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Background: The cerebrospinal fluid (CSF) levels of tau phosphorylated at threonine 217 (p217tau) or 181 (p181tau), and neurofilament light chain (NfL) are definite biomarkers of tauopathy and neurodegeneration in Alzheimer's disease (AD). Objective: To validate their utility in excluding other neurological diseases and age-related changes in clinical settings. Methods: We developed monoclonal antibodies against p217tau and NfL, established novel ELISAs, and analyzed 170 CSF samples from patients with AD or other neurological diseases. Results: In AD, p217tau is a more specific and abundant CSF component than p181tau. However, CSF NfL levels increase age-dependently and to a greater extent in central and peripheral nervous diseases than in AD. Conclusions: CSF p217tau correlates better with AD neurodegeneration than other tau-related biomarkers and the major phosphorylated tau species. The clinical usage of NfL as a neurodegeneration biomarker in AD requires exclusion of various central and peripheral neurological diseases. [ABSTRACT FROM AUTHOR]

Abstract Background Autoantibodies against GPIHBP1, the endothelial cell transporter for lipoprotein lipase (LPL), cause severe hypertriglyceridemia ("GPIHBP1 autoantibody syndrome"). Affected patients have low serum GPIHBP1 and LPL levels. We report the development of a sensitive and specific ELISA, suitable for routine clinical use, to detect GPIHBP1 autoantibodies in serum and plasma. Methods Serum and plasma samples were added to wells of an ELISA plate that had been coated with recombinant human GPIHBP1. GPIHBP1 autoantibodies bound to GPIHBP1 were detected with an HRP-labeled antibody against human immunoglobulin. Sensitivity, specificity, and reproducibility of the ELISA was evaluated with plasma or serum samples from patients with the GPIHBP1 autoantibody syndrome. Results A solid-phase ELISA to detect and quantify GPIHBP1 autoantibodies in human plasma and serum was developed. Spiking recombinant human GPIHBP1 into the samples reduced the ability of the ELISA to detect GPIHBP1 autoantibodies. The ELISA is reproducible and sensitive; it can detect GPIHBP1 autoantibodies in samples diluted by >1000-fold. Conclusion We have developed a sensitive and specific ELISA for detecting GPIHBP1 autoantibodies in human serum and plasma; this assay will make it possible to rapidly diagnose the GPIHBP1 autoantibody syndrome. Highlights • GPIHBP1 autoantibodies cause severe hypertriglyceridemia (chylomicronemia). • An ELISA to measure GPIHBP1 autoantibodies in serum or plasma was developed. • The ELISA is sensitive, capable of detecting GPIHBP1 autoantibodies. • This assay is useful for diagnosing the GPIHBP1 autoantibody syndrome. [ABSTRACT FROM AUTHOR]

Background: Recent genetic and pathological studies have suggested that a lipoprotein receptor, LR11, is intricately implicated in the pathogenesis of Alzheimer disease (AD). We have recently established a novel sandwich ELISA, which enabled the sensitive quantification of a soluble LR11 (sLR11). By this ELISA, we attempted to determine the difference in the levels of CSF sLR11 in AD patients. Methods: We examined CSF from 29 AD patients, 20 frontotemporal lobar degeneration patients and 27 age-matched control subjects. The CSF sLR11 level as well as the levels of tau and β-amyloid42 (Aβ42) were determined by sandwich ELISA. Results: The CSF tau level and tau/Aβ42 ratio were significantly increased (p < 0.01) in the AD patients. The CSF sLR11 level in the AD patients was significantly higher (p < 0.01) than that of the frontotemporal lobar degeneration patients and the controls. The APOE-ε4-positive AD patients have higher sLR11 levels than the APOE-ε4-negative patients (p < 0.01). Conclusions: These results suggest that the quantification of CSF sLR11 may serve as a biomarker of AD, although the diagnostic value for individual patients is limited. An elevated CSF sLR11 level in AD patients may be relevant to AD pathogenesis. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Connective tissue growth factor (CTGF) may be a potential marker of fibrosis. However, platelet-derived CTGF may be released into the plasma by platelet activation during or after blood collection, thereby interfering with accurate determination of the true plasma CTGF level. Plasma CTGF exists as the N-terminal CTGF fragment (N-fragment), composed of modules 1 and 2, whereas platelet CTGF exists as full-length CTGF (full-length), composed of modules 1-4. We perceived the need to develop a method for distinguishing between the N-fragment and full-length CTGF levels, so that the true plasma and serum CTGF (N-fragment) levels could be accurately determined. Methods: Full-length levels were determined by a sandwich enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies recognizing modules 1 and 4, respectively (M1/4 ELISA). Total CTGF (full-length CTGF plus N-terminal CTGF) levels were determined by a sandwich ELISA using two monoclonal antibodies recognizing modules 1 and 2, respectively (M1/2 ELISA). N-terminal CTGF levels were determined by subtracting the full-length levels from the total CTGF levels. Results: Both the M1/2 and M1/4 ELISAs showed good analytical performance. When the CTGF levels of plasma and serum collected simultaneously from the same subject were compared, the N-fragment levels determined by the subtraction method were the same, in spite of the fact that full-length CTGF was present in the sample. Conclusion: N-fragment levels in plasma and serum can be accurately determined by this subtraction method, even if full-length CTGF in platelets is released during or after blood collection. [ABSTRACT FROM AUTHOR]

Abstract: Preβ1-HDL, a putative discoid-shaped high-density lipoprotein (HDL) is known to participate in the retrieval of cholesterol from peripheral tissues. In this study, to clarify potential sources of this lipoprotein, we conducted heparin injection on four Japanese volunteer men and found that serum triglyceride (TG) level decreased in parallel with the increase in serum nonesterified fatty acids and plasma lipoprotein lipase (LPL) protein mass after heparin injection. Plasma preβ1-HDL showed considerable increases at 15min after the heparin injection in all of the subjects. In contrast, serum HDL-C levels did not change. Gel filtration with fast protein liquid chromatography system (FPLC) study on lipoprotein profile revealed that in post-heparin plasma, low-density lipoprotein and αHDL fractions did not change, whereas there was a considerable decrease in very low-density lipoprotein (VLDL) fraction and an increase in preβ1-HDL fraction when compared with those in pre-heparin plasma. We also conducted in vitro analysis on whether preβ1-HDL was produced during VLDL lipolysis by LPL. One hundred microliters of VLDL extracted from pooled serum by ultracentrifugation was incubated with purified bovine milk LPL at 37°C for 0–120min. Preβ1-HDL concentration increased in a dose dependent manner with increased concentration of added LPL in the reaction mixture and with increased incubation time, indicating that preβ1-HDL was produced during lipolysis of VLDL by LPL. Taken these in vivo and in vitro analysis together, we suggest that lipolysis of VLDL particle by LPL is an important source for formation of preβ1-HDL. [Copyright &y& Elsevier]

Abstract: Background: We reported that the soluble LDL receptor relative with 11 ligand-binding repeats (sLR11) is a promising biomarker for follicular lymphoma (FL). In this study, we evaluated the fluctuations in serum sLR11 levels compared with those of serum soluble urokinase-type plasminogen activator receptor (suPAR) and soluble interleukin-2 receptor (sIL-2R) in patients with non-Hodgkin's lymphoma (NHL). Methods: Serum sLR11, suPAR, and sIL-2R levels were measured using ELISA in 175 NHL patients and 57 healthy controls. The levels at diagnosis and at remission were evaluated in 64 paired samples. Results: Serum sLR11 levels were significantly increased in FL, diffuse large B-cell lymphoma (DLBCL), and peripheral T-cell lymphoma patients compared with healthy controls. Serum sLR11 levels revealed significant positive correlations with serum suPAR and sIL-2R levels. Serum sLR11 levels at remission were decreased compared with those at diagnosis, and the declines at remission expressed a slope of approximately −1 with an intercept near that of controls. The receiver operating characteristic–area under the curve of serum sLR11 concentrations was equivalent to that of serum suPAR and sIL-2R concentrations in an early-stage DLBCL and FL. Conclusions: sLR11 may be a novel soluble receptor indicative of early-stage NHL, with potential use for evaluating therapeutic efficacy. [Copyright &y& Elsevier]

Abstract Background Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) play a central role in triglyceride-rich lipoprotein metabolism by catalyzing the hydrolysis of triglycerides. Quantification of LPL and HTGL activity is useful for diagnosing lipid disorders, but there has been no automated method for measuring these lipase activities. Methods The automated kinetic colorimetric method was used for assaying LPL and HTGL activity in the post-heparin plasma using the natural long-chain fatty acid 2-diglyceride as a substrate. LPL activity was determined with apoCII and HTGL activity was determined without apoCII with 2 channel of auto-analyzer. Results The calibration curve for dilution tests of the LPL and HTGL activity assay ranged from 0.0 to 500 U/L. Within-run CV was obtained within a range of 5%. No interference was observed in the testing of specimens containing potentially interfering substances. The measurement range of LPL activity in the post-heparin plasma was 30–153 U/L, while HTGL activity was 135–431 U/L in normal controls. Conclusions The L PL and HTGL activity assays are applicable to quantitating the LPL and HTGL activity in the post-heparin plasma. This assay is more convenient and faster than radiochemical assay and highly suitable for the detection of lipid disorders. Highlights • Automated and simultaneous LPL and HTGL activity assay in plasma was developed. • The L PL and HTGL activity is suitable to detect in 15 min in post-heparin plasma. • Both activities decreased to basal levels in 90 min after heparin administration. • Both activities were detectable in patient plasma under cardiac catheter test. [ABSTRACT FROM AUTHOR]

Abstract: Preβ1-HDL, a minor HDL subfraction consisting of apolipoprotein A-I (apoA-I), phospholipids and unesterified cholesterol, plays an important role in reverse cholesterol transport. Plasma preβ1-HDL levels have been reported to be increased in patients with coronary artery disease (CAD) and dyslipidemia. To clarify the clinical significance of measuring plasma preβ1-HDL levels, we examined those levels in 112 patients with CAD, consisting of 76 patients with stable CAD (sCAD) and 36 patients with unstable angina pectoris (uAP), and in 30 patients without CAD as controls. The preβ1-HDL levels were determined by immunoassay using a specific monoclonal antibody (Mab55201) that we established earlier. The mean preβ1-HDL level in the CAD patients was significantly higher than the level in the controls (34.8±12.9mg/L vs. 26.6±6.9mg/L, p <0.001). In addition, the mean preβ1-HDL level was markedly higher in the uAP subgroup than in the sCAD subgroup (43.1±11.5mg/L vs. 30.9±11.7mg/L, p <0.0001). These tendencies remained even after excluding dyslipidemic subjects. These results suggest that elevation of the plasma preβ1-HDL level is associated with the atherosclerotic phase of CAD and may be useful for identifying patients with uAP. [Copyright &y& Elsevier]

• Novel ELISA systems for t-tau and p181tau developed here showed good correlations with widely used ELISA systems. • Serum and hemoglobin contamination in CSF samples did not decrease ELISA sensitivity. • Significant increases in t-tau and p181tau levels in Alzheimer's disease were confirmed. • These ELISA systems are reliable assays for measuring CSF t-tau and p181tau levels. Cerebrospinal fluid (CSF) total tau (t-tau) and tau protein phosphorylated at threonine 181 (p181tau) are established biomarkers for Alzheimer's disease (AD). Herein, we measured t-tau and p181tau to evaluate novel enzyme-linked immunosorbent assays (ELISAs) using 72 CSF samples including from patients with AD with dementia (ADD) and various neurodegenerative diseases. Our assay system showed good correlations with widely used ELISA systems for t-tau and p181tau and showed that serum and hemoglobin contamination in CSF samples did not decrease sensitivity. Significant increases in both t-tau and p181tau levels were observed in ADD. These findings suggested that our ELISAs were reliable assays for CSF t-tau and p181tau similar to commonly used ELISAs. [ABSTRACT FROM AUTHOR]

There is no established method for measuring human hepatic triglyceride (TG) lipase (HTGL) concentration in serum. In this study, we developed new monoclonal Abs (MoAbs) (9A1 mouse MoAb and 141A1 rat MoAb) that react with HTGL both in serum and in postheparin plasma (PHP) and established a novel ELISA system for measuring serum HTGL and PHP-HTGL concentrations. To confirm the specificity of MoAbs, we performed immunoprecipitation-immunoblotting analysis. Both 9A1 mouse MoAb and 141A1 rat MoAb were able to immunoprecipitate not only recombinant HTGL and PHP-HTGL but also serum HTGL, demonstrating that HTGL exists in serum obtained without heparin injection. This method yielded intra- and interassay coefficients of variation of <6% and showed no cross-reactivity with LPL or endothelial lipase. In clinical analysis on 42 male subjects with coronary artery disease, there were strong positive correlations of serum HTGL concentration to PHP-HTGL concentration ( r = 0.727, P < 0.01). Serum HTGL concentrations showed positive correlations to serum TGs ( r = 0.314, P < 0.05) and alanine aminotransferase ( r = 0.406, P < 0.01), and tendencies toward positive correlations to LDL cholesterol, small dense LDL, and γGTP. These results suggest that this new ELISA method for measuring serum HTGL is applicable in daily clinical practice., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preβ1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preβ1-HDL ELISA. Here, we purified preβ1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preβ1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preβ1-HDL and original plasma preβ1-HDL, or between the preβ1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preβ1-HDL MAbs, reactivity with intact plasma preβ1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preβ1-HDL is lipid-free monomolecular apoA-1.

Background: Vascular smooth muscle cells (SMCs) migrate from the arterial media to the intima in the progression of atherosclerosis, and dysfunction of SMCs leads to enhanced atherogenesis. A soluble form of the LDL receptor relative with 11 ligand-binding repeats (sLR11) is produced by the intimal SMCs, and the circulating concentrations of sLR11 likely reflect the pathophysiological condition of intimal SMCs. Furthermore, polymorphism of the LR11 gene has been found to be related to the onset of Alzheimer disease. This study describes the development of a sandwich immunoassay for quantifying sLR11 in human serum and cerebrospinal fluid., Methods: We used synthetic peptides or DNA immunization to produce monoclonal antibodies (MAbs) A2-2-3, M3, and R14 against different epitopes of LR11., Results: sLR11 was immunologically identified as a 250-kDa protein in human serum and cerebrospinal fluid by SDS-PAGE separation, and was purified from serum by use of a receptor-associated protein and MAb M3. An immunoassay for quantification of sLR11 with a working range of 0.25-4.0 microg/L was developed using the combination of MAbs M3 and R14. Treatment of serum with 5.25% n-nonanoyl-N-methyl-d-glucamine reduced the matrix effects of serum on the absorbance detection in the ELISA system. The linear dynamic range of the ELISA spanned the variation of circulating sLR11 concentrations in individuals with atherosclerosis., Conclusions: A sandwich ELISA was established for quantifying sLR11 in serum and cerebrospinal fluid. This technique provides a novel means for assessing the pathophysiology of atherosclerosis, and possibly neurodegenerative diseases.

Prebeta1-HDL is a minor HDL subfraction that acts as an efficient initial acceptor of cell-derived free cholesterol. During 37 degrees C incubation, plasma prebeta1-HDL decreases over time due to its conversion to alpha-migrating HDL by lecithin:cholesterol acyltransferase (LCAT). This conversion may be delayed in hemodialysis patients who have decreased LCAT activity. To clarify whether LCAT-dependent conversion of prebeta1-HDL to alpha-migrating HDL is delayed in hemodialysis patients, prebeta1-HDL concentrations were determined in 45 hemodialysis patients and 45 gender-matched control subjects before and after 37 degrees C incubation with and without the LCAT inhibitor. It was found that the baseline prebeta1-HDL concentration in hemodialysis patients was more than twice that in the controls (44.9 +/- 21.4 versus 19.8 +/- 6.7 mg/L apoAI; P < 0.001). After 2-h incubation, the LCAT-dependent decrease in prebeta1-HDL in hemodialysis patients was about one-third of that in the controls (30 +/- 27 versus 97 +/- 17% of baseline; P < 0.01). The LCAT-dependent rate of decrease in prebeta1-HDL levels (DR(prebeta1)) was the same for samples from hemodialysis patients exhibiting normal (> or =1.03 mmol/L) and low HDL-cholesterol levels (32 +/- 32 versus 28 +/- 23% of baseline; NS). DR(prebeta1) was positively correlated with LCAT activity (r = 0.617; P < 0.001). In conclusion, the LCAT-dependent conversion of prebeta1-HDL to alpha-migrating HDL is severely delayed in hemodialysis patients. The impaired catabolism of prebeta1-HDL may accelerate atherosclerosis in hemodialysis patients.

We have established an immunoassay for pre beta 1-HDL (the initial acceptor of cellular cholesterol) using a monoclonal antibody, MAb55201. Because pre beta 1-HDL is unstable during storage, fresh plasma must be used for pre beta 1-HDL measurements. In this study, we describe a method of stabilizing pre beta 1-HDL, and evaluate the analytical performance of the immunoassay for pre beta 1-HDL. Fresh plasma was stored under various conditions with or without a pretreatment consisting of a 21-fold dilution into 50% (v/v) sucrose. Pre beta 1-HDL concentration was measured by immunoassay. In nonpretreated samples, pre beta 1-HDL decreased significantly from the baseline after 6 h at room temperature. Although pre beta 1-HDL was more stable at 0 degrees C than at room temperature, it increased from 30.2 +/- 8.5 (SE) to 56.5 +/- 5.5 mg/l apolipoprotein A-I (apoA-I) (P < 0.001) in hyperlipidemics, and from 18.4 +/- 1.2 to 37.9 +/- 3.3 mg/l apoA-I (P < 0.001) in normolipidemics after 5-day storage. After 30-day storage at -80 degrees C, pre beta 1-HDL increased from 29.0 +/- 4.0 to 38.0 +/- 5.7 mg/l apoA-I (P < 0.001) in hyperlipidemics, whereas it did not change in normolipidemics. In pretreated samples, pre beta 1-HDL concentration did not change significantly under any of the above conditions. Moreover, pre beta 1-HDL concentrations determined by immunoassay correlated with those determined by native two-dimensional gel electrophoresis (n = 24, r = 0.833, P < 0.05). An immunoassay using MAb55201 with pretreated plasma is useful for clinical measurement of pre beta 1-HDL.

Objective: The role of pre-beta1-high density lipoprotein (pre-beta1-HDL) in cholesterol efflux was investigated by separating human plasma into purified pre-beta1-HDL and pre-beta1-HDL-deficient plasma by using a monoclonal antibody specifically reacting with pre-beta1-HDL., Methods and Results: When compared with whole plasma, pre-beta1-HDL-deficient plasma was equally efficient in promoting cholesterol efflux from human skin fibroblasts and THP-1 human macrophage cells. When added at the same apolipoprotein A-I concentration, pre-beta1-HDL was less effective than whole plasma in promoting cholesterol efflux from fibroblasts but equally effective in promoting cholesterol efflux from THP-1 cells. However, pre-beta1-HDL-deficient plasma reconstituted with 16% pre-beta1-HDL was more active than whole plasma, demonstrating that pre-beta1-HDL does promote cholesterol efflux actively. The amount of cellular cholesterol present in reisolated pre-beta1-HDL was 1.5- to 2-fold greater after incubation of the cells with whole plasma than after incubation of the cells with pre-beta1-HDL-deficient plasma or plasma treated with the anti-pre-beta1-HDL antibody. However, the anti-pre-beta1-HDL antibody did not inhibit cholesterol efflux., Conclusions: We conclude that whereas pre-beta1-HDL is capable of taking up cellular cholesterol, its presence in plasma is not essential for cholesterol efflux, at least in vitro. Instead, pre-beta1-HDL may be the first product of apolipoprotein A-I lipidation during the formation of HDL but may not play a major role in transferring cellular cholesterol to HDL.