor Fab fragments isolated from thymus-derived phage display libraries from myasthenia gravis patients reflect predominant specificities in serum and block the action of pathogenic serum antibodies. J Immunol. 1997; 158(4):1919-29. PMID: 9029134
Tzartos SJ, Changeux JP. High affinity binding of alpha-bungarotoxin to the purified alpha-subunit and to its 27,000-dalton proteolytic peptide from Torpedo marmorata acetylcholine receptor. Requirement for sodium dodecyl sulfate. EMBO J. 1983; 2(3):381–387. PMID: 11894953
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