Normal Human Serum Samples Research Articles

Triple-recognition strategy for one-pot detection of single nucleotide variants by aligner-mediated cleavage-triggered exponential amplification

The detection of single nucleotide variants (SNVs) is important for the diagnosis and treatment of cancer. To date, researchers have devised several methods to detect SNVs, but most of them are complex and time-consuming. To improve SNVs detection specificity and sensitivity, we developed a triple-recognition strategy, which facilitates aligner-mediated cleavage-triggered exponential amplification (Trec-AMC-EXPAR) for the rapid, specific, and one-pot detection of SNV. Under optimized conditions, Trec-AMC-EXPAR detected two clinically significant SNVs, PIK3CAH1047R and EGFR L858R within 80 min, with a reliable detection of 0.1% SNV in the wide type, which is lower than that of allele-specific PCR (AS-PCR) for detecting SNV. Finally, by spiking into normal human serum samples, mutants mixed with the wild-type targets in different ratios were analyzed, resulting in the relative standard deviation (RSD) of recovery ratios <3%. The findings suggested the potential application of Trec-AMC-EXPAR in clinical disease diagnosis. In summary, the proposed Trec-AMC-EXPAR technique provides a novel fast and convenient method for one-pot detection of SNV with high sensitivity and specificity.

Method validation of a bridging immunoassay in combination with acid-dissociation and bead treatment for detection of anti-drug antibody

Anti-drug antibody (ADA) positivity is correlated with disease relapse risk when treated with monoclonal antibody (mAb) therapeutics. ADA evaluation can assist with interpreting pharmacokinetic, pharmacological, and toxicology results. Here, we established an ADA assay based on two steps of acid dissociation combined with a bridging immunoassay to provide a comprehensive validation strategy. The three-tiered sample analysis process included screening, confirmation, and titration assays using therapeutic HLX26 (targeting lymphocyte activation gene-3 [LAG-3]) as an example. The cut points were determined by testing 50 individual normal human serum samples, including screening cut point (SCP) (SNR: 1.08), confirmatory cut point (CCP) (% inhibition: 12.65), and titration cut point (TCP) (sample-to-noise ratio [SNR]: 1.17). The assay sensitivity, low positive control (LPC), and high positive control (HPC) titer acceptable range were also set up as 33.0 ng/mL, 41.0 ng/mL, and 320–1280, respectively. After full validation, both the intra-assay and inter-assay precision testing passed with coefficient of variations (CVs) < 20%. The assay enabled excellent drug tolerance up to 768.0 μg/mL at the HPC level and 291.0 μg/mL at the LPC level, while the tolerance of target interference was up to 74.0 ng/mL of soluble LAG3. Moreover, no false-positive results were observed in the presence of 5% hemolyzed serum samples and 150 mg/dL of triglyceride in the serum samples, no hook effect was observed, and the stability performed normally under room temperature for 24 h, 2–8 °C for 7 d, and six freeze/thaw cycles. In summary, this ADA assay is feasible and could be used for evaluating the immunogenicity of HLX26 in clinical trials.

A strategy of “fluorescence-visualization dual channel” for highly sensitive and rapid detection of phosphate biomarkers based on stimulus-responsive infinite coordination polymers

The novel stimulus-responsive host-guest nanoparticles were prepared based on lanthanide infinite coordination polymer ([email protected]) for rapid and point-of-care analysis of phosphate (Pi) biomarkers. The obtained [email protected], which have the host-guest structure, were applied to high-sensitive detection of Pi biomarkers by the means of ratiometric “fluorescence-visualization dual channel”. The established method has the advantages of low detection limit (51 nM), wide linear range (0.25–5 μM) and rapid response to Pi within 3 min. The developed sensor was used to detect Pi in normal human urine and serum samples, with the recoveries of 96.04–106.3%. Furthermore, the fluorescent color changes of [email protected] from blue to red can be used to visually inspect Pi by the naked eyes. More importantly, a portable test strip based on [email protected] was fabricated and successfully applied to the rapid visual detection of Pi in human urine and serum samples.

A Paper-Based Analytical Device Integrated with Smartphone: Fluorescent and Colorimetric Dual-Mode Detection of β-Glucosidase Activity.

In this work, indoxyl-glucoside was used as the substrate to develop a cost-effective, paper-based analytical device for the fluorescent and colorimetric dual-mode detection of β-glucosidase activity through a smartphone. The β-glucosidase can hydrolyze the colorless substrate indoxyl-glucoside to release indoxyl, which will be self-oxidized to generate green products in the presence of oxygen. Meanwhile, the green products emit bright blue-green fluorescence under ultraviolet–visible light irradiation at 365 nm. Fluorescent or colorimetric images were obtained by a smartphone, and the red-green-blue channels were analyzed by the Adobe Photoshop to quantify the β-glucosidase activity. Under the optimum conditions, the relative fluorescent and colorimetric signals have a good linear relationship with the activity of β-glucosidase, in the range of 0.01–1.00 U/mL and 0.25–5.00 U/mL, and the limits of detection are 0.005 U/mL and 0.0668 U/mL, respectively. The activities of β-glucosidase in a crude almond sample measured by the fluorescent and colorimetric methods were 23.62 ± 0.53 U/mL and 23.86 ± 0.25 U/mL, respectively. In addition, the spiked recoveries of normal human serum and crude almond samples were between 87.5% and 118.0%. In short, the paper-based device, combined with a smartphone, can provide a simple, environmentally friendly, and low-cost method for the fluorescent and colorimetric dual-mode detection of β-glucosidase activity.

Hepatocyte-derived MASP1-enriched small extracellular vesicles activate HSCs to promote liver fibrosis.

Liver fibrosis is a chronic disease characterized by different etiological agents; dysregulated interactions between hepatocytes and HSCs contribute to this disease. β-arrestin 1 (ARRB1) plays an important role in liver fibrosis; however, the effect of ARRB1 on the crosstalk between hepatocytes and HSCs in liver fibrosis is unknown. The aim of this study is to investigate how ARRB1 modulates hepatocyte and HSC activation during liver fibrosis. Normal and fibrotic human liver and serum samples were obtained. CCl 4 -induced liver fibrosis and methionine-choline deficiency-induced NASH models were constructed. Primary hepatocytes and HSCs were isolated, and human hepatic LO2 and stellate LX2 cells were used. Small extracellular vesicles (EVs) were purified, and key proteins were identified. ARRB1 was up-regulated in hepatocytes and associated with autophagic blockage in liver fibrosis. ARRB1 increased the release of hepatocyte-derived small EVs by inhibiting multivesicular body lysosomal degradation and activating Rab27A, thereby activating HSCs. Proteomic analyses showed that mannan-binding lectin serine protease 1 (MASP1) was enriched in hepatocyte-derived small EVs and activated HSCs via p38 mitogen-activated protein kinase (MAPK)/activating transcription factor 2 (ATF2) signaling. ARRB1 up-regulated MASP1 expression in hepatocytes. MASP1 promoted liver fibrosis in mice. Clinically, MASP1 expression was increased in the serum and liver tissue of patients with liver fibrosis. ARRB1 up-regulates the release of hepatocyte-derived MASP1-enriched small EVs by regulating the autophagic-lysosomal/multivesicular body pathway and Rab27A. Hepatocyte-derived MASP1 activates HSCs to promote liver fibrogenesis through p38 MAPK/ATF2 signaling. Thus, MASP1 is a pivotal therapeutic target in liver fibrosis.

Synergistic enhanced rolling circle amplification based on mutS and radical polymerization for single-point mutation DNA detection

The detection of nucleic acids in biofluids is essential for changing the paradigm of disease diagnosis. As there are very few nucleic acids present in human biofluids, a high sensitivity method is required to detect nucleic acids for disease diagnosis. The Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation is associated with non-small cell lung cancer. It is a point mutation and requires a highly selective detection technique. In this study, high sensitivity and selectivity were achieved for the detection of KRAS mutation using rolling circle amplification (RCA), atomic transfer radical polymerization (ATRP), mutS enzyme, and electrochemical sensors. Although RCA can isothermally amplify DNA, it has low selectivity for detecting single-base mismatch DNA, and its sensitivity is not suitable for circulating tumor DNA detection. The selectivity of RCA was improved by using mutS, which can bind specifically to point mutations. In addition, as a method of isothermal radical polymerization, ATRP was used to amplify the weak signal of RCA. Since RCA and ATRP reactions occur simultaneously, detection time was reduced, and the calculated detection limit was 3.09 aM. Computational and experimental analyses were conducted to verify each detection step and the combination of mutS, ATRP, and RCA. The experiment was performed using normal human serum samples for biological application, and the proposed detection method was confirmed to have excellent potential for diagnosing cancer patients.

Sandwich-type microRNA biosensor based on graphene oxide incorporated 3D-flower-like MoS2 and AuNPs coupling with HRP enzyme signal amplification.

A sandwich electrochemical biosensing strategy for ultrasensitive detection of miRNA-21 was developed by using graphene oxide incorporated 3D-flower-like MoS2 (3D MoS2-rGO) nanocomposites as the substrate and horseradish peroxidase (HRP)-functionalized DNA strand 1 (S1)-gold nanoparticles (S1-AuNPs-HRP) as signal amplification probes. Herein, 3D MoS2-rGO nanocomposites not only had a large specific surface area and excellent conductivity, but also provided more attachment sites for electrodepositing AuNPs. In the presence of target miRNA, a sandwich structure was formed, and the determination of the miRNA-21 was carried out by measuring the DPV response of H2O2 mediated by hydroquinone (HQ) at a potential of + 0.052V (vs AgCl reference electrode). Under the optimal experimental conditions, the as-prepared biosensor enabledthe ultrasensitive detection of miRNA-21 from 5 fM to 0.5μM with the low detection limit of 0.54 fM (S/N = 3), comparable or lower than previous reported methods for miRNA-21 detection, which benefited from the synergistic amplification of 3D MoS2-rGO and AuNPs-HRP. Theprepared biosensor showed satisfactory selectivity, reproducibility, and stability towards miRNA-21 detection. Thebiosensor was feasible for accurate and quantitative detection of miRNA-21 in normal human serum samples with RSD below 5.86%, which showed a great potential in clinical analysis and disease diagnosis.

Highly sensitive non-enzymatic glucose sensor based on carbon nanotube microelectrode set

In this work, a carbon nanotube microelectrode set (CNT µ-ES) was modified, in which three electrodes based on highly densified carbon nanotube fiber (HD-CNTf) cross-sections (length ~40 µm) were embedded in an inert polymer matrix with exposed open-ended CNTs at the interface. An HD-CNTf cross-section (∼40 µm diameter) electrochemically modified with copper nanoparticles (CuNPs) was used as the working electrode; a bare HD-CNTf cross-section (∼94 µm diameter) was used as the counter electrode; and an HD-CNTf cross-section (∼94 µm diameter) electroplated with Ag/AgCl and then coated with Nafion™ was used as a quasi-reference electrode. The electrochemical activity of the CuNPs/HD-CNTf microsensor for glucose electrooxidation was examined by cyclic voltammetry and amperometry in 0.1 M NaOH solution. As shown by amperometry studies, the proposed nonenzymatic CuNPs/HD-CNTf microsensor had a remarkably low limit of detection (28 nM) and a wide linear quantification range with an excellent sensitivity (1942 nA·µM−1·cm−2). This sensitivity can be attributed to the synergetic effect of electrocatalytic CuNPs and aligned HD-CNTf, which provide excellent conductivity. The electrooxidation of glucose on the developed microsensor was free from chloride poisoning and independent of the oxygen concentration. The developed microsensor demonstrated insignificant interference from the oxidation of common interfering species and carbohydrate compounds at their physiological concentrations. Finally, the CuNPs/ HD-CNTf microsensor was successfully applied for the quantification of glucose in normal human serum and diabetic patient urine samples, demonstrating the applicability of this strategy for commercial nonenzymatic glucose sensors.

Determination of the biomarker L-tryptophan level in diabetic and normal human serum based on an electrochemical sensing method using reduced graphene oxide/gold nanoparticles/18-crown-6.

A novel nanocomposite-modified electrode based on reduced graphene oxide (rGO) decorated with 18-crown-6 (Cr.6) and gold nanoparticles (GNPs) on the surface of a glassy carbon electrode (GCE) was successfully fabricated to investigate the electrochemical sensing of the biomarker L-tryptophan (L-Trp) in the presence of dopamine (DA), ascorbic acid (AA), urea, and glucose. The rGO-GNPs-Cr.6/GCE displayed high electrochemical catalytic activity for L-Trp determination using square-wave voltammetry (SWV). The electrochemical behavior of L-Trp at the rGO-GNPs-Cr.6/GCE displayed higher oxidation current and potential (oxidation peak current of 40 μA at 0.85 V) than rGO-GNPs/GCE, Cr.6/GCE, GNPs/GCE, rGO/GCE, and bare GCE. The SWV demonstrated a linear range of L-Trp concentration from 0.1 to 2.5μM. A low limit of detection (LOD) was found for L-Trp, with LOD of about 0.48 μM and 0.61 μM in diabetic and normal serum, respectively. The fabricated sensor demonstrated high selectivity and sensitivity, and good stability and reproducibility for L-Trp sensing. Finally, the nanocomposite (rGO-GNPs-Cr.6)-modified GCE was applied for the determination of L-Trp in normal and diabetic human serum samples, and displayed excellent LOD and recoveries higher than 91.8%. Graphical Abstract.

Electrodeposition of Three‐Dimensional Network Nanostructure PEDOT/PANI for Simultaneous Voltammetric Detection of Ascorbic Acid, Dopamine and Uric Acid

AbstractIn this study, poly(3,4‐ethylenedioxythiophene) (PEDOT) and polyaniline (PANI) were electrodeposited via cyclic voltammetry (CV) on glass carbon electrode (GCE) layer by layer. The prepared electrode (GCE/PEDOT/PANI) was used for the simultaneous sensitive determination of ascorbic acid (AA), dopamine (DA) and uric acid (UA) at different oxidation potentials. The peak distances of AA and DA, DA and UA, AA and UA were 216 mV, 104 mV, 320 mV, respectively. For individual detection of AA, DA and UA, the linear ranges were 1×10−4‐1×10−2 M, 3×10−5‐1×10−3 M and 7×10−7‐1×10−4 M with detection limits of 2.42×10−5 M, 4.58×10−6 M and 2.38×10−7 M (S/N=3), respectively. For simultaneous detection of AA, DA, and UA, the linear response ranges were 1×10−4‐1×10−3 M, 3×10−4‐1×10−2 M, and 3×10−6‐1×10−4 M with detection limits of 6.91×10−5 M, 3.66×10−5 M and 4.13×10−7 M (S/N=3), respectively. The GCE/PEDOT/PANI displayed good selectivity, reproducibility and storage stability, and was further applied to the detection of AA, DA, and UA in 5% normal human serum samples.

Pozelimab, a Human Antibody Against Complement Factor C5, Demonstrates Robust Inhibition of Alternative Complement Activity Both in Normal Human Serum and in Phase I Normal Healthy Volunteers

INTRODUCTION: Blockade of complement factor C5 has demonstrated benefit in paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, generalized myasthenia gravis and neuromyelitis optica. We have completed a Phase I study of pozelimab, a fully human anti-C5 IgG4, in healthy volunteers. Pozelimab was well tolerated and resulted in dose-dependent inhibition of hemolytic activity through the classical complement pathway in normal healthy volunteers. Complete inhibition of hemolytic activity was maintained over a 4-week dosing period by a weekly subcutaneous (SC) regimen following an intravenous (IV) loading dose (ASH2018 abstract). OBJECTIVE: To further characterize the impact of pozelimab on the alternative complement pathway activity, we investigated the effect of pozelimab on alternative pathway-mediated hemolysis using an AH50 assay in the completed first-in-human (FIH) study. In addition, we compared the effect of pozelimab in both alternative and classical pathway hemolysis assays with those of in-house eculizumab and in-house ravulizumab in pooled normal human serum (NHS) samples, ex vivo. METHODS: In total, 56 subjects were randomized to 4 sequential ascending IV single dose cohorts plus 2 sequential ascending SC single dose cohorts followed by 1 multiple dose cohort (consisting of an IV loading dose and weekly SC doses). Each cohort consisted of 8 subjects randomized to receive pozelimab or placebo (6 active: 2 placebo). Serum collected at multiple time-points was utilized to assess the effect of pozelimab on alternative pathway activity. For ex vivo spike experiments, pooled NHS was used to compare the hemolytic function of pozelimab, in-house produced eculizumab and in-house produced ravulizumab. Comparator antibodies were synthesized from published sequence. The alternative pathway (AP) and classical pathway (CP) hemolysis assays were performed based on lysis of rabbit red blood cells (RBCs) and sensitized sheep RBCs, respectively. Both assays measure the amount of hemoglobin released from red blood cells at 412 nm. RESULTS: In the FIH study, baseline AH50 was comparable across treatment groups with a mean of 110 U/mL (standard deviation = 19, n = 56). Pozelimab exposure led to dose-dependent inhibition of AH50. In all 4 IV dosing cohorts, peak suppression of hemolysis was observed at end of infusion (EOI). Maximal suppression of hemolysis was approximately −85% change from baseline. This was achieved with the 30 mg/kg IV group and the repeat dose 15 mg/kg IV + 400 mg SC QW group. In the 2 SC cohorts, peak suppression of hemolysis was observed 3-7 days post dosing, which was consistent with observed peak concentrations of pozelimab in serum. In an ex vivo spike study, pozelimab, in-house eculizumab and in-house ravulizumab were spiked into 10, 25 or 48% pooled NHS for AP, and 5, 10 or 25% for CP. The results from AP hemolysis assays showed that, for a given concentration of spiked antibody, the maximal suppression of hemolysis for all the antibodies decreased with increased percentage of serum (Figure). The maximal suppression of hemolysis was consistently higher (32-169%) for pozelimab compared with in-house eculizumab, and lower for in-house ravulizumab compared with pozelimab and in-house eculizumab at all serum percentages tested. The results from CP hemolysis assays showed that, although the maximal suppression of hemolysis was similar for all antibodies tested, in-house ravulizumab was required to be at least a log higher in concentration to achieve a similar effect as the other two anti-C5 antibodies. CONCLUSIONS: Ex vivo studies with pooled NHS demonstrate that pozelimab robustly blocks both CP and AP hemolysis. In-house ravulizumab appeared to be less potent compared with in-house eculizumab in both CP and AP hemolysis assays. The Phase I healthy volunteer study of pozelimab demonstrated dose-dependent and significant inhibition of alternative pathway hemolysis, with the maximal suppression of hemolysis approximately −85% change from baseline. Figure Disclosures Devalaraja-Narashimha: Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Ni:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Huang:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Wang:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Chaudhari:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Prasad:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Harari:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Rankin:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Morton:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Weyne:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. OffLabel Disclosure: The drug is pozelimab and it is a fully human anti-C5 IgG4 being tested a Phase 1 study in healthy volunteers.

Voltammetric layer-by-layer biosensor featuring purine nucleoside phosphorylase and chitosan for inosine in human serum solutions

A highly sensitive and selective voltammetric biosensing platform for the determination of inosine (INO) concentrations and further applications to biological sample solutions was developed. The biosensor configures a layer-by-layer (LbL) self-assembly of the positively charged chitosan (CS) and negatively charged purine nucleoside phosphorylase (PNP) on screen-printed carbon electrode (SPCE); PNP was chosen to improve the selectivity since it is INO selective while CS was used for the enzyme PNP loading. The electrocatalytic reaction of INO and PNP was studied using cyclic voltammetry (CV) and square wave voltammetry (SWV). Under the optimal condition with two-bilayer of the PNP and CS on the modified electrode (PNP-CS)2-SPCE, a linear dynamic range from 2 to 90 μM INO with a limit of detection of 0.3 μM was achieved when using SWV method. Finally, the developed biosensing platform was applied to the detection of INO in both normal human serum sample and myocardial infarction (MI) patient serum sample in addition to INO dietary supplements. The sensing results of normal human and MI patient serum samples were further compared to those obtained from liquid chromatography-mass spectrometry (LC-MS) and a commercial inosine assay kit.

TNT009 Prevents Erythrocyte C3 Fragment Opsonization and Rescues Reticulocytes from Destruction in Patients with Cold Agglutinin Disease

▪Autoantibody mediated classical complement pathway (CP) activation has been hypothesized to drive hemolytic anemia in cold agglutinin disease (CAD) patients. Red blood cell (RBC) destruction is believed to occur as a result of C3 opsonin mediated extravascular hemolysis in the liver. We recently reported that TNT009, a humanized monoclonal antibody targeting the CP specific serine protease C1s, rapidly restores hemoglobin levels in severely anemic CAD patients. Here we describe the pharmacodynamic changes in the complement profile of patients to provide a mechanistic understanding of the hematological responses and therapeutic benefit observed following TNT009 treatment.In a Phase 1b trial, we enrolled 5 female CAD patients with severe anemia, one of whom had a lymphoplasmacytic lymphoma with >70% bone marrow infiltration and no measurable CP activity prior to dosing. This patient did not respond to TNT009 while on study and will be omitted from subsequent analyses. Patients were given an initial 10 mg/kg test dose of TNT009 on Day 1, followed by four weekly doses of 60 mg/kg on Day 2 or Day 5. Patients were followed for 4 weeks following the last dose (washout). Plasma and serum samples were collected throughout the study to measure TNT009 concentrations and to monitor serological markers of anemia and hemolysis. Additionally, futhan-containing plasma samples were collected to assess the levels of CP specific components including C1s, C1s-C1INH, and C1q by ELISA. RBCs were collected to monitor cell surface complement deposition (C3 fragments) via flow cytometry. Finally, C4 levels and an ELISA-based readout of CP activity were examined as measures of the pharmacodynamic effect of TNT009.Baseline levels of circulating C4 were either low or undetectable in CAD patients. Accordingly, serum CP activity was reduced compared to normal human serum samples. Following the first 60 mg/kg TNT009 dose, CP activity was immediately and completely inhibited within 15 minutes of dosing in all patients and remained inhibited for 3 weeks after the last dose. During this period of inhibition, C4 levels rose from a median circulating concentration of <90 mcg/mL (range: <70 - 145) to 251 mcg/mL (range: 238 - 353; p < .001). Plasma C1s levels, on the other hand, decreased from a median plasma concentration of 53.3 mcg/mL (range: 49.4 - 60.3) to a nadir of <3.13 mcg/mL, the lower limit of quantification (LLOQ), (p < .001). Similarly, C1s-C1INH decreased from a median value of 4.5 mcg/mL (range: 4.2 - 5.3) to a nadir of <0.16 mcg/mL (LLOQ; p < .001). Notably, circulating plasma C1q levels were unaffected.Classical pathway inhibition led to a significant increase in reticulocytes in all patients by, on average, 69% within 24 hours of dosing (p < .05). Interestingly, within 1 week after the first TNT009 dose, reticulocyte counts returned to pre-treatment levels and continued to decrease throughout the study, as expected when hemoglobin normalizes. Similarly, within 24 hours of the first dose of TNT009, bilirubin levels dropped from a median value of 2.1 mg/dL (range 1.6 - 3.8) to 0.7 mg/dL (range 0.6 - 1.2), resulting in an average reduction of 66% from baseline levels (p < .05) and returning to pre-treatment levels following washout. Finally, we monitored in vivo complement activation by staining for C3 fragment deposition on RBCs. In general, we observed a gradual reduction in the percentage of C3 fragment positive RBCs over the course of the study from a median value of 49% (range: 37 - 81) to 29% (range: 20 - 38) before washout of TNT009. The decrease in opsonized RBCs was concomitant with the rise in hemoglobin (example shown in Figure 1).Figure 1: Elevation of hemoglobin is associated with a decrease in C3 fragment coated erythrocytesHere we report that TNT009 administration depletes circulating C1s and immediately halts in vivo CP activity, normalizing plasma C4 levels in CAD patients. The abrupt increase in reticulocyte count within 24 hours of dosing suggests that cold agglutinin mediated complement activation affects reticulocyte survival, preventing their maturation into erythrocytes. The observed reduction in C3 opsonized RBCs suggests that TNT009 ameliorates anemia by preventing complement mediated hepatic RBC sequestration, supported by the immediate normalization of circulating bilirubin levels. These results provide a mechanistic interpretation of the therapeutic effects of TNT009 in CAD patients. [Display omitted] DisclosuresPanicker:True North Therapeutics, Inc.: Employment, Equity Ownership. Hussain:True North Therapeutics, Inc.: Employment, Equity Ownership. Parry:Truenorth Therapeutics, Inc.: Employment, Equity Ownership. Gilbert:Truenorth Therapeutics, Inc.: Employment, Equity Ownership. Jaeger:Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses.

A Human Serum-Based Enzyme-Free Continuous Glucose Monitoring Technique Using a Needle-Type Bio-Layer Interference Sensor

The incidence of diabetes is continually increasing, and by 2030, it is expected to have increased by 69% and 20% in underdeveloped and developed countries, respectively. Therefore, glucose sensors are likely to remain in high demand in medical device markets. For the current study, we developed a needle-type bio-layer interference (BLI) sensor that can continuously monitor glucose levels. Using dialysis procedures, we were able to obtain hypoglycemic samples from commercial human serum. These dialysis-derived samples, alongside samples of normal human serum were used to evaluate the utility of the sensor for the detection of the clinical interest range of glucose concentrations (70–200 mg/dL), revealing high system performance for a wide glycemic state range (45–500 mg/dL). Reversibility and reproducibility were also tested over a range of time spans. Combined with existing BLI system technology, this sensor holds great promise for use as a wearable online continuous glucose monitoring system for patients in a hospital setting.

Harmonised calculation of osmolal gap using the kiss principle

Introduction Calculated osmolal gap (OG) is useful to estimate the presence of unmeasured osmotically active compounds in serum. At least 34 formulae have been proposed but the literature1 suggests the Smithline2 formula (2xNa + glucose + urea), which is one of the simplest and most popular, performs as well as any other. We have used this formula to determine its performance when applied to the RCPAQAP normal human serum samples. Methods Using the Smithline formula we determined the SD of the calculated osmolality (CO) and OG when applied to results from laboratories using different analytical systems. Results The Smithline formula gave a CO of 293 mmol/L (SD 3.2) and OG of –1 mmol/L (SD 4.8). Conclusion Calculations in clinical pathology necessarily are limited by the combination of errors in the individual components. Nevertheless we can recommend use of the Smithline formula which would produce CO with an SD of around 3 and OG close to zero. If all laboratories adopt this approach we can indeed keep it straight and simple!

An electrochemiluminescence (ECL)-based assay for the specific detection of anti-drug antibodies of the IgE isotype

To address a possible linkage between the occurrence of the hypersensitivity reactions and the induction of IgE anti-drug-antibodies (ADA), a drug specific IgE ADA assay was developed using electrochemiluminescence (ECL) technology. In the assay a drug-specific IgE isotype chimeric antibody was generated and used as an ADA positive control. The biotinylated drug X (an antibody) and ruthenium-labeled omalizumab (an anti-human IgE antibody) were used as capture and detection reagents, respectively.The binding affinities of the chimeric IgE isotype positive control have been shown to be highly comparable to drug X and drug Y (drug X is the 2nd generation of drug Y), indicating that it could serve as a highly useful control to compare and contrast the relative ability of the two generations of drug to elicit IgE ADA responses. The assay cut point factor (CPF) was estimated to be 1.13. The cut point factor derived from normal human serum samples was statistically equivalent to the cut point factor determined from targeted population samples. The assay could detect less than 250ng/mL of IgE antibodies in the presence of 300μg/mL drug X. The assay sensitivity was <0.2ng/mL. A minimal prozone was observed at 100μg/mL IgE ADA, but the sample remained highly detectable. The inter-assay precision was within 12%. The assay was not susceptible to non-specific matrix effects. The performance specifications ensured that the assay was suitable for validation. The combination of the chimeric IgE positive control and the detection antibody (ruthenium-labeled omalizumab) used for the assay could potentially provide a general bioanalytical approach for other biopharmaceuticals for the detection of IgE ADA responses.

Determination of Iron(II), Iron(III) and Total Iron in Some β-Thalassemia Patients Using Different Analytical Techniques

There are many well-known analytical methods for determination of iron (II) and iron (III). A mong these methods: gravimetric, titrimetric, potentiometric, conductometric, batch and flow – injection spectrophotometric methods. In the present study, two batch spectrophotometric, atomic absorption spectrometric and biolabo kit methods have been used for determination of iron (II), iron (III) and total iron. The present methods have the advantages of high sensitivity, low cost reagent, low operation cost, simplicity, speed and their applications for determination of iron (II) and iron (III) in some serum samples of normal human and β-thallasemia patients in Erbil city. For the first time especially in Erbil city attempts were made to use zero, first and second derivative spectra to identify the serum samples of some β-thallasemia patients from the normal human serum samples due to the appearance and resolution of peaks in both cases.

Free glycans derived from glycoproteins present in human sera

During the course of studies on the analysis of O-glycans in biological samples, we found that significant amount of free glycans are present in normal human serum samples. The most abundant free glycan was disialo-biantennary glycan typically observed in transferrin which is one of the abundant glycoproteins found in sera. Minor glycans were also considered to be mainly due to transferrin, but some glycans were derived from mucin-type O-glycans, although the amount was quite minute. However, high mannose-type glycans could not be detected at all. Although there have been many reports on the presence of intracellular "free" N-glycans (mainly derived from high mannose-type glycans) generated either from lipid-linked oligosaccharides or from misfolded glycoproteins through endoplasmic-reticulum associated protein degradation pathway, there is little information on the presence of free glycans in extracellular matrix and biological fluids such as serum. This report is the first one which demonstrates the presence of free glycans due to glycoproteins in sera.

Determination of Iron (II), Iron (III) and Total Iron in Some β-Thalassemia Patients using Different Analytical Techniques

There are many well-known analytical methods for determination of iron (II) and iron (III). A mong these methods: gravimetric, titrimetric, potentiometric, conductometric, batch and flow – injection spectrophotometric methods. In the present study, two batch spectrophotometric, atomic absorption spectrometric and biolabo kit methods have been used for determination of iron (II), iron (III) and total iron. The present methods have the advantages of high sensitivity, low cost reagent, low operation cost, simplicity, speed and their applications for determination of iron (II) and iron (III) in some serum samples of normal human and β-thallasemia patients in Erbil city. For the first time especially in Erbil city attempts were made to use zero, first and second derivative spectra to identify the serum samples of some β-thallasemia patients from the normal human serum samples due to the appearance and resolution of peaks in both cases.

Multiplex multi-antigen, multi-species, microsphere-based ELISA to detect antibodies to three human Plasmodium species

Background Multiplex ELISA that detect antibodies against more than one Plasmodium species while allowing species differentiation would be highly valuable for epidemiology and vaccine studies in areas of mixed infections and identification of malaria-exposed blood donors in non-endemic countries. Here we report a highly sensitive, multiplex ELISA based on recombinant proteins from Plasmodium falciparum, P. vivax and P. malariae malaria for panspecies and species-differentiating detection of antibodies in malaria-positive reference samples and in samples from individuals living in a malaria endemic region in Ghana, Africa. Materials and methods Multiplex ELISA was developed utilizing the Luminex xMAP technology that allows the simultaneous detection of antibodies of different specificities that react with antigenic epitopes on multiple beads (microspheres) of different dye intensity. Seven recombinant Plasmodium antigens (P. falciparum: CSP, AMA-1, LSA-1 and MSP119, P. vivax :A MA-1 and MSP119 and P. malariae: MSP119) were covalently coupled to carboxylated magnetic beads. The dilutions of human plasma/serum were incubated with 3000-5000 antigen-conjugated beads in 96-well plate. Following incubation with a biotin-labeled human antiIgG conjugate, a streptavidin-PE conjugated fluorescent substrate was added and the plates were read on Bio-Rad BioPlex 200 reader. The reader was set to read a minimum of 50 beads with identical unique detection signal, the results were expressed as median-fluorescent intensity and cut-off titers were established using a pool of normal human serum samples from the US blood donors. Results Multiplex ELISA detected 100% of the confirmed malaria reference samples belonging to P. falciparum, P. vivax and P. malariae infected patients. The inclusion of multiple antigens in the multiplex assay makes the test more sensitive than the conventional plate ELISA. The assay was capable to detect differential antibody reactivity to seven Plasmodium antigens in serum samples from 75 adults from malaria endemic area in Ghana who had no demonstrable parasitemia by microscopy. The assay also successfully distinguished between the mixed P. falciparum and P. malariae infections in imported malaria samples obtained in United States. Conclusions