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P1‐104: DIFFERENCES IN SALIVARY Aβ LEVELS AND ORAL MICROFLORA IN ALZHEIMER'S DISEASE MOUSE MODELS
Author(s) -
Floden Angela M.,
Sohrabi Mona,
Nookala Suba,
Manocha Gunjan D.,
Combs Colin K.
Publication year - 2018
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2018.06.107
Subject(s) - saliva , endocrinology , presenilin , salivary gland , alzheimer's disease , amyloid precursor protein , medicine , secretion , amyloid (mycology) , biology , disease , pathology
various forms and might contain some unstable forms, it is difficult to quantify AbO in degenerative status, such as SDS-PAGE. Therefore, quantitative method of AbO has not been established. Previously, mouse monoclonal anti-AbO form antibody (m6H4) was generated by Matsubara and colleagues (Life Sciences, 2012). In this study, we establish the quantitative measure method of AbO by dot blot assay using m6H4 specific to AbO form in undegenerated status. Methods: Synthetic Ab1–40 monomer and 5-Carboxytetramethylrhodamine (5-TAMRA)-labeled Ab1–40 monomer dissolved in 0.1% ammonium solution (100 mM) were ultra-centrifuged at 540,000 g for 20 h at 4 C to obtain seed-free supernatant. The supernatant fromAb1–40 and 5-TAMRA-labeledAb1–40monomerswere co-incubated to synthesize AbOs at 37 C for 0, 1, 4 and 20 h. Preparing soluble AbOs, the incubated AbO solution were ultra-centrifuged at 100,000 g for 1 h at 4 C. For dot blot assay, 1 mL of soluble AbOs solutions (0, 5, 10, 25 and 50 mM) were spotted on nitrocellulose membrane, and incubated with m6H4 and mouse monoclonal antiAb antibody (4G8) reactive to 17-24 amino acid residue of Ab followed byHRP-labeled secondary antibody and the chemiluminescent substrate. Chemiluminescent signals were visualized using ChemiDoc Touch and quantified using Image Lab software (Bio-Rad Laboratories, Inc.). Results:Signal intensity of AbOs detected by m6H4 demonstrated time-dependent and concentration-dependent upward trends, enabled to make calibration curves which correlation coefficient at each time points were above 0.83 to 0.99. In contrast, signal intensity of Ab monomer and oligomer detected by 4G8 was stable over the period, and detected as concentration-dependent upward trends. The correlation coefficients of calibration curves by 4G8 at each timewere above 0.99. These results suggested that dot blot assay usingm6H4was able to detect AbO specific time dependent and concentration-dependent changes. Conclusions: Production levels of AbOs detected by m6H4 might show time-dependent and concentration-dependent increase. Dot blot assay using our newly developed AbOs specific antibody could be able to quantitate synthetic AbOs.