Development of an Immunoassay for the Detection of Amyloid Beta 1-42 and Its Application in Urine Samples.

Amyloid beta peptides (Aß1-42) have been found to be associated with the cause of Alzheimer's disease (AD) and dementia. Currently, methods for detecting Aß1-42 are complicated and expensive. The present study is aimed at developing an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect Aß1-42 by using a polyclonal antibody from alpaca, an application used in urine samples. The serum was collected from the alpaca after immunizing it with Aß1-42 at 500 µg/injection 5 times. The ic-ELISA was developed and showed a half-maximal inhibitory concentration (IC50) of 103.20 ng/ml. The limit of detection (LOD) was 0.39 ng/100 µl. The cross-reactivity was tested with Aß1-40 and 8 synthesized peptides that had sequence similarities to parts of Aß1-42. The cross-reactivity of Aß1-40 and peptide 1 (DAEFRHDSGYE) was 55% and 69.4%, respectively. The ic-ELISA was applied to analyze Aß1-42 in the urine and precipitated protein urine samples. This method can be used for detecting a normal level of total soluble Aß (approximately 1 ng in 5 mg of precipitated urine protein) and can be used for detecting the early stages of AD. It is considered to be an easy and inexpensive method for monitoring and diagnosing AD.

A Pilot Study of Urinary Exosomes in Alzheimer's Disease.

BACKGROUND: Exosomes are nano-sized extracellular vesicles secreted by most cell types and abundantly present in body fluids, including blood, saliva, urine, cerebrospinal fluid, and breast milk. Exosomes can spread toxic amyloid-beta (Aß) and hyperphosphorylated tau between cells, contributing to neuronal loss in Alzheimer's disease (AD). OBJECTIVE: To explore changes in the morphology, number, and pathological protein levels of urinary exosomes in AD patients compared with age-matched healthy subjects. METHODS: In this study, enzyme-linked immunosorbent assay was used to detect the levels of Aß1-42 and P-S396-tau (normalized by CD63) in urinary exosomes of AD patients and matched healthy subjects. We used transmission electron microscopy and nanoparticle tracking analysis to observe the exosomes. RESULTS: We found that the levels of Aß1-42 and P-S396-tau in the urinary exosomes of AD patients were higher than those of matched healthy controls. Exosomes taken from AD patients were more numerous. CONCLUSION: The differences in levels of Aß1-42 and P-S396-tau and the quantity of urinary exosomes between AD patients and healthy controls may provide a basis for early diagnosis of AD.

Evaluation of dementia by acrolein, amyloid-ß and creatinine.

Plasma, urine and cerebrospinal fluid (CSF) were examined for biochemical markers of dementia. Protein-conjugated acrolein (PC-Acro) and the amyloid-ß (Aß)40/42 ratio in plasma can be used to detect mild cognitive impairment (MCI) and Alzheimer's disease (AD). In plasma, PC-Acro and the Aß40/42 ratio in MCI and AD were significantly higher relative to non-demented subjects. Furthermore, urine acrolein metabolite, 3-hydroxypropyl mercapturic acid (3-HPMA)/creatinine (Cre) and amino acid-conjugated acrolein (AC-Acro)/Cre in AD were significantly lower than MCI. It was also shown that reduced urine 3-HPMA/Cre correlated with increased plasma Aß40/42 ratio in dementia. The Aß40/PC-Acro ratio in CSF, together with Aß40 and Aß40/42 ratio, was lower in AD than MCI. Increased plasma PC-Acro and Aß40/42 ratio and decreased urine 3-HPMA/Cre correlated with cognitive ability (MMSE). These results indicate that the measurements of acrolein derivatives together with Aß and Cre in biologic fluids is useful to estimate severity of dementia.

Protein misfolding, congophilia, oligomerization, and defective amyloid processing in preeclampsia.

Preeclampsia is a pregnancy-specific disorder of unknown etiology and a leading contributor to maternal and perinatal morbidity and mortality worldwide. Because there is no cure other than delivery, preeclampsia is the leading cause of iatrogenic preterm birth. We show that preeclampsia shares pathophysiologic features with recognized protein misfolding disorders. These features include urine congophilia (affinity for the amyloidophilic dye Congo red), affinity for conformational state-dependent antibodies, and dysregulation of prototype proteolytic enzymes involved in amyloid precursor protein (APP) processing. Assessment of global protein misfolding load in pregnancy based on urine congophilia (Congo red dot test) carries diagnostic and prognostic potential for preeclampsia. We used conformational state-dependent antibodies to demonstrate the presence of generic supramolecular assemblies (prefibrillar oligomers and annular protofibrils), which vary in quantitative and qualitative representation with preeclampsia severity. In the first attempt to characterize the preeclampsia misfoldome, we report that the urine congophilic material includes proteoforms of ceruloplasmin, immunoglobulin free light chains, SERPINA1, albumin, interferon-inducible protein 6-16, and Alzheimer's ß-amyloid. The human placenta abundantly expresses APP along with prototype APP-processing enzymes, of which the α-secretase ADAM10, the ß-secretases BACE1 and BACE2, and the γ-secretase presenilin-1 were all up-regulated in preeclampsia. The presence of ß-amyloid aggregates in placentas of women with preeclampsia and fetal growth restriction further supports the notion that this condition should join the growing list of protein conformational disorders. If these aggregates play a pathophysiologic role, our findings may lead to treatment for preeclampsia.

Improving memory and decreasing cognitive impairment in Tg-APPswe/PSEN1dE9 mice with Aß3-10 repeat fragment plasmid by reducing Aß deposition and inflammatory response.

The present study aimed to investigate the effects of Aß3-10 repeat fragment plasmid for the treatment of Tg-APPswe/PSEN1dE9 (Tg) mice. The plasmid pcDNA3.1-(Aß3-10)10-CpG was constructed and intramuscularly injected into 12-month-old Tg mice. Through the use of behavioral tests, anti-Aß antibody and Aß assays, cytokine assay, Aß deposition, and astrocytes analysis results demonstrated that Aß3-10 repeat fragment plasmid exhibited immunogenicity and reduced memory impairment in Tg mice via clearance of cerebral Aß deposition, without significant side effects. Aß3-10 repeat fragment plasmid immunization reduced Th1 cell-mediated immunity, secretion of interferon-γ, and stimulation to astrocytes. These data showed that the Aß3-10 repeat fragment plasmid improved memory and decreased cognitive impairment in Tg mice by reducing Aß deposition and inflammatory responses.

Complex pharmacokinetics of a humanized antibody against human amyloid beta peptide, anti-abeta Ab2, in nonclinical species.

PURPOSE: Anti-Aß Ab2 (Ab2) is a humanized monoclonal antibody against amino acids 3-6 of primate (but not rodent) amyloid ß (Aß) and is being evaluated for the treatment of Alzheimer's disease (AD). This study was conducted to predict the human pharmacokinetics of Ab2. METHODS: In vivo PK profile of Ab2 in preclinical species and in vitro mechanistic studies in preclinical and human systems were used for pharmacokinetic predictions. RESULTS: In Tg2576 and PSAPP mice that have ~100-fold higher circulating levels of human Aß compared to humans, elimination of Ab2 was target-mediated, such that exposure was 5-10 fold lower compared to wild-type rodents or to PDAPP mice that have human Aß concentrations in plasma similar to humans. In cynomolgus monkeys, the t(1/2) of Ab2 was faster (<2.5 days) compared to that of the control antibody (~13 days). The fast elimination of Ab2 in cynomolgus monkeys was linked to off-target binding to cynomolgus monkey fibrinogen that was also causing incomplete recovery of Ab2 in cynomolgus monkey serum in blood partitioning experiments. Ab2 had significantly weaker to undetectable binding to human (and mouse) fibrinogen and had good recovery in human serum in blood partitioning experiments. CONCLUSIONS: These data predict that elimination of Ab2 in healthy or AD humans is expected to be slow, with t(1/2) similar to that observed for other humanized antibodies.

Pittsburgh compound-B and Alzheimer's disease biomarkers in CSF, plasma and urine: An exploratory study.

