Serum antibody specific for mis-disordered truncated tau (151–391/2N4R) was detected by enzyme-linked immunosorbent assay (ELISA). Ninety-six-well plates (Dynex Technologies, Chantilly, VA, USA) were coated with 250 ng of recombinant full-length tau isoform 2N4R or pathological truncated tau (151–391/2N4R) per well at 4 °C overnight, then washed twice with PBS and blocked with 3% bovine serum albumin (in 0.05%PBS with Tween-20) for 2 hours at 37 °C. After blocking, the plates were incubated with serial dilutions of the serum (100 μl/well in twofold or fivefold dilution steps) for 1 hour at 37 °C. The bound serum antibodies were detected with horseradish peroxidase (HRP)-conjugated goat antimouse immunoglobulin G (IgG) (Zhongshan Golden Bridge Biotechnology Co., Beijing, China) and chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (Thermo Fisher Scientific).
To determine the isotypes of the specific antibodies produced in response to vaccine, mis-disordered truncated tau (151–391/2N4R) was coated onto ELISA plates, and sera from immunized mice were diluted at 1:6000 and added to the plate, followed by the addition of HRP-conjugated IgG1, IgG2a, IgG2b, and IgG3 (Abcam, Cambridge, UK).
To determine the isotypes of the specific antibodies produced in response to vaccine, mis-disordered truncated tau (151–391/2N4R) was coated onto ELISA plates, and sera from immunized mice were diluted at 1:6000 and added to the plate, followed by the addition of HRP-conjugated IgG1, IgG2a, IgG2b, and IgG3 (Abcam, Cambridge, UK).