Dilution Of Human Serum Research Articles

Investigating the antimicrobial activity, cytotoxicity, and action mechanism of acylated and amidated derivatives of AurH1 antifungal peptide

BackgroundThe increasing growth of microbial resistance threatens the health of human societies. Therefore, the discovery and design of new antibiotics seem necessary. Today, antimicrobial peptides (AMPs) are receiving attention due to their unique properties. In our previous studies, exclusive antifungal effects of AurH1, which is a truncated and modified form of Aurein1.2, were synthesized. In this study, AurH1 antifungal peptide was synthesized into acylated (Ac-AurH1) and amidated (AurH1-NH2) derivatives, and their antifungal activity, cytotoxicity, anticancer activity, hemolytic effects were investigated. Finally, the time- of killing, the action mechanism of amidated and acylated peptides, and the effects of salts and human serum on their antimicrobial potency were determined. All the results obtained about these peptides were compared with the AurH1 without chemical modifications.ResultsThe results showed that amidation at the C-terminal of AurH1 compared to acylation at the N-terminal of it can improve the antifungal properties and cytotoxicity of AurH1. The results showed that AurH1 amidation can maintain the antifungal activity of this peptide in the culture medium containing specific dilutions of human serum compared to the intact AurH1. Also, the amidation of the C-terminal of AurH1 could not affect the mechanism of action and its time -of killing.ConclusionAs a result, the amidation of the C-terminal of the AurH1 is a suitable strategy to improve its antifungal properties and cytotoxicity. This modification can enhance its properties for animal studies.

Carbon dots hybrid for dual fluorescent detection of microRNA-21 integrated bioimaging of MCF-7 using a microfluidic platform

BackgroundMicroRNAs have short sequences of 20 ~ 25-nucleotides which are similar among family members and play crucial regulatory roles in numerous biological processes, such as in cell development, metabolism, proliferation, differentiation, and apoptosis.ResultsWe reported a strategy for the construction of a dual-emission fluorescent sensor using carbon dots (CDs) and confirmed their applications for ratiometric microRNA-21 sensing and bioimaging of cancer cells in a microfluidic device. The composition of blue CDs (B-CDs) and yellow CDs (Y-CDs) depicts dual-emission behavior which is centered at 409 and 543 nm under an excitation wavelength of 360 nm. With increasing microRNA-21 concentration, the robust and specific binding of DNA probe functionalized B-CDs to complementary microRNA-21 target induced perturbations of probe structure and led to changing fluorescence intensity in both wavelengths. Consequently, the ratio of turn-on signal to turn-off signal is greatly altered. With monitoring of the inherent ratiometric fluorescence variation (ΔF540nm/ΔF410nm), as-prepared BY-CDs were established as an efficient platform for ratiometric fluorescent microRNA-21 sensing, with a wide linear range of 0.15 fM to 2.46 pM and a detection limit of 50 aM.ConclusionsFurthermore, the proposed assay was applied for detecting microRNA-21 in dilute human serum samples with satisfactory recovery and also in MCF-7 cell lines in the range 3000 to 45,000 (cell mL−1) with a detection limit (3 cells in 10 μL), demonstrating the potential of the assay for clinic diagnosis of microRNA-associated disease. More importantly, the images revealed that MCF-7 cells well labeled with BY-CDs could exhibit the applicability of the proposed microfluidic system as an effective cell trapping device in bioimaging.Graphical

Nanowired dual-electrodes surface to monitor cerebral ischemia by current-volt measurements.

The level of clotting protein 'factor IX' (FIX) is highly associated with cerebral ischemia, and this research work has developed a sensitive detection of FIX on dielectrode sensor by current-volt measurement. Sensing area was grown with zinc oxide nanowire to attach more probe for FIX interaction. Aptamer was utilized as the detection probe and attached on the sensing electrode surface through amine-aldehyde chemical linkage. In addition, biotin-streptavidin interaction was utilized to attach the higher number aptamers on the electrode surface connected with dual-probe station. FIX detection limit was found as 10 fM in the phosphate buffer saline spiked samples and 1:320 dilution of human serum. The linear ranges were as 10 fM to 100pM and 1:320 to 1:80, respectively. With a good determination co-efficient [y = 2.6813x-3.8467; R 2 = 0.9479] this biosensing strategy helps to quantify FIX and monitor the condition of cerebral ischemia.

Label-free detection of C-Reactive protein using bioresponsive hydrogel-based surface relief diffraction gratings

Responsive hydrogel-based surface relief gratings have demonstrated great performances as transducers in optical sensing. However, novel and smart designs of hydrogels are needed for the appropriate detection of analytes and biomolecules since the existing materials are very limited to specific molecules. In this work, a biosensing system based on surface relief gratings made of bioresponsive hydrogels has been developed. In particular, the hydrogel contains phosphocholine moieties to specifically recognize C-Reactive protein (CRP). The CRP-Sensing hydrogel capacity to selectively detect CRP was fully demonstrated. Using Direct Laser Interference Patterning, micro-gratings were created on CRP-Sensing hydrogel substrates and applied for the label-free sensing of CRP using a simple laser-based homemade optical setup. Limits of detection (LOD) and quantification (LOQ) in human serum dilutions of 1.07 and 8.92mgL-1, respectively, were reached. These results demonstrate that the biosensing system allows the selective label-free detection of CRP within concentration ranges around those related to risks of cardiovascular diseases and sepsis. Besides, amplification strategies have been carried out improving the sensitivity, widening the linear range, and reaching better LOD and LOQ (0.30mgL-1 and 4.36mgL-1). Finally, all the approaches were tested for the quantification of CRP in certified human serum with recoveries of around 100%.

Distinct differences in analytical performance of two commercially available assays for specific IgE to egg white and house dust mite allergens

BackgroundMeasurements of allergen-specific IgE antibodies with different manufacturers’ assays show modest or poor agreement. This study compares analytical performance of specific IgE tests for whole allergen extracts and individual allergen components of two assay systems, IMMULITE and ImmunoCAP, using human sera as well as monoclonal antibodies.MethodsComparisons were performed for specific IgE to house dust mite (HDM, n = 44), egg white (EW, n = 36) and the allergen components rDer p 1, rDer p 2, nGal d 1, nGal d 2 and nGal d 4 in human sera and with monoclonal mouse/human chimeric IgE antibodies specific for the same allergen components. Competitive interference with IgE measurement was investigated using allergen-specific monoclonal IgG and IgG4 antibodies.ResultsMeasurements of IgE to HDM and EW in serial dilutions of human sera revealed weaker dilution linearity with IMMULITE than with ImmunoCAP. Analysis of five different monoclonal IgE antibodies with total and specific IgE assays, expected to return similar levels, gave an average specific/total IgE ratio of 0.96 (range 0.71–1.14) with ImmunoCAP and 1.89 (range 0.76–2.85) with IMMULITE, indicating overestimation of specific IgE by IMMULITE. With the EW IgE tests of both assay systems, measurements of a chimeric anti-Gal d 2 IgE antibody were unaffected by a competing mouse IgG antibody. While the same was true for measurement of a chimeric anti-Der p 1 IgE antibody using the HDM test in ImmunoCAP, a suppression of measured concentrations by up to 42% was observed in IMMULITE. Similarly, measurement of HDM-specific IgE in human sera by ImmunoCAP was unaffected by a competing monoclonal anti-Der p 2 IgG4 antibody while IMMULITE displayed a reduction of HDM-specific IgE values by up to 30%.ConclusionsIn this evaluation of analytical performance of two widely used assay systems, ImmunoCAP showed higher accuracy in quantitation of specific IgE and greater resistance against competing allergen-specific non-IgE antibodies which may arise through natural or dietary exposure, or as a result of allergen immunotherapy treatment.

Highly Sensitive MicroRNA Detection by Coupling Nicking-Enhanced Rolling Circle Amplification with MoS2 Quantum Dots.

In this work, a label-free and highly sensitive fluorescence assay was constructed for microRNA detection. Nicking-enhanced rolling circle amplification (RCA) induced by G-quadruplex formation is coupled with inner filter effect (IFE)-based quenching effects of MoS2 quantum dots (MoS2 QDs). The padlock probe contains a recognition sequence to target microRNA and an accessible nicking site. The padlock probe is cyclized upon hybridization with target microRNA. Sequentially, amplification initiates a production of a long-concatenated sequence of circular probes. Abundant G-quadruplex sequences are produced via the nicking process and then used as the trigger to initiate the next RCA. In the presence of hemin, numerous hemin/G-quadruplex DNAzymes are formed, which catalyze the oxidation of o-phenylenediamine (OPD) into the colored product 2,3-diaminophenazine, resulting in quenching of the fluorescence of MoS2 QDs. This sensing strategy enables detection of microRNA let-7a with high selectivity and a detection limit of 4.6 fM. The as-prepared sensor was applied for detecting microRNA let-7a in dilute human serum samples and achieved a satisfactory recovery rate, demonstrating its potential in clinic diagnosis of microRNA-associated disease and biochemical research.

A sensitive and selective electrochemical sensor based on N, P-Doped molybdenum Carbide@Carbon/Prussian blue/graphite felt composite electrode for the detection of dopamine

In this paper, a composite electrode of N,P-doped Mo2C@C/Prussian blue (PB)/graphite felt (N,P–Mo2C@C/PB/GF) was prepared by a simple method and used for sensitive and effective detection of dopamine (DA). N,P-doped Mo2C nanospheres were prepared by using phosphomolybdic acid (PMo12) as an initiator to promote the polymerization of polypyrrole. Such nanospheres were used to accelerate the deposition process of PB from K3[Fe(CN)6] and FeCl3 in solution. The N,P–Mo2C@C/PB nanohybrid was then anchored to GF in order to obtain the electrochemical sensor. Two linear ranges were extrapolated for dopamine detection: from 0.18 to 30 μmol L−1 with a sensitivity of 0.268 μA μmol−1, and from 30 to 280 μmol L−1 with a sensitivity of 0.045 μA μmol−1. The device showed a detection limit as low as 0.011 μmol L−1, an excellent selectivity to DA over common interfering analytes, and a favorable long-time stability. Finally, the sensor was used for quantitative analysis of DA in the 10-fold dilution of human serum (10%) and exhibited a satisfactory recovery.

Molecularly imprinted polymer based hybrid structure SiO2@MPS-CdTe/CdS: a novel fluorescence probe for hepatitis A virus

A novel designed fluorescence molecularly imprinted polymer (MIP) probe made from CdTe/CdS quantum dot (QD)-based silica nanoparticles (SiO2@MPS-CdTe/CdS) was successfully created via a sol−gel process. The target virus - hepatitis A virus (HAV) was selectively captured by imprinted polymer layer, resulting in the fluorescence quenching of the QDs within 20 min, which could be explained by the energy transfer mechanism. Under optimized conditions, the limit of detection for the SiO2@MPS-CdTe/CdS MIP was as low as 88 pmol · L−1, and excellent linearity was obtained from 0.2 to 1.4 nM. Additionally, the nanosensor was successfully used to detect the HAV from a dilution of human serum, in which recoveries were in the range of 96.7%–103.8%. Overall, the current work proposes a novel and cost-effective method to synthesize SiO2@MPS-CdTe/CdS MIPs for use as a tool to rapidly and efficiently detect HAV, and it also provides promising perspectives to further advance virus imprinting research.

Characterization of therapeutic antibodies in the presence of human serum proteins by AU-FDS analytical ultracentrifugation

The preclinical characterization of biopharmaceuticals seeks to determine the stability, state of aggregation, and interaction of the antibody/drug with other macromolecules in serum. Analytical ultracentrifugation is the best experimental method to understand these factors. Sedimentation velocity experiments using the AU-FDS system were performed in order to quantitatively characterize the nonideality of fluorescently labeled therapeutic antibodies in high concentrations of human serum proteins. The two most ubiquitous serum proteins are human serum albumin, HSA, and γ-globulins, predominantly IgG. Tracer experiments were done pairwise as a function of HSA, IgG, and therapeutic antibody concentration. The sedimentation coefficient for each fluorescently labeled component as a function of the concentration of the unlabeled component yields the hydrodynamic nonideality (ks). This generates a 3x3 matrix of ks values that describe the nonideality of each pairwise interaction. The ks matrix is validated by fitting both 2:1 mixtures of HSA (1–40 mg/ml) and IgG (0.5–20 mg/ml) as serum mimics, and human serum dilutions (10–100%). The data are well described by SEDANAL global fitting with the ks nonideality matrix. The ks values for antibodies are smaller than expected and appear to be masked by weak association. Global fitting to a ks and K2 model significantly improves the fits.

A magnetic molecularly imprinted optical chemical sensor for specific recognition of trace quantities of virus.

A magnetic resonance light scattering (RLS) sensor based on the molecularly imprinted polymer (MIP) technique was developed for specific recognition of trace quantities of hepatitis A virus (HAV). Through a surface imprinting technique, the virus-magnetic-MIPs (virus-MMIPs) were prepared as the specific identification element, which was based on the effective synthesis of biomimetic polydopamine (PDA) inspired by mussels on the surface of Fe3O4 magnetic nanoparticles. The preparation process of the virus-magnetic-MIPs was simple and rapid under an applied magnetic field. The surface of the magnetic-MIP captured viruses through specific recognition in water, which caused changes to the particle size and shape, and subsequently resulted in changes in the intensity of the RLS. The sensor was applied to determine the amount of HAV in the linear concentration range of 0.02–1.40 nmol L−1, with a low detection limit of 6.2 pmol L−1, and it was successfully applied for the immediate detection of added HAV from a 2000-fold dilution of human serum. More importantly, the proposed strategy addressed the difficulty of virus-MIP detection in the elution process and it is rapid, easy, sensitive, and eco-friendly.

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay). As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers.

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay). As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

Development of a thermosensitive molecularly imprinted polymer resonance light scattering sensor for rapid and highly selective detection of hepatitis A virus in vitro

In this study, a thermosensitive imprinted polymer coating SiO2 was developed for a resonance light scattering (RLS) thermosensitive virus-affinity sensor, which exhibited high selectivity for virus monitoring. In this sensor, N-isopropylacrylamide was a temperature-sensitive element. The recognition performance of the nanosensor for viruses was regulated by temperature control for the specific capture of the target virus at 40°C, resulting in the increase of RLS intensity and its release at 20°C. Moreover, the RLS intensity of the nanosensor significantly increased within 30min. Accordingly, a very good detection limit of 1.1pmolL−1 was obtained. The nanosensor was successfully used to detect the additional hepatitis A virus from a dilution of human serum, and recoveries in the range of 90.8% to 108.3% were attained at three spiking levels of viruses. Overall, this study addressed the problems of high nonspecific adsorption and long duration of detection process. Furthermore, a facile, rapid, and efficient tool for virus detection was provided.

A virus resonance light scattering sensor based on mussel-inspired molecularly imprinted polymers for high sensitive and high selective detection of Hepatitis A Virus

We described a novel resonance light scattering (RLS) sensor for the specific recognition of trace quantities of Hepatitis A Virus (HAV); the sensor was based on a mussel-inspired hepatitis molecularly imprinted polymer. As a recognition element, polydopamine (PDA)-coated totivirus-imprinted polymer was introduced on the surface of SiO2 nanoparticles (virus-imprinted SiO2@PDA NPs) using an efficient one-step synthesis method. The target virus was selectively captured by the imprinted polymer films, thereby increasing the RLS intensity. A simple fluorescence spectrophotometer was employed to measure the changes in the intensity. The enhanced RLS intensity (∆IRLS) was proportional to the concentration of HAV in the range of 0.04–6.0nmol∙L−1, with a low limit of detection of 8.6pmol∙L−1. The selectivity study confirmed that the resultant HAV-imprinted SiO2@PDA NPs possessed high selectivity for HAV. The sensor was successfully applied for the direct detection of additional HAV from a 20,000-fold dilution of human serum. The proposed strategy is simple, eco-friendly, highly selective, and sensitive.

VEGF Cancer Biomarker Protein Detection in Real Human Serum Using Capacitive Label‐Free Aptasensor

SummaryVascular Endothelial Growth Factor (VEGF) is a signalling protein known as a serum biomarker for a number of diseases including cancer. Therefore, it is necessary to develop a label‐free, rapid and sensitive method for detecting VEGF in its native forms in human serum. In this work, we have demonstrated the proof‐of‐concept of a sensitive label‐free biosensor based on capacitance changes induced by capturing of VEGF in human serum using anti‐VEGF aptamers functionalized on interdigitated (IDE) capacitor arrays (aptasensors). Limit of detection of VEGF protein using this aptasensor was in the range 0.5–2 ng/mL in dilute human serum. The label‐free capacitive aptasensing strategy provides a simple, reliable and effective means of biosensing serum VEGF levels for early cancer diagnosis.

New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.

Label-free capacitance based aptasensor platform for the detection of HER2/ErbB2 cancer biomarker in serum

In this study, a label-free capacitive aptasensor was developed based on capturing of human epidermal growth factor receptor 2 (HER2) protein by anti-HER2 ssDNA aptamers functionalized on interdigitated microelectrodes of capacitor as bio-recognition elements. The aptasensor response was measured by non-Faradaic Impedance Spectroscopy (nFIS) method. Capacitance signal specific to target HER2 protein is based on changes occurred due to charge distribution upon interaction of aptamer–protein molecules against to the applied AC frequency (50–350MHz). HER2 protein in spiked human serum was successfully detected through concentration dependent changes in impedance/capacitance values as a result of the formation of aptamer–HER2 protein complex (0.2–2ngmL−1 of HER2) on capacitor microelectrodes. The label-free capacitive aptasensing is a versatile and promising approach for early detection of cancer biomarkers in dilute human serum and the method potentially be extended to a wide variety of diseases biomarkers.

Serum-Dependent Enhancement of Coxsackievirus B4-Induced Production of IFNα, IL-6 and TNFα by Peripheral Blood Mononuclear Cells

Only a few reports have been published on the interactions between Coxsackievirus B4 (CVB4) and human peripheral blood mononuclear cells (PBMC) but have not been extensively documented. Human serum containing non-neutralizing anti-CVB4 antibodies increased CVB4-induced synthesis of IFNα by PBMC. In this study, we determined if CVB4 and human serum have the ability to activate inflammatory cytokines in addition to IFNα in PBMC cultures. PBMC from healthy donors were inoculated with infectious, inactivated CVB4 or with CVB4 incubated with dilutions of human serum or polyvalent IgG with anti-CVB4 activity. Levels of IFNα, TNFα, IL-6, IL-12, IFNγ and IL-10 in the cell-free supernatants of PBMC cultures were measured using ELISA. Infection was assessed by real-time PCR. PBMC inoculated with CVB4 produced inflammatory cytokines but not IFNα. When CVB4 was incubated with serum or IgG, IFNα was detected in the culture supernatants, and high concentrations of TNFα and IL-6 were measured. The concentrations of TNFα and IL-6 were not reduced in cultures inoculated with inactivated CVB4, whereas the IgG-dependent enhancement of IFNα, IL-6 and TNFα production with inactivated virus was suppressed. The potentiation of IFNα production was associated with a high intracellular viral load. Infectious and non-infectious CVB4 can induce the production of inflammatory cytokines but not IFNα by PBMC. High levels of IFNα, in addition to TNFα and IL-6, in culture supernatants were obtained when infectious CVB4 was combined with immune serum or IgG, and they were associated with high amounts of intracellular viral RNA.

Development, validation and use of ELISA for antibodies to human alpha-1 antitrypsin

AbstractEvaluation of human antibody responses to alpha-1 antitrypsin (AAT) in clinical trials and clinical practice has been limited by the lack of a validated assay. Here we describe the development and validation of an ELISA method for quantification of human and nonhuman primate antibody responses to human AAT. A reference anti-human AAT serum standard was generated using sera from a cynomolgus macaque injected with a recombinant adeno-associated virus vector expressing human AAT. The ELISA was validated for use with human serum dilutions as low as 1:10 and was able to distinguish between specific responses in cynomolgus serum and non-specific increases in apparent antibody titer in serum from subjects in a clinical trial of an AAT gene therapy vector.

An aptamer-capture based chromogenic assay for thrombin

A simple chromogenic assay for human alpha thrombin is developed through aptamer affinity capture and a subsequent enzyme reaction. Thrombin is captured on the aptamer-modified magnetic beads, and catalyzes the conversion of chromogenic substrates to optically measured products. The measurement of the generated products by an absorbance spectrometer allows for the final quantification of thrombin. This assay shows high sensitivity by taking advantage of sample enrichment and enzyme amplification, and exhibits good specificity by involving the selective aptamer binding and the specific enzyme reaction. A concentration detection limit of 40fM can be reached when the tripeptide substrate of tosyl-Gly-Pro-Arg-p-nitroanilide is used in a 24h enzyme reaction, and the use of 2h enzyme reaction in the assay enables the detection of 400fM thrombin for a rapid analysis. This assay can be applied to detect thrombin in dilute human serum.