Detection of AD-specific four repeat tau with deamidated asparagine residue 279-specific fraction purified from 4R tau polyclonal antibody
N-peptide corresponding the RD4 epitope (VQII*NKKLDLSNVQSKC) and d-peptide, deamidated at N279D (VQII*DKKLDLSNVQSKC), were custom synthesized by Eurofin (Tokyo, Japan, purity > 99%).
Formalin-fixed, paraffin-embedded blocks were obtained from four patients of PSP (69–77 yo), a patient of CBD (68 yo), and 4 patients of AD (78–98 yo). Permission of autopsy and of subsequent use for research was obtained from the next to kin of the patients and this study was approved by ethics committee of Tokyo Metropolitan Institute of Medical Science (16-9).
For RD4, serial pretreatment was performed with 0.25% potassium permanganate; KMnO4, for 15 min, 2% oxalic acid for 3 min, > 99% formic acid for 30 min and autoclaved in 0.05 M citrate buffer for 20 min at 120 °C. For 4R tau polyclonal antibody (TIP-4RT-P01, Cosmo bio, Tokyo, Japan) , autoclaved in 0.05 M citrate buffer for 10 min at 120 °C and > 99% formic acid for 10 min . Specificity of immunolabeling was confirmed by its disappearance in the presence of the immunogen peptides.
After washing formyl cellufine in the column, 200 µg of 4R-D peptide was mixed with 0.6 ml of formyl cellufine in 0.6 ml of coupling buffer (50 mM Na2CO3·NaHCO3, pH 8.5) at room temperature on rotating shaker. After adding 3 mg of sodium cyanoborohydride (NaBH3CN), the formyl cellufine was further shaked at 4 °C for 8–12 h, washed with a blocking buffer (0.1 M monoethanolamine; MEA + 50 mM Tris–HCl buffer, pH 8.0) and mixed with 3 mg of NaBH3CN. This 4R-D peptide primed cellufine was washed with the elution buffer (0.1 M glycine–HCl, pH 2.5) and then with washing buffer (1 M NaCl 1% Triton-X100, pH 7.5) and stored at 4 °C until use. Because 4R tau immunoreactivity on AD brain with the anti-4R tau antibody (1:30,000) was absorbed at the peptide concentration around 1.0 × 10−7 µmol/µl, 50 µl of the anti-4R tau antibody was diluted × 8 with 10% bovine serum albumin and mixed with 289.5 µg of 4R-N peptide, equivalent to 1.0 × 10−7 µmol/µl at its final concentration. This mixture was reacted with the cellufine primed with 4R-D peptide at 4 °C for 8 h on a rotating shaker. After washing, the bound antibody was eluted with the elution buffer to obtain 4R-D fraction.
The peptide (N- or d-) was serially diluted to 1.0 × 10−12~4 μmol/μl in 50 mM Tris–HCl, pH 8.8 and coated onto microtiter plates (SUMILON) at 4 °C for 16 h. The plates were blocked with 10% fetal bovine serum for 2 h. They were reacted with the purified 4R-D fraction for 90 min at RT followed by incubation with HRP-anti-rabbit IgG made in goat (Bio-Rad) at 1:1000 dilution, and reacted with the substrate, 0.4 mg/ml o-phenylenediamine, in citrate buffer (24 mM citric acid, 51 mM Na2HPO4), The absorbance at 490 nm was measured using Plate Chameleon (HIDEX) as described.
This work was supported by Grant-in-Aid for JSPS KAKENHI Grant numbers 17H03555 (TU), 16K14572 (TU).
- 1.Dan A, Takahashi M, Masuda-Suzukake M, Kametani F, Nonaka T, Kondo H et al (2013) Extensive deamidation at asparagine residue 279 accounts for weak immunoreactivity of tau with RD4 antibody in Alzheimer’s disease brain. Acta Neuropathol Comm 1:54. https://doi.org/10.1186/2051-5960-1-54 CrossRefGoogle Scholar
- 2.de Silva R, Lashley T, Gibb G, Hanger D, Hope A, Reid A et al (2003) Pathological inclusion bodies in tauopathies contain distinct complements of tau with three or four microtubule-binding repeat domains as demonstrated by new specific monoclonal antibodies. Neuropathol Appl Neurobiol 29(3):288–302CrossRefGoogle Scholar
- 3.Flament S, Delacourte A, Verny M, Hauw JJ, Javoy-Agid F (1991) Abnormal tau proteins in progressive supranuclear palsy. Similarities and differences with the neurofibrillary degeneration of the Alzheimer type. Acta Neuropathol 81(6):591–596Google Scholar
- 5.Hasegawa M, Morishima-Kawashima M, Takio K, Suzuki M, Titani K, Ihara Y (1992) Protein sequence and mass spectrometric analyses of tau in the Alzheimer’s disease brain. J Biol Chem 267(24):17047–17054Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.