Choline transporter-like protein 2 (CTL2), a 68–72 kDa inner-ear membrane glycoprotein, is a candidate target antigen in autoimmune hearing loss (AIHL). The objective of this study was to test recombinant human CTL2 as a potential target for the detection of human autoantibodies in patients with AIHL.
In vitro assay development.
Human inner ear CTL2 mRNA was cloned into baculovirus and used to infect insect cells. Immunofluorescence and western blotting were used to determine optimal expression of recombinant human CTL2 (rHuCTL2) in insect cells. AIHL patient sera of known reactivity with guinea pig inner ear were tested for antibodies to purified rHuCTL2 on western blots. Sera from normal hearing donors were used as controls.
The rHuCTL2 protein migrated as three bands: a core protein of 62kDa and two N-glycosylated bands at 66 and 70 kDa. Sera from 6/12 (50%) of AIHL patients with antibody to the 68–72 kDa inner-ear protein or to supporting cells also have antibody to rHuCTL2. Four of the four patients with antibody to rHuCTL2 responded to corticosteroids, whereas 4/8 that lacked antibody to rHuCTL2 did not. Among normal human sera, 80% were negative; binding was barely detectable in 3/15 (20%).
The rHuCTL2 protein can be produced efficiently and used as a substrate for testing human sera. Antibodies to rHuCTL2 were detected in 50% of inner-ear–reactive AIHL sera. Additionally, circulating antibody to rHuCTL2 is with associated response to corticosteroids in some AIHL patients. Laryngoscope, 2009