Rapid, scalable assay of amylin‐β amyloid co‐aggregation in brain tissue and blood

Abstract

AbstractBackgroundIslet amyloid polypeptide (amylin) secreted from the pancreas crosses the blood brain barrier into the brain parenchyma and interacts with Alzheimer’s disease (AD) associated cerebral ß‐amyloid (Aß) plaques, which are found in both sporadic and early‐onset familial AD. Immunoprecipitated amylin from human AD brain tissue analyzed by Western blot using an anti‐Aß antibody detects Aß immunoreactivity, which indicates that the amylin and Aß peptides aggregate in the brains of humans with AD, forming amylin‐Aß hetero‐oligomers that are sodium dodecyl sulfate (SDS)‐soluble. We report the development of an ELISA to detect amylin‐Aß hetero‐oligomers in brain tissue and blood.MethodThe amylin‐Aß ELISA relies on a monoclonal anti‐Aß middomain antibody (detection) and a polyclonal anti‐amylin antibody (capture) designed to recognize an epitope that is distinct from the high affinity amylin‐Aß binding sites. We screened temporal cortex homogenates obtained from 13 cognitively unaffected (CU) humans and 47 persons with sporadic AD (sAD) for amylin‐Aß hetero‐oligomers. Individuals were of similar age (sAD, 85.65 ± 7.04 years vs. CU, 87.22 ± 7.30 years; p = 0.482). Brain tissue amylin and Aß concentrations were measured using conventional ELISAs. Amylin‐Aß oligomer levels were also measured in whole blood lysates from APP/PS1 rats and APP/PS1 rats expressing amyloid forming amylin in the pancreas (APP/PS1/HIP rats; all rats age‐matched, 16‐months).ResultThe brains of most AD patients had detectable amylin‐Aß hetero‐oligomer concentrations, whereas about half of those CU had brain tissue amylin‐Aß hetero‐oligomers below the detection limit (0.02 ng/mg total protein; one‐way ANOVA, P < 0.0001). Amylin‐Aß hetero‐oligomers were more common in brains with frequent neuritic plaques (CERAD C). Higher brain tissue amylin‐Aß hetero‐oligomer levels were associated with greater brain tissue amylin concentrations (r = 0.38; P < 0.05). There was no correlation between cerebral amylin‐Aß hetero‐oligomer accumulation and age in the analysis of samples from sAD and CU groups (r = ‐0.22; P > 0.05). Levels of amylin‐Aß hetero‐oligomers in whole blood lysates obtained from APP/PS1/HIP rats were higher than those from APP/PS1 littermates (P < 0.05).ConclusionStudies are needed to determine the extent to which amylin‐Aß hetero‐oligomerization can be detected in human blood.