Neuraminidase from arthrobacter ureafaciens

Six control subjects and seven atopic keratoconjunctivitis patients were recruited. Clinical parameters other than diagnosis, such as age and sex information were not collected. The recruitment was announced for consecutive patients who were visiting the Department of Ophthalmology Juntendo University, Juntendo and Urayasu Hospital, Tokyo and Chiba, Japan. When conjunctival samples were collected from AKC patients, there was no treatment of eye drops to these patients. Healthy volunteers who did not show signs of allergic conjunctivitis or corneal diseases were recruited. Written informed consent was received prior to participation. Impression cytology specimens were obtained using EYEPRIM (OPIA Technologies SAS, France) as shown in Fig. 1a. The cells were fixed with 4% paraformaldehyde in PBS, rinsed with PBS, and embedded in O.C.T. compound. The sections were subjected to AB and PAS staining, HID staining, or immunostaining. In some cases, sections were treated with neuraminidase from Arthrobacter ureafaciens (#24229-74, Nacalai Tesque, Japan) before staining.

Purified IgA1 (5 µg) was digested sequentially with exoglycosidases at 37°C under the following conditions: neuraminidase from Arthrobacter ureafaciens (Nacalai Tesque, Kyoto, Japan), 5 mU in 25 µL of 80 mM sodium acetate buffer (pH 5.0) for 18 h; β-galactosidase from bovine testes (Sigma), 2.2 mU in 5 µL of 80 mM sodium acetate buffer (pH 5.0) overnight; β1,4-galactosidase from Streptococcus pneumoniae (Calbiochem, La Jolla, CA), 3 mU in 5 µL of 80 mM sodium acetate buffer (pH 5.0) overnight.