Anti human igg hrp

Nocodazole and cytochalasin D were purchased from Sigma (St. Louis, MI, USA). Primary antibodies used were mouse anti-MAP2b and mouse anti-EEA1 from BD Biosciences (USA), mouse anti-β tubulin-1 and mouse anti-golgi 58 from Sigma (St. Louis, MI, USA), and mouse anti-ER from Molecular Probes (Eugene, OR, USA). Phalloidin BODIPY 558/568 and secondary antibodies anti-mouse isotype specific (IgG2a) Alexa Fluor 488, anti-mouse isotype specific (IgG1) Alexa Fluor 594, and anti-mouse isotype specific (IgG2b) Alexa Fluor 594 were all purchased from Molecular Probes (Eugene, OR, USA). Mouse monoclonal antibodies, MV12/1/C2-2/1 (IgG2a) and MV12/2/A5-1/5 (IgG3) specific for JEV E and NS1, respectively, were supplied by Venture Technologies (Malaysia). The flavivirus reactive mouse monoclonal antibody 4G2 was produced in-house. Anti-human IgG HRP and anti-mouse IgG HRP were purchased from DAKO Cytomation (Glostrup, Denmark).

The recombinant protein (1 mg l⁻¹) was coated on the ELISA plates (NUNC-Immuno Plate; Nalge Nunc International, Denmark) and diluted in PBS overnight at 4 °C. The plates were washed twice with PBS and 0.05% Tween-20 and blocked by 0.5% BSA in PBS. The sera were diluted 10 times and loaded into the wells. The plates were incubated for 1 h at 37 °C and washed four times. The HRP(Horseradish Peroxidase) conjugate was added (anti-human IgG/HRP) (Dakocytomation), 1:10 000 dilution in reagent buffer (Tris-buffered saline, 0.05% Tween-20). The plates were incubated for 1 h at room temperature. A final washing step (four times) was followed by addition of 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich, St Louis, MO, USA). After 15 min incubation at room temperature, the reaction was stopped using 1.8 N H₂SO₄. Optical density(OD) value was read using an ELISA reader at 450 nm, with correction values at 540 nm. The titer of the autoantibodies was calculated based on the OD value.