The fabrication of magnetic particle-based chemiluminescence immunoassay for human epididymis protein-4 detection in ovarian cancer

The magnetic particles have a significant influence on the immunoassay detection and cancer therapy. Herein, the chemiluminescence immunoassay combined with the magnetic particles (MPCLIA) was presented for the clinical determination and analysis of human epididymis protein 4 (HE4) in the human serum. Under the optimized experiment conditions, the secure MPCLIA method can detect HE4 in the broader range of 0–1000 pmol/L, with a lower detection limit of 1.35 pmol/L. The satisfactory recovery rate of the method in the serum ranged from 83.62% to 105.10%, which was well within the requirement of clinical analysis. Moreover, the results showed the good correlation with enzyme-linked immunosorbent assay (ELISA), with the correlation coefficient of 0.9589. This proposed method has been successfully applied to the clinical determination of HE4 in the human serum.     

A magnetic particles-based chemiluminescence enzyme immunoassay for rapid detection of ovalbumin

Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles–chemiluminescence enzyme immunoassay (MPs–CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol–H₂O₂ chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs–CLEIA method had a linear range from 0.31 to 100 ng/ml with a detection limit of 0.24 ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA.     

Coumarin tethered cyclic imides as efficacious glucose uptake agents and investigation of hit candidate to probe its binding mechanism with human serum albumin

A series of novel coumarin-cyclic imide conjugates (1a–1j) were designed and synthesized to evaluate their glucose uptake activity by insulin resistant liver hepatocyte carcinoma (HepG2) cells through 2-NBDG uptake assay. Compounds (1a–1j) were characterised using various analytical methods such as ¹H NMR, ¹³C NMR, IR, GC–MS, elemental and single-crystal X-ray diffraction techniques. Compounds (1a–1j) exhibited 85.21 – 65.80% of glucose uptake and showed low level of cytotoxicity towards human embryonic kidney cells (HEK-293) indicating good selectivity and safety profile. Compound 1f was identified as a hit candidate exhibiting 85.21% of glucose uptake which was comparable with standard antidiabetic drug Metformin (93.25% glucose uptake). Solution stability study under physiological pH conditions ≈ (3.4 – 8.7), indicates that compound 1f is sufficiently stable at varied pH conditions and thereby compatible with bio-physiological environments. Interaction of 1f with human serum albumin (HSA) were also studied which quantifies that compound 1f binds with HSA efficiently through facile binding reaction in solution. Fluorescence, UV–vis spectrophotometry and molecular modeling methodologies were employed for studying the interaction mechanism of compound 1f with protein.     

Gd(III) complexes as contrast agents for magnetic resonance imaging: a proton relaxation enhancement study of the interaction with human serum albumin

 The non-covalent interaction between human serum albumin (HSA) and DOTA-like Gd(III) complexes containing hydrophobic benzyloxymethyl (BOM) substituents has been thoroughly investigated by measuring the solvent proton relaxation rates of their aqueous solutions. The binding association constants (K _A) to HSA are directly related to the number of hydrophobic substituents present on the surface of the complexes. Furthermore, an estimation of ΔH° and ΔS° has been obtained by the temperature dependence of K _A. Assays performed with the competitor probes warfarin and ibuprofen established that the complexes interact with HSA through two nearly equivalent binding sites located in the subdomains IIA and IIIA of the protein. Strong relaxation enhancements, promoted by the formation of slowly tumbling paramagnetic adducts, have been measured at 20 MHz for complexes containing two and three hydrophobic substituents. The macromolecular adduct with the latter species has a relaxivity of 53.2±0.7 mM^–1 s^–1, which represents the highest value so far reported for a Gd(III) complex. The temperature dependence of the relaxivity for the paramagnetic adducts with HSA indicates long exchange lifetimes for the water molecules dipolarly interacting with the paramagnetic centre. This is likely to be related to the formation, upon hydrophobic interaction of the complexes with HSA, of a clathrate-like, second-coordination-sphere arrangement of water molecules. Besides affecting the dissociative pathway of the coordinated water molecule, this water arrangement may itself significantly contribute to enhancement of the bulk solvent relaxation rate. Received: 6 November 1995 / Accepted: 17 April 1996     

Contrast agents for magnetic resonance angiographic applications: 1H and 17O NMR relaxometric investigations on two gadolinium(III) DTPA-like chelates endowed with high binding affinity to human serum albumin

N,N′,N″,N‴ -pentaacetic acid) bearing different substituents for binding to human serum albumin (HSA) are compared. In spite of the structural differences of the recognition synthon and of the residual electric charge, the two chelates display an analogous binding affinity for the serum protein. Upon formation of the adducts with HSA, the exchange rates of the coordinated water appear slowed down by an amount corresponding to ca. 50% of the rates found for the free complexes. The relaxivity of [Gd(BOM)₃DTPA (H₂O)]^2 − is significantly higher than that of MS-325 either in the free complex or in the macromolecular adduct. Finally, the effect of pH on the stability of the HSA adducts and on the values of their relaxivities has been investigated. Received: 11 June 1999 / Accepted: 15 September 1999     

Spectroscopic and thermodynamic determination of three distinct binding sites for Co(II) ions in human serum albumin

Human serum albumin (HSA) is the most abundant protein of blood serum, involved in the transport of metal ions, including Co(II). Using circular dichroism spectroscopic titrations we characterized three distinct Co(II) binding sites in HSA. Applying Cu(II), Ni(II) and Cd(II) ions as competitors we determined that these sites are identical with three binding sites known for other metal ions. We ordered these sites according to their binding affinities as cadmium site B (CdB) > multi-metal binding site (MBS) > N-terminal binding site (NTS). Using isothermal titration calorimetry (ITC) we confirmed the presence of these three binding sites and determined their conditional binding constants at pH 7.4 as 9 ± 5, 1.1 ± 0.5, and 0.9 ± 0.3 × 10⁴ M⁻¹, respectively. The impact of these results on the albumin cobalt binding (ACB) clinical assay for myocardial ischemia is discussed.     

Synchronous fluorescence determination and molecular modeling of 5-Aminosalicylic acid (5-ASA) interacted with human serum albumin

In this paper, we proposed a new method for the determination of either human serum albumin (HSA) or 5-Aminosalicylic acid (5-ASA) by synchronous fluorescence spectra and examined the interaction between them using the molecular modeling method under simulative physiological conditions. The optimum conditions of synchronous fluorometric determination of HSA were investigated and the method was successfully applied to the determination of 5-ASA added to serum, urine, and saliva samples. The linear range of the determination of HSA and 5-ASA were 1.60 – 414 μg mL⁻¹ and 0.76 –22.95 μg mL⁻¹, the detection limits were 0.552 μg mL⁻¹ and 0.38 μg mL⁻¹, respectively. In addition, the effect of various common ions on the determination of HSA with 5-ASA was also discussed at room temperature. Figure The salicylic acid moiety is located within the binding pocket. The ring of 5-ASA was inserted in the hydrophobic cavity of site I, and it is important to note that the residue ARG-218 and the trptophan residue of HSA (Trp214) are in close proximity to the ring of 5-ASA suggesting the existence of hydrophobic interaction between them.     

Interaction of ochratoxin A with human serum albumin. Binding sites localized by competitive interactions with the native protein and its recombinant fragments

Competitive interactions of ochratoxin A (OTA) and several other acidic compounds were utilized to gain insight into the localization of binding sites and the nature of binding interactions between anionic species and human serum albumin (HSA). Depolarization of OTA fluorescence in the presence of a competing anion was used to quantify ligand-protein interactions. The results obtained were rationalized in terms of OTA displacement from its major binding site. Based on their ability to displace OTA, two distinct groups of the anionic ligands were revealed. The first group contained structurally diverse compounds that shared a common binding site in subdomain IIA (Sudlow Site I). The second group consisted of three non-steroidal anti-inflammatory drugs, which showed much lower affinity to Site I than the OTA dianion. The major site for these drugs was located in domain III. Fluorescence spectroscopy measurements of OTA, warfarin (WAR) and naproxen (NAP) complexes with recombinant proteins corresponding to the domains of HSA (D1-D3) revealed binding to all domains but with different affinities. The binding constants for OTA and WAR decreased in the series D2z.Gt;D3>D1. In contrast, NAP showed the most favorable interaction with D3 and comparable affinities to the two remaining domains. The OTA binding constant for D2, 7.9 x 10(5) M(-1), was smaller than the largest constant for HSA by a factor of approximately 7. The binding constant for OTA with D3, 1.1 x 10(5) M(-1), was very close to that of the secondary binding site for HSA.     

The fluorescence and circular dichroism of proteins in reverse micelles: application to the photophysics of human serum albumin and N-acetyl-l-tryptophanamide

Evidence is presented that a compartmentalised protein exists in its native state only within a particular size of aqueous cavity. This behaviour is shown to exist in AOT reverse micelles using fluorescence quenching and circular dichroism (CD) studies of human serum albumin (HSA). In particular, far ultraviolet CD measurements show that a reduction in quencher accessibility to the fluorophore is consistent with the protein being nearest to its native conformation at a waterpool size of around 80 Å diameter. We also show that the biexponential fluorescence decay of N-acetyl-l-tryptophanamide (NATA) in AOT reverse micelles arises from the probe being located in two distinct sites within the interfacial region. The more viscous of these two sites is located on the waterpool side of the interface and the other is located on the oil side of the interface.     

Development of a rapid and high‐performance chemiluminescence immunoassay based on magnetic particles for protein S100B in human serum

Protein S100B is a clinically useful non‐invasive biomarker for brain cell damage. A rapid chemiluminescence immunoassay (CLIA) for S100B in human serum has been developed. Fluorescein isothiocyanate (FITC) and N‐(aminobutyl)‐N‐(ethylisoluminol) (ABEI) are used to label two different monoclonal antibodies of anti‐S100B. Protein S100B in serum combines with labeled antibodies and can form a sandwiched immunoreaction. A simplified separation procedure based on the use of magnetic particles (MPs) that were coated with anti‐FITC antibody is performed to remove the unwanted materials. After adding the substrate solution, the relative light unit (RLU) of ABEI is measured and is found to be directly proportional to the concentration of S100B in serum. The relevant variables involved in the CLIA signals are optimized and the parameters of the proposed method are evaluated. The results demonstrate that the method is linear to 25 ng/mL S100B with a detection limit of 0.02 ng/mL. The coefficient of variation (CV) is < 5% and < 6% for intra‐ and interassay precision, respectively. The average recoveries are between 97 and 107%. The linearity–dilution effect produces a linear correlation coefficient of 0.9988. Compared with the commercial kit, the proposed method shows a correlation of 0.9897. The proposed method displays acceptable performance for quantification of S100B and is appropriate for use in clinical diagnosis. Copyright © 2013 John Wiley & Sons, Ltd.     

Capillary electrophoresis coupled with end‐column electrochemiluminescence for the determination of ephedrine in human urine,and a study of its interactions with three proteins

A tris(2,2‐bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺)‐based electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE) method has been established for the sensitive determination of ephedrine for the first time. Under the optimized conditions [ECL detection at 1.15 V, 25 mmol/L phosphate buffer solution (PBS), pH 8.0, as running buffer, separation voltage 12.5 kV, 5 mmol/L Ru(bpy)₃²⁺ with 60 mmol/L PBS, pH 8.5, in the detection cell] linear correlation (r = 0.9987) between ECL intensity and ephedrine concentration was obtained in the range 6.0 × 10^–8–6.0 × 10^–6 g/mL. The detection limit was 4.5 × 10^–9 g/mL (S:N = 3). The developed method was successfully applied to the analysis of ephedrine in human urine and the investigation of its interactions with three proteins, including bovine serum albumin (BSA), cytochrome C (Cyt‐C) and myoglobin (Mb). The number of binding sites and the binding constants between ephedrine and BSA, Cyt‐C and Mb were 8.52, 12.60, 10.66 and 1.55 × 10⁴ mol/L, 6.58 × 10³ mol/L and 1.59 × 10⁴ mol/L, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

A method for estimating free Ca within human red blood cells,with an application to the study of their Ca-dependent K permeability

Summary Murphy, Coll, Rich and Williamson (J. Biol. Chem. 255:6600–6608, 1980) described a null-point method for estimating intracellular free Ca in liver cells. They used digitonin to lyse the cells in solutions of varying Ca concentration. This method has been adapted for use with human red cells. The values found are about 0.4 m Ca in fresh cells, and from 0.4 to 0.7 m Ca in blood-bank cells, at pH 7.2 and 37°C. These are likely to be overestimates, and the errors and limitations of the method are discussed. Red cells may be loaded with Ca by metabolic depletion in Ca-containing solutions. Such cells have an elevated K permeability, and the relationships between free Ca, total Ca and K permeability were investigated, using⁸⁶Rb as a tracer for K.⁸⁶Rb flux studies show that the affinity of the K channel for Ca is the same in cells as in resealed ghosts where intracellular Ca can be controlled with Ca buffers, but the rate of tracer equilibration is 3–6 times faster in ghosts than in cells.     

A validated method for rapid analysis of ethane in breath and its application in kinetic studies in human volunteers

Oxidative stress may initiate lipid peroxidation that generates ethane. Ethane, at low concentrations, is eliminated by pulmonary exhalation. Previous methods have not allowed frequent sampling, thus ethane kinetics has not been studied in man. A validated method over the range 3.8-100,000 ppb with a limit of quantitation of 3.8 ppb (CV 9.3%) based on cryofocusing technique of a 60 ml breath sample allowed frequent sampling. Due to a rapid analytical procedure batches of more than 100 samples may be analyzed. In human volunteers (24-55 years) uptake was studied for up to 23 min (n=9), elimination was studied for 210 min (n=9). Ethane was inhaled (concentrations varied from 16 to 29 ppm (parts per million)) through a non-rebreathing system; sampling was performed with short intervals from the expiratory limb. Samples were also drawn from the inhalatory limb. Ninety-five percent of steady state (inspired) concentration was reached within 1.75 min. Five percent of the initially inhaled concentrations was found in exhaled air 1.5 min after termination of inhalation. A terminal mean half life of 31 min for ethane was also observed. The data indicate that frequent sampling will be necessary to capture relevant changes in breath ethane.     

Antiferromagnetic interactions through phenoxo bridges and lattice water: Synthesis, structure, and magnetic properties of new Mn(III) Schiff base complexes in combination with thiocyanate ligand

The Schiff base ligands, N,N′-bis(2-hydroxyacetophenone)-1,2-diaminoethane (acphenH₂) and N,N′-bis(2-hydroxyacetophenone)-1,3-diaminopropane (acphpnH₂), prepared in situ were used to synthesise two new Mn(III) complexes which were characterised by crystallography and variable temperature magnetic measurements. [Mn(acphen)NCS]₂ is a phenoxo-bridged dimeric compound with the thiocyanate coordinating in the usual bent mode (Mn-N-C angle, 152°) and is weakly antiferromagnetic. Since there are no significant inter-dimer contacts in the crystal, the low temperature magnetic behaviour is influenced by single ion zero-field splitting. Exact diagonalisation of the spin Hamiltonian was performed to derive the following parameters: J = −0.7 cm⁻¹, D = −0.6 cm⁻¹. Mn(acphpn)(H₂O)NCS is monomeric with an unusual linearly coordinated thiocyanate (Mn-N-C angle, 178°). Two lattice water molecules link the Mn(III) complex molecules through hydrogen bonds to form one-dimensional chains in the crystal. Magnetic exchange along the chain makes this compound also weakly antiferromagnetic with J ∼ -2 cm⁻¹.     

Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.     

Stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography--mass spectrometry for measurement of phenolic xenoestrogens in human urine samples

A high-sensitivity analytical method that uses stir bar sorptive extraction (SBSE) with in situ derivatization and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS) for the simultaneous measurement of trace amounts of phenolic xenoestrogens (PXs), such as 2,4-dichlorophenol (DCP), 4-tert-butylphenol (BP), 4-tert-octylphenol (OP), 4-nonylphenol technical isomers (NP), pentachlorophenol (PCP) and bisphenol A (BPA), in human urine samples was developed. The urine sample (1 ml) was de-conjugated by adding beta-glucuronidase and sulfatase. Then, protein precipitation was performed by the addition of acetonitrile. After centrifugation, the supernatant was diluted with purified water and subjected to SBSE with in situ derivatization and TD-GC-MS. The detection limits of DCP, BP, OP, NP, PCP and BPA in the urine samples were 20, 10, 10, 50, 20 and 20 pg ml-1 (ppt), respectively. The calibration curves for PXs were linear and had correlation coefficients higher than 0.99. The average recoveries of those analytes in the urine samples were higher than 95% (RSD: <10%, n=6) with correction using the added surrogate standards. This simple, accurate, sensitive and selective method can be used in the determination of PXs in human urine samples.     

A rapid and sensitive resonance Rayleigh scattering spectra method for the determination of quinolones in human urine and pharmaceutical preparation

A new method based on resonance Rayleigh scattering (RRS) was proposed for the determination of quinolones (QNS) at the nanogram level. In pH 3.3–4.4 Britton–Robinson buffer medium, quinolones such as ciprofloxacin, pipemidic acid (PIP), lomefloxacin (LOM), norfloxacin (NOR) and sarafloxacin (SAR) were protonated and reacted with methyl orange (MO) to form an ion‐pair complex, which then further formed a six‐membered ring chelate with Pd(II). As a result, new RRS spectra appeared and the RRS intensities were enhanced greatly. RRS spectral characteristics of the MO–QNS–Pd(II) systems, the optimum conditions for the reaction, and the influencing factors were investigated. Under optimum conditions, the scattering intensity (∆I) increments were directly proportional to the concentration of QNS with in certain ranges. The method had high sensitivity, and the detection limits (3σ) ranged from 6.8 to 12.6 ng/mL. The proposed method had been successfully applied for the determination of QNS in pharmaceutical formulations and human urine samples. In addition, the mechanism of the reaction system was discussed based on IR, absorption and fluorescence spectral studies. The reasons for the enhancement of scattering spectra were discussed in terms of fluorescence‐scattering resonance energy transfer, hydrophobicity and molecular size. Copyright © 2014 John Wiley & Sons, Ltd.     

T细胞IFN-g表达水平检测方法的建立及其在马传染性 贫血疫苗免疫机理研究中的应用

[目的]马传染性贫血病毒(EIAV)弱毒疫苗致弱机制和免疫保护机理的研究可以为慢病毒疫苗的研究提供重要的模型.为探讨IFN-γ表达水平与疫苗保护性免疫的关系,本研究旨在建立一种准确、有效地检测EIAV感染马不同T细胞亚型表达IFN-γ水平的方法.[方法]我们将分离的马传贫弱毒疫苗免疫马(FDDV)、强毒感染马(LV)和健康马的外周血单核细胞(PBMC),体外分别经病毒(FDDV)和PMA/Inomycin激活、 BFA 阻断蛋白分泌、荧光标记马的特异性表面抗体和IFN-γ抗体等过程后,进行流式检测.[结果]疫苗免疫马产生的特异性IFN-γ水平为CD4 1.7(0.9%/CD8 6.1(1.2%,而强毒组则为CD4 0.6(0.1%/CD8 2.4(0.9%.[结论]本研究建立的多荧光参数流式细胞术同时检测细胞内IFN-γ染色和淋巴细胞亚型的方法,具有良好的特异性,稳定性和重复性.为研究EIAV弱毒疫苗免疫保护机制奠定了基础.     

Binding of Leishmania spp with gold nanoparticles supported polyethylene glycol and its application for the sensitive detection of infectious photogenes in human plasma samples: A novel biosensor

The management of pathogen detection using a rapid and cost‐effective method presents a major challenge to the biological safety of the world. The field of pathogen detection is nascent and therefore, faces a dynamic set of challenges as the field evolves. Visceral leishmaniasis (VL), or kala‐azar is the most severe form of leishmaniasis. Delay to the accurate diagnosis and treatment is likely to lead to fatality. The reliable, fast and sensitive detection is closely linked to safe and effective treatment of Leishmania spp. Despite several routine and old method for sensitive and specificity detection of Leishmania spp, there is highly demand for developing modern and powerfully system. In this study a novel ultra‐sensitive DNA‐based biosensor was prepared for detection of Leishmania spp. For the first time, the specific and thiolated sequences of the Leishmania spp genome (5′‐SH‐[CH₂]₆ ATCTCGTAAGCAGATCGCTGTGTCAC‐3′) were recognized by electrochemical methods. Also, selectivity of the proposed bioassay was examined by three sequences that were mismatched in 1, 2, and 3 nucleotides. The linear range (10^?6 to 10^?21 M) and limit of detection (LLOQ = 1 ZM) obtained are remarkable in this study. Also, simple and cost‐effective construction of genosensors was another advantage of the proposal DNA‐based assay. The experimental results promise a fast and simple method in detection of kala‐azar patients with huge potential of the nanocomposite‐based probe for development of ideal biosensors.     

Clinical characteristics and molecular epidemiology of human metapneumovirus in children with acute lower respiratory tract infections in China, 2017 to 2019: A multicentre prospective observational study

Human metapneumovirus (HMPV) infection is one of the leading causes of hospitalization in young children with acute respiratory illness. In this study, we prospectively collected respiratory tract samples from children who were hospitalized with acute lower respiratory tract infection in six hospitals in China from 2017 to 2019. HMPV was detected in 145 out of 2733 samples (5.3%) from the hospitalized children. The majority of HMPV-positive children were under the age of two (67.6%), with a median age of one year. HMPV can independently cause acute lower respiratory tract infection in young children, while all patients showed mild clinical symptoms. Of all the co-infected patients, HMPV was most commonly detected with enterovirus (EV) or rhinovirus (RhV) (38.0%, followed by respiratory syncytial virus (RSV) (32.0%). The highest detection rate occurred from March to May in both northern and southern China. Out of 145 HMPV positive samples, 48 were successfully typed, of which 36 strains were subgrouped into subtypes A2c (75%), eight strains were included in subtype B1 (16.7%), and four strains were included in subtype B2 (8.3%). Moreover, 16 A2c strains contained 111-nucleotide duplications in the G gene. Twenty-seven complete HMPV genomes were successfully obtained, and 25 (92.6%) strains belonged to subtype A2c, whereas one strain was included in subgroup B1 and another was included in subgroup B2. A total of 277 mutations were observed in the complete genomes of 25 A2c strains. All results presented here improve our understanding of clinical characteristics and molecular epidemiology of HMPV infection in children.