Nanoparticle-based immunomagnetic assay of plasma biomarkers for differentiating dementia and prodromal states of Alzheimer's disease - A cross-validation study.

Blood-based biomarker assays of plasma ß-amyloid (Aß) and tau have the advantages of cost-effective and less invasive for the diagnosis of Alzheimer's disease (AD). We used two independent cohorts to cross-validate the clinical use of the nanoparticle-based immunomagnetic assay of plasma biomarkers to assist in the differential diagnosis of early AD. There were in total 160 subjects in the derivation cohort, and 242 in the validation cohort both containing controls, mild cognitive impairment due to AD and AD dementia diagnosed according to the 2011 NIA-AA guidelines. The cutoff value for plasma Aß1-42 (16.4 pg/ml) performed the best in differentiating between controls and patients with prodromal or clinical AD, with 92.5% for positive percent agreement (PPA), negative percent agreement (NPA), and overall rate of agreement (ORA). Aß1-42 × tau (642.58) was useful for separating patients with dementia and prodromal states of AD, with 84.9% PPA, 78.8% NPA and 83% ORA.

Plasma and Serum Alpha-Synuclein as a Biomarker of Diagnosis in Patients With Parkinson's Disease.

Background: Parkinson's disease (PD) is the second most common neurodegenerative disease, and α-synuclein plays a critical role in the pathogenesis of PD. Studies have revealed controversial results regarding the correlation between motor severity and α-synuclein levels in peripheral blood from patients with PD. Objective: We examined α-synuclein levels in plasma or serum in patients with PD and investigated the relationship between plasma or serum α-synuclein level and motor symptom severity. Methods: We recruited 88 participants (48 patients with PD and 40 healthy controls). Clinical information was collected, and venous blood was drawn from each participant to be processed to obtain plasma or serum. The plasma or serum α-synuclein level was detected using monoclonal antibodies with magnetic nanoparticles, and was measured through immunomagnetic reduction. Plasma or serum α-synuclein levels were quantitatively detected. Results: In patients with PD, the means of plasma and serum α-synuclein level were 3.60 ± 2.53 and 0.03 ± 0.04 pg/mL, respectively. The areas under the receiver operating characteristic curve of plasma and serum α-synuclein for distinguishing patients with PD from healthy controls were 0.992 and 0.917, respectively. The serum α-synuclein level also showed a significant correlation with patients in H-Y stages 1-3 (r = 0.40, p = 0.025), implying that the serum α-synuclein level may be a potential marker of motor symptom severity in patients with early PD. Conclusions: Our data suggest that the α-synuclein level in serum or plasma can differentiate between healthy controls and patients with PD. Serum α-synuclein levels moderately correlate with motor severity in patients with early PD.

Plasma Biomarkers Differentiate Parkinson's Disease From Atypical Parkinsonism Syndromes.

Objective: Parkinson's disease (PD) has significant clinical overlaps with atypical parkinsonism syndromes (APS), which have a poorer treatment response and a more aggressive course than PD. We aimed to identify plasma biomarkers to differentiate PD from APS. Methods: Plasma samples (n = 204) were obtained from healthy controls and from patients with PD, dementia with Lewy bodies (DLB), multiple system atrophy, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), or frontotemporal dementia (FTD) with parkinsonism (FTD-P) or without parkinsonism. We measured plasma levels of α-synuclein, total tau, p-Tau181, and amyloid beta 42 (Aß42) by immunomagnetic reduction-based immunoassay. Results: Plasma α-synuclein level was significantly increased in patients with PD and APS when compared with controls and FTD without parkinsonism (p < 0.01). Total tau and p-Tau181 were significantly increased in all disease groups compared to controls, especially in patients with FTD (p < 0.01). A multivariate and receiver operating characteristic curve analysis revealed that a cut-off value for Aß42 multiplied by p-Tau181 for discriminating patients with FTD from patients with PD and APS was 92.66 (pg/ml)2, with an area under the curve (AUC) of 0.932. An α-synuclein cut-off of 0.1977 pg/ml could separate FTD-P from FTD without parkinsonism (AUC 0.947). In patients with predominant parkinsonism, an α-synuclein cut-off of 1.388 pg/ml differentiated patients with PD from those with APS (AUC 0.87). Conclusion: Our results suggest that integrated plasma biomarkers improve the differential diagnosis of PD from APS (PSP, CBD, DLB, and FTD-P).

Plasma Amyloid-ß (Aß42) Correlates with Cerebrospinal Fluid Aß42 in Alzheimer's Disease.

The 42 amino acid form of amyloid-ß (Aß42) plays a key role in the pathogenesis of Alzheimer's disease (AD) and is a core biomarker for the diagnosis of AD. Numerous studies have shown that cerebrospinal fluid (CSF) Aß42 concentrations are decreased in AD, when measured by enzyme-linked immunosorbent assay (ELISA) and other conventional immunoassays. While most studies report no change in plasma Aß42, independent studies using the immunomagnetic reduction (IMR) technique report an increase in plasma Aß42 levels in AD. To confirm the opposite changes of Aß42 levels in CSF and plasma for AD, we assayed the levels of Aß42 in plasma of subjects with known CSF Aß42 levels. In total 43 controls and 63 AD patients were selected at two sites: the VU University Medical Center (n = 55) and Sahlgrenska University Hospital (n = 51). IMR and ELISA were applied to assay Aß42 in plasma and CSF, respectively. We found a moderately negative correlation between plasma and CSF Aß42 levels in AD patients (r = -0.352), and a weakly positive correlation in controls (r = 0.186). These findings further corroborate that there are opposite changes of Aß42 levels in CSF and plasma in AD. The possible causes for the negative correlation are discussed by taken assay technologies, Aß42 transport from brain to peripheral blood, and sample matrix into account.

Assay of Plasma Phosphorylated Tau Protein (Threonine 181) and Total Tau Protein in Early-Stage Alzheimer's Disease.

The feasibility of assaying plasma phosphorylated tau protein (threonine 181), denoted p-tau181, using immunomagnetic reduction (IMR) is explored. The reagent for assaying p-tau181 with IMR was synthesized, and its analytic performances were characterized. Seventy-three subjects were recruited. Each participant was examined with neuropsychological tests, magnetic resonance imaging, and IMR assay for plasma p-tau181. Using commercially available IMR kits, the plasma total tau protein (T-tau) of each subject was assayed. The dynamic range for assaying p-tau181 using IMR was 1.96×10-2 pg/ml to 104 pg/ml. There was no significant interference from total tau protein in the assay of p-tau181. The measured concentrations of plasma p-tau181 were 2.46±1.09 pg/ml for healthy controls, 4.41±1.85 pg/ml for MCI due to AD, and 6.14±1.59 pg/ml for very mild AD. Meanwhile, the measured concentrations of plasma T-tau were 18.85±10.16 pg/ml for healthy controls, 32.98±10.18 pg/ml for MCI due to AD, and 37.54±12.29 pg/ml for very mild AD. A significant difference in plasma p-tau181 was observed between healthy controls and MCI due to AD (p < 0.001) and between MCI due to AD and very mild AD (p < 0.001). However, for the plasma T-tau concentration, a significant difference existed only between healthy controls and MCI due to AD (p < 0.001). This implies that the plasma p-tau181 level is correlated more to AD severity than plasma T-tau is. Additionally, p-tau181 was observed as approximately 14% of T-tau in human plasma.

Analytical performance of reagent for assaying tau protein in human plasma and feasibility study screening neurodegenerative diseases.

Immunomagnetic reduction (IMR), which involves the use of antibody-functionalized magnetic nanoparticles to specifically label target biomarkers, was utilized to develop an assay for total tau protein in human plasma. The analytic properties of the IMR assay on tau protein were investigated. The limit of detection was found to be 0.026 pg/ml. Other properties such as Hook effect, assay linearity, dilution recovery range, reagent stability, interference test, and spiked recovery were also characterized. The ultra-sensitive IMR assay was applied to detect the plasma tau protein levels of subjects with prevalent neurodegenerative diseases, such as Alzheimer's disease (AD), mild cognitive impairment (MCI) due to AD, Parkinson's disease (PD), frontotemporal dementia (FTD) and vascular dementia (VD). The concentrations of plasma tau protein in patients with VD, PD, MCI due to AD, FTD, and AD patients were higher than that of healthy controls. Using an ROC curve analysis, the cutoff value for discriminating dementia patients from healthy controls was 17.43 pg/ml, resulting in 0.856 and 0.727 for clinical sensitivity and specificity, respectively. The area under the ROC curve was 0.908. These results imply that the IMR plasma tau assay would be useful to screen for prevalent neurodegenerative diseases.

Plasma Levels of Aß42 and Tau Identified Probable Alzheimer's Dementia: Findings in Two Cohorts.

The utility of plasma amyloid beta (Aß) and tau levels for the clinical diagnosis of Alzheimer's disease (AD) dementia has been controversial. The main objective of this study was to compare Aß42 and tau levels measured by the ultra-sensitive immunomagnetic reduction (IMR) assays in plasma samples collected at the Banner Sun Health Institute (BSHRI) (United States) with those from the National Taiwan University Hospital (NTUH) (Taiwan). Significant increase in tau levels were detected in AD subjects from both cohorts, while Aß42 levels were increased only in the NTUH cohort. A regression model incorporating age showed that tau levels identified probable ADs with 81 and 96% accuracy in the BSHRI and NTUH cohorts, respectively, while computed products of Aß42 and tau increased the accuracy to 84% in the BSHRI cohorts. Using 382.68 (pg/ml)2 as the cut-off value, the product achieved 92% accuracy in identifying AD in the combined cohorts. Overall findings support that plasma Aß42 and tau assayed by IMR technology can be used to assist in the clinical diagnosis of AD.

Plasma α-synuclein predicts cognitive decline in Parkinson's disease.

OBJECTIVE: α-Synuclein is critical to the pathogenesis of Parkinson's disease (PD). Few studies examined the plasma levels of α-synuclein due to the exceptionally low level of α-synuclein in plasma compared with cerebrospinal fluid. We aimed to investigate plasma α-synuclein in patients with PD of different disease severity. METHODS: There were total 114 participants, including 80 patients with PD and 34 controls, in the study. Participants received a complete evaluation of motor and non-motor symptoms, including cognitive function. We applied immunomagnetic reduction-based immunoassay to measure plasma levels of α-synuclein. RESULTS: Plasma levels of α-synuclein were significantly higher in patients with PD compared with controls (median: 1.56 pg/mL, 95% CI 1.02 to 1.98 pg/mL vs 0.02 pg/mL, 95% CI 0.01 to 0.03 pg/mL; p<0.0001). Although there was a significant increase in plasma α-synuclein levels in PD patients with a higher Hoehn-Yahr (H-Y) stage, there was no correlation with motor symptom severity, as assessed by Unified Parkinson's Disease Rating Scale part III scores, after confounders (age, gender, and disease duration) were taken into account. However, plasma α-synuclein levels were significantly higher in PD patients with dementia (PDD) than in PD patients with mild cognitive impairment (PD-MCI) or normal cognition (0.42 pg/mL, (95% CI 0.25 to 0.93) for PD with normal cognition; 1.29 pg/mL (95% CI 0.76 to 1.93) for PD-MCI and 4.09 pg/mL (95% CI 1.99 to 6.19) for PDD, p<0.01) and were negatively correlated with Mini-Mental State Examination scores (R2-adjusted=0.3004, p<0.001), even after confounder adjustment. CONCLUSIONS: Our data suggest that plasma α-synuclein level correlates with cognitive decline but not motor severity in patients with PD. Plasma α-synuclein could serve as a surrogate biomarker for patients at risk of cognitive decline.

Development of an ultra-high sensitive immunoassay with plasma biomarker for differentiating Parkinson disease dementia from Parkinson disease using antibody functionalized magnetic nanoparticles.

BACKGROUND: It is difficult to discriminate healthy subjects and patients with Parkinson disease (PD) or Parkinson disease dementia (PDD) by assaying plasma α-synuclein because the concentrations of circulating α-synuclein in the blood are almost the same as the low-detection limit using current immunoassays, such as enzyme-linked immunosorbent assay. In this work, an ultra-sensitive immunoassay utilizing immunomagnetic reduction (IMR) is developed. The reagent for IMR consists of magnetic nanoparticles functionalized with antibodies against α-synuclein and dispersed in pH-7.2 phosphate-buffered saline. A high-Tc superconducting-quantum-interference-device (SQUID) alternative-current magnetosusceptometer is used to measure the IMR signal of the reagent due to the association between magnetic nanoparticles and α-synuclein molecules. RESULTS: According to the experimental α-synuclein concentration dependent IMR signal, the low-detection limit is 0.3 fg/ml and the dynamic range is 310 pg/ml. The preliminary results show the plasma α-synuclein for PD patients distributes from 6 to 30 fg/ml. For PDD patients, the concentration of plasma α-synuclein varies from 0.1 to 100 pg/ml. Whereas the concentration of plasma α-synuclein for healthy subjects is significantly lower than that of PD patients. CONCLUSIONS: The ultra-sensitive IMR by utilizing antibody-functionalized magnetic nanoparticles and high-Tc SQUID magnetometer is promising as a method to assay plasma α-synuclein, which is a potential biomarker for discriminating patients with PD or PDD.

A Novel Detection Platform for Shrimp White Spot Syndrome Virus Using an ICP11-Dependent Immunomagnetic Reduction (IMR) Assay.

Shrimp white spot disease (WSD), which is caused by white spot syndrome virus (WSSV), is one of the world's most serious shrimp diseases. Our objective in this study was to use an immunomagnetic reduction (IMR) assay to develop a highly sensitive, automatic WSSV detection platform targeted against ICP11 (the most highly expressed WSSV protein). After characterizing the magnetic reagents (Fe3O4 magnetic nanoparticles coated with anti ICP11), the detection limit for ICP11 protein using IMR was approximately 2 x 10(-3) ng/ml, and the linear dynamic range of the assay was 0.1~1 x 10(6) ng/ml. In assays of ICP11 protein in pleopod protein lysates from healthy and WSSV-infected shrimp, IMR signals were successfully detected from shrimp with low WSSV genome copy numbers. We concluded that this IMR assay targeting ICP11 has potential for detecting the WSSV.

Development of antibody functionalized magnetic nanoparticles for the immunoassay of carcinoembryonic antigen: a feasibility study for clinical use.

BACKGROUND: Magnetic nanoparticles functionalized antibodies are used for in-vitro assays on bio-markers. This work demonstrates the synthesis of high-quality magnetic nanoparticles coated with antibodies against carcinoembryonic antigen (CEA). Various characterizations, such as particle size, particle suspension, bio-activity and the stability of bio-magnetic nanoparticles suspended in liquid, are studied. The properties for the assay of CEA molecules in serum are also studied. The assay method used is so-called immunomagnetic reduction. RESULTS: The results show that the effects of common materials in serum that interfere with detected signals are not significant. The low-detection limit is 0.21 ng/ml, which is well below the clinical threshold of 2.5 ng/ml. CONCLUSIONS: The dynamic range for the assay of CEA molecules in serum is 500 ng/ml. By assaying serum CEA molecules from 24 normal controls and 30 colorectal-cancer patients, the threshold for the serum-CEA concentration to diagnose colorectal cancer is 4.05 ng/ml, which results in a clinical sensitivity of 0.90 and specificity of 0.87.

Combined plasma biomarkers for diagnosing mild cognition impairment and Alzheimer's disease.

A highly sensitive immunoassay, the immunomagnetic reduction, is used to measure several biomarkers for plasma that is related to Alzheimer's disease (AD). These biomarkers include Aß-40, Aß-42, and tau proteins. The samples are composed of four groups: healthy controls (n=66), mild cognitive impairment (MCI, n=22), very mild dementia (n=23), and mild-to-serve dementia, all due to AD (n=22). It is found that the concentrations of both Aß-42 and tau protein for the healthy controls are significantly lower than those of all of the other groups. The sensitivity and the specificity of plasma Aß-42 and tau protein in differentiating MCI from AD are all around 0.9 (0.88-0.97). However, neither plasma Aß-42 nor tau-protein concentration is an adequate parameter to distinguish MCI from AD. A parameter is proposed, which is the product of plasma Aß-42 and tau-protein levels, to differentiate MCI from AD. The sensitivity and specificity are found to be 0.80 and 0.82, respectively. It is concluded that the use of combined plasma biomarkers not only allows the differentiation of the healthy controls and patients with AD in both the prodromal phase and the dementia phase, but it also allows AD in the prodromal phase to be distinguished from that in the dementia phase.

Use of immunomagnetic reduction for C-reactive protein assay in clinical samples.

BACKGROUND: Magnetic nanoparticles biofunctionalized with antibodies are able to recognize and bind to the corresponding antigens. In this work, anti-C-reactive protein (CRP) antibody was covalently conjugated onto the surface of magnetic nanoparticles to label CRP specifically in serum. METHODS: The level of serum CRP was detected by immunomagnetic reduction (IMR) assay, which identifies the changes in the magnetic signal representing the level of interaction between antibody-conjugated magnetic nanoparticles and CRP proteins. To investigate the feasibility of IMR for clinical application, pure CRP solutions and 40 human serum samples were tested for IMR detection of CRP to characterize sensitivity, specificity, and interference. RESULTS: In comparison with the immunoturbidimetry assay, the results of the IMR assay indicated higher sensitivity and had a high correlation with those of the current immunoturbidimetry assay. CONCLUSION: We have developed a novel and promising way to assay CRP in human serum using immunomagnetic reduction in clinical diagnosis.

Feasibility studies for assaying alpha-fetoprotein using antibody-activated magnetic nanoparticles.

Some previous reports have already shown the characterizations of immunomagnetic reduction (IMR). The assay technology involves the utilities of biofunctionalized magnetic nanoparticles to label target biomolecules. However, the detection threshold and interference tests for IMR have not been investigated in detail. In this study, alpha-fetoprotein (AFP) was used as a target biomolecule. The signals for AFP solutions of various concentrations, or with interfering materials, were detected via IMR. These samples were also used for characterizing the detection threshold and interference with enzyme-linked immunosorbent assay (ELISA). The results of assaying AFP level with IMR and ELISA were compared. The detection threshold for assaying AFP with IMR was found to be 3 ng/mL, which is 15 times lower than that of ELISA, and definitely suppresses false negative. For the interfering materials noted commonly in serum such as hemoglobin, bilirubin, triglyceride, and vascular endothelial growth factor, there was no detectable interfering effect when assaying AFP with IMR. Several serum samples from normal people and liver-tumor-bearing patients were used for the detections of AFP concentration via IMR. These results reveal the feasibilities of assaying AFP in blood using IMR, as well as achieving high-sensitive and high-specific assay for AFP.

Exploration of the relationship between the tumor burden and the concentration of vascular endothelial growth factor in liver-cancer-bearing animals using immunomagnetic reduction assay.

To achieve early-stage diagnosis, a high-sensitivity assay method is needed. As a biomarker, vascular endothelial growth factor (VEGF) has played a growing role in diagnosing and treating hepatocellular carcinoma (HCC). In this work, an immunomagnetic reduction (IMR) through bio-functionalized magnetic nanoparticles and a high-temperature superconducting-quantum-interference-device magnetometer were utilized for quantitative detection of low-concentration VEGF in serum from rats with HCC. The precision and accuracy of IMR on VEGF were characterized. Further, the results of assaying VEGF in the serum of rats were compared with those of using enzyme-linked immunosorbant assay (ELISA). It was found the correlations between the detected VEGF concentration in the rat serum and tumor burdens were 0.99 and 0.90 for IMR and ELISA, respectively, within the range from 2 pg/ml to 8000 pg/ml of VEGF concentration.

Structural basis for RNA trimming by RNase T in stable RNA 3'-end maturation.

RNA maturation relies on various exonucleases to remove nucleotides successively from the 5' or 3' end of nucleic acids. However, little is known regarding the molecular basis for substrate and cleavage preference of exonucleases. Our biochemical and structural analyses on RNase T-DNA complexes show that the RNase T dimer has an ideal architecture for binding a duplex with a short 3' overhang to produce a digestion product of a duplex with a 2-nucleotide (nt) or 1-nt 3' overhang, depending on the composition of the last base pair in the duplex. A 'C-filter' in RNase T screens out the nucleic acids with 3'-terminal cytosines for hydrolysis by inducing a disruptive conformational change at the active site. Our results reveal the general principles and the working mechanism for the final trimming step made by RNase T in the maturation of stable RNA and pave the way for the understanding of other DEDD family exonucleases.

Biofunctionalized magnetic nanoparticles for specifically detecting biomarkers of Alzheimer's disease in vitro.

Magnetic nanoparticles biofunctionalized with antibodies against ß-amyloid-40 (Aß-40) and Aß-42, which are promising biomarkers related to Alzheimer's disease (AD), were synthesized. We characterized the size distribution, saturated magnetizations, and stability of the magnetic nanoparticles conjugated with anti-Aß antibody. In combination with immunomagnetic reduction technology, it is demonstrated such biofunctionalized magnetic nanoparticles are able to label Aßs specifically. The ultralow-detection limits of assaying Aßs in vitro using the magnetic nanoparticles via immunomagnetic reduction are determined to a concentration of ∼10 ppt (10 pg/mL). Further, immunomagnetic reduction signals of Aß-40 and Aß-42 in human plasma from normal samples and AD patients were analyzed, and the results showed a significant difference between these two groups. These results show the feasibility of using magnetic nanoparticles with Aßs as reagents for assaying low-concentration Aßs through immunomagnetic reduction, and also provide a promising new method for early diagnosis of Alzheimer's disease from human blood plasma.

Structural and biochemical characterization of CRN-5 and Rrp46: an exosome component participating in apoptotic DNA degradation.

Rrp46 was first identified as a protein component of the eukaryotic exosome, a protein complex involved in 3' processing of RNA during RNA turnover and surveillance. The Rrp46 homolog, CRN-5, was subsequently characterized as a cell death-related nuclease, participating in DNA fragmentation during apoptosis in Caenorhabditis elegans. Here we report the crystal structures of CRN-5 and rice Rrp46 (oRrp46) at a resolution of 3.9 A and 2.0 A, respectively. We found that recombinant human Rrp46 (hRrp46), oRrp46, and CRN-5 are homodimers, and that endogenous hRrp46 and oRrp46 also form homodimers in a cellular environment, in addition to their association with a protein complex. Dimeric oRrp46 had both phosphorolytic RNase and hydrolytic DNase activities, whereas hRrp46 and CRN-5 bound to DNA without detectable nuclease activity. Site-directed mutagenesis in oRrp46 abolished either its DNase (E160Q) or RNase (K75E/Q76E) activities, confirming the critical importance of these residues in catalysis or substrate binding. Moreover, CRN-5 directly interacted with the apoptotic nuclease CRN-4 and enhanced the DNase activity of CRN-4, suggesting that CRN-5 cooperates with CRN-4 in apoptotic DNA degradation. Taken together all these results strongly suggest that Rrp46 forms a homodimer separately from exosome complexes and, depending on species, is either a structural or catalytic component of the machinery that cleaves DNA during apoptosis.

Degradation of human thymidine kinase is dependent on serine-13 phosphorylation: involvement of the SCF-mediated pathway.

The expression level of human thymidine kinase (hTK) is regulated in a cell-cycle-dependent manner. One of the mechanisms responsible for the fluctuation of TK expression in the cell cycle can be attributed to protein degradation during mitosis. Given the facts that cell-cycle-dependent proteolysis is highly conserved in all eukaryotes and yeast cells are an excellent model system for protein-degradation study, here we report on the use of Saccharomyces cerevisiae and Schizosaccharomyces pombe to investigate the degradation signal and mechanism required for hTK degradation. We found that the stability of hTK is significantly reduced in mitotic yeasts. Previously, we have observed that Ser-13 is the site of mitotic phosphorylation of hTK in HeLa cells [Chang, Huang and Chi (1998) J. Biol. Chem. 273, 12095-12100]. Here, we further provide evidence that the replacement of Ser-13 by Ala (S13A) renders hTK stable in S. pombe and S. cerevisiae. Most interestingly, we demonstrated that degradation of hTK is impaired in S. cerevisiae carrying a temperature-sensitive mutation in the proteasomal gene pre1-1 or the Skp1-Cullin-1/CDC53-F-box (SCF) complex gene cdc34 or cdc53, suggesting the contribution of the SCF-mediated pathway in hTK degradation. As phosphorylation is a prerequisite signal for SCF recognition, our results implied that phosphorylation of Ser-13 probably contributes to the degradation signal for hTK via the SCF-mediated proteolytic pathway.