BACKGROUND: The positron emission tomography (PET) radiotracer Pittsburgh Compound-B (PIB) is an in vivo ligand for measuring beta-amyloid (Abeta) load. Associations between PET PIB and cerebrospinal fluid (CSF) Abeta1-42 and apolipoprotein E epsilon4 (APOE epsilon4) have been observed in several studies, but the relations between PIB uptake and other biomarkers of Alzheimer's disease (AD) are less investigated. METHOD: PET PIB, PET 18Fluoro-2-deoxy-D-glucose and different AD biomarkers were measured twice in CSF, plasma and urine 12 months apart in 10 patients with a clinical diagnosis of mild to moderate AD. RESULTS: PIB retention was constant over 1 year, inversely related to low CSF Abeta1-42 (p = 0.01) and correlated positively to the numbers of the APOE epsilon4 allele (0, 1 or 2) (p = 0.02). There was a relation between mean PIB retention and CSF ApoE protein (r = -0.59, p = 0.07), and plasma cystatin C (r = -0.56, p = 0.09). CONCLUSION: PIB retention is strongly related to CSF Abeta1-42, and to the numbers of the APOE epsilon4 allele.

Oxidative damage increased in presenilin1/presenilin2 conditional double knockout mice.

OBJECTIVE: This report aims to describe the oxidative damage profile in brain of presenilin1 and presenilin2 conditional double knockout mice (dKO) at both early and late age stages, and to discuss the correlation between oxidative stress and the Alzheimer-like phenotypes of dKO mice. METHODS: The protein level of Abeta(42) in dKO cortex and free 8-OHdG level in urine were measured by ELISA. Thiobarbituric acid method and spectrophotometric DNPH assay were used to determine the lipid peroxidation and protein oxidation in cortex, respectively. SOD and GSH-PX activities were assessed by SOD Assay Kit-WST and GSH-PX assay kit, separately. RESULTS: Significant decrease of Abeta(42) was verified in dKO cortex at 6 months as compared to control mice. Although lipid peroxidation (assessed by MDA) was increased only in dKO cortex at 3 months and protein oxidation (assessed by carbonyl groups) was basically unchanged in dKO cortex, ELISA analysis revealed that free 8-OHdG, which was an indicator of DNA lesion, was significantly decreased in urine of dKO mice from 3 months to 12 months. Activities of SOD and GSH-PX in dKO and control cortices showed no statistical difference except a significant increase of GSH-PX activity in dKO mice at 9 months. CONCLUSION: Oxidative damage, especially DNA lesion, was correlated with the neurodegenerative symptoms that appeared in dKO mice without the deposition of Abeta(42). Triggers of oxidative damage could be the inflammatory mediators released by activated microglia and astrocytes.

Detection of amyloid beta protein in the urine of Alzheimer's disease patients and healthy individuals.

To seek for a new valid biomarker using non-invasive specimens for the diagnosis of Alzheimer's disease (AD) and mild cognitive impairment (MCI), we carried out the detection of amyloid beta (Abeta) protein in urine. Ten-millilitre urine samples were first sedimented with trichloroacetic acid, and the pellets were resuspended for further analysis by Western blotting with anti-Abeta antibody. The detection sensitivity of the method was 40pg/ml. Rates of subjects positive for monomeric Abeta according to their clinical dementia rating (CDR) were 11.1% for CDR 0, 62.5% for CDR 0.5, 83.3% for CDR 1, 54.5% for CDR 2 and 0% for CDR 3. A single Abeta band relative to the CDR score reflects an alteration in the production, solubility and clearance of Abeta in the brain. Thus, the method could be used as both a diagnostic and monitoring tool in assessing AD and MCI patients during disease-modifying therapies.

Alzheimer's soluble amyloid beta is a normal component of human urine.

Soluble A beta (Sa beta) is normally present at a low concentration in human plasma and cerebrospinal fluid. Although the factors involved in the regulation of Sa beta plasma levels are still unknown, we have explored its excretion in the urine as one of the possible homeostatic mechanisms. The presence of Sa beta in the urine was investigated via immunoprecipitation experiments with anti-A beta antibodies followed by detection and identification by immunoblot, MALDI mass spectrometry and sequence analysis. Soluble A beta (4.3 kDa) immunoreactivity was present in the urine of normal donors, Down's syndrome individuals as well as in patients with renal disorders exhibiting glomerular or mixed proteinuria. Edman degradation of the immunoprecipitated material yielded the intact A beta N-terminus and mass spectra analysis indicated the existence of a major component at mlz 4327, corresponding to the molecular mass of A beta1-40. Semiquantitative data obtained from the immunoprecipitation experiments indicate that under normal conditions the daily excretion of intact Sa beta in the urine represents less than 1% of the circulating pool.