Direct Immunoassay Research Articles

Influence of the Mediterranean Diet on 25-Hydroxyvitamin D Levels in Adults

The Mediterranean diet (MD) is a dietary pattern effective in terms of prevention of obesity-related diseases, and represents the gold standard in preventive medicine, due to the synergistic action of many nutrients with antioxidant and anti-inflammatory properties. In addition, excess body weight significantly increases the risk of hypovitaminosis D, a well-recognized common feature of individuals with obesity. It is well-known that there is a clear gender difference in the adherence to the MD. The aim of this study was to investigate the association between adherence to the MD and 25-hydroxyvitamin D (25OHD) levels in adults, according to gender. Study population consisted of 617 participants; 296 were males and 321 were females, matched by age and body mass index (BMