Fully automated chemiluminescence enzyme immunoassays showing high correlation with immunoprecipitation mass spectrometry assays for ß-amyloid (1-40) and (1-42) in plasma samples.

Blood based ß-amyloid (Aß) assays that can predict amyloid positivity in the brain are in high demand. Current studies that utilize immunoprecipitation mass spectrometry assay (IP-MS), which has high specificity for measuring analytes, have revealed that precise plasma Aß assays have the potential to detect amyloid positivity in the brain. In this study, we developed plasma Aß40 and Aß42 immunoassays using a fully automated immunoassay platform that is used in routine clinical practice. Our assays showed high sensitivity (limit of quantification: 2.46 pg/mL [Aß40] and 0.16 pg/mL [Aß42]) and high reproducibility within-run (coefficients of variation [CVs]: <3.7% [Aß40] and <2.0% [Aß42]) and within-laboratory (CVs: <4.6% [Aß40] and <5.3% [Aß42]). The interference from plasma components was less than 10%, and the cross-reactivity with various lengths of Aß peptides was less than 0.5%. In addition, we found a significant correlation between the IP-MS method and our immunoassay (correlation coefficients of Pearson's r: 0.91 [Aß40] and 0.82 [Aß42]). Our new method to quantify plasma Aß40 and Aß42 provides clinicians and patients with a way to continuously monitor disease progression.

Evaluation of the effect of polymeric microneedle arrays of varying geometries in combination with a high-velocity applicator on skin permeability and irritation.

Polymeric microneedles offer the advantages of being both mass-producible and inexpensive. However, their weakness lies in the fact that they are not adequate for sharp fabrication of a needle tip, which is an important factor for effective penetration. We hypothesized that effective penetration can be achieved using a high-velocity application system. Therefore, in the present study, we investigated the influence of various polymeric microneedle array geometries on skin permeability and irritation using such a system. Volar forearms of 16 healthy volunteers were treated using the microneedle system with four different parameters: applicator velocity (4.3, 6, and 8.5 m/s), tip radius (10, 15, and 20 µm), length (100, 200, and 300 µm), and number of needles (189 and 305 on a 50-mm(2) area). A higher velocity of piercing clearly enhanced skin permeability and damage. A larger tip radius resulted in lower skin permeability and irritation at an applicator velocity of 4.3 m/s but did not have an effect at 6 m/s. Skin permeability was positively variable, ranging from 100 to 200 µm of needle length, and needle number showed no influence in the range investigated. In conclusion, a faster application speed could significantly enhance skin permeability and damage and compensate for insufficient penetration of the larger tip radius and shorter needles, which are also important factors for effective insertion.

Fully automated and highly specific plasma ß-amyloid immunoassays predict ß-amyloid status defined by amyloid positron emission tomography with high accuracy.

BACKGROUND: Clinicians, researchers, and patients alike would greatly benefit from more accessible and inexpensive biomarkers for neural ß-amyloid (Aß). We aimed to assess the performance of fully automated plasma Aß immunoassays, which correlate significantly with immunoprecipitation mass spectrometry assays, in predicting brain Aß status as determined by visual read assessment of amyloid positron emission tomography (PET). METHODS: The plasma Aß42/Aß40 ratio was measured using a fully automated immunoassay platform (HISCL series) in two clinical studies (discovery and validation studies). The discovery and validation sample sets were retrospectively and randomly selected from participants with early Alzheimer's disease (AD) identified during screening for the elenbecestat Phase 3 program. RESULTS: We included 197 participants in the discovery study (mean [SD] age 71.1 [8.5] years; 112 females) and 200 in the validation study (age 70.8 [7.9] years; 99 females). The plasma Aß42/Aß40 ratio predicted amyloid PET visual read status with areas under the receiver operating characteristic curves of 0.941 (95% confidence interval [CI] 0.910-0.973) and 0.868 (95% CI 0.816-0.920) in the discovery and validation studies, respectively. In the discovery study, a cutoff value of 0.102 was determined based on maximizing the Youden Index, and the sensitivity and specificity were calculated to be 96.0% (95% CI 90.1-98.9%) and 83.5% (95% CI 74.6-90.3%), respectively. Using the same cutoff value, the sensitivity and specificity in the validation study were calculated to be 88.0% (95% CI 80.0-93.6%) and 72.0% (95% CI 62.1-80.5%), respectively. CONCLUSIONS: The plasma Aß42/Aß40 ratio measured using the HISCL series achieved high accuracy in predicting amyloid PET status. Since our blood-based immunoassay system is less invasive and more accessible than amyloid PET and cerebrospinal fluid testing, it may contribute to the diagnosis of AD in routine clinical practice.

Alternative splicing in human transcriptome: functional and structural influence on proteins.

Alternative splicing is a molecular mechanism that produces multiple proteins from a single gene, and is thought to produce variety in proteins translated from a limited number of genes. Here we analyzed how alternative splicing produced variety in protein structure and function, by using human full-length cDNAs on the assumption that all of the alternatively spliced mRNAs were translated to proteins. We found that the length of alternatively spliced amino acid sequences, in most cases, fell into a size shorter than that of average protein domain. We evaluated comprehensively the presumptive three-dimensional structures of the alternatively spliced products to assess the impact of alternative splicing on gene function. We found that more than half of the products encoded proteins which were involved in signal transduction, transcription and translation, and more than half of alternatively spliced regions comprised interaction sites between proteins and their binding partners, including substrates, DNA/RNA, and other proteins. Intriguingly, 67% of the alternatively spliced isoforms showed significant alterations to regions of the protein structural core, which likely resulted in large conformational change. Based on those findings, we speculate that there are a large number of cases that alternative splicing modulates protein networks through significant alteration in protein conformation.

A minimally invasive system for glucose area under the curve measurement using interstitial fluid extraction technology: evaluation of the accuracy and usefulness with oral glucose tolerance tests in subjects with and without diabetes.

BACKGROUND: Recent studies have highlighted the importance of managing postprandial hyperglycemia, but adequate monitoring of postprandial glucose remains difficult because of wide variations in levels. We have therefore developed a minimally invasive system to monitor postprandial glucose area under the curve (AUC). This system involves no blood sampling and uses interstitial fluid glucose (IG) AUC (IG-AUC) as a surrogate marker of postprandial glucose. This study aimed to evaluate the usefulness of this system by comparing data with the findings of oral glucose tolerance tests (OGTTs) in subjects with and without diabetes. SUBJECTS AND METHODS: The glucose AUC monitoring system was validated by OGTTs in 37 subjects with and 10 subjects without diabetes. A plastic microneedle array was stamped on the forearm to extract IG. A hydrogel patch was then placed on the pretreated area to accumulate IG. Glucose and sodium ion concentrations in the hydrogel were measured to calculate IG-AUC at 2-h postload glucose. Plasma glucose (PG) levels were measured every 30 min to calculate reference PG-AUC. RESULTS: IG-AUC correlated strongly with reference PG-AUC (r=0.93) over a wide range. The level of correlation between IG-AUC and maximum PG level was also high (r=0.86). The painless nature of the technique was confirmed by the response of patients to questionnaires. CONCLUSIONS: The glucose AUC monitoring system using IG provided good estimates of reference PG-AUC and maximum PG level during OGTTs in subjects with and without diabetes. This system provides easy-to-use monitoring of glucose AUC, which is a good indicator of postprandial glucose.

Measurement of glucose area under the curve using minimally invasive interstitial fluid extraction technology: evaluation of glucose monitoring concepts without blood sampling.

BACKGROUND: Monitoring postprandial hyperglycemia is crucial in treating diabetes, although its dynamics make accurate monitoring difficult. We developed a new technology for monitoring postprandial hyperglycemia using interstitial fluid (ISF) extraction technology without blood sampling. The glucose area under the curve (AUC) using this system was measured as accumulated ISF glucose (IG) with simultaneous calibration with sodium ions. The objective of this study was to evaluate this technological concept in healthy individuals. METHODS: Minimally invasive ISF extraction technology (MIET) comprises two steps: pretreatment with microneedles and ISF accumulation over a specific time by contact with a solvent. The correlation between glucose and sodium ion levels using MIET was evaluated in 12 subjects with stable blood glucose (BG) levels during fasting. BG and IG time courses were evaluated in three subjects to confirm their relationship while BG was fluctuating. Furthermore, the accuracy of glucose AUC measurements by MIET was evaluated several hours after a meal in 30 subjects. RESULTS: A high correlation was observed between glucose and sodium ion levels when BG levels were stable (R=0.87), indicating that sodium ion is a good internal standard for calibration. The variation in IG and BG with MIET was similar, indicating that IG is an adequate substitute for BG. Finally, we showed a strong correlation (R=0.92) between IG-AUC and BG-AUC after a meal. CONCLUSIONS: These findings validate the adequacy of glucose AUC measurements using MIET. Monitoring glucose using MIET without blood sampling may be beneficial to patients with diabetes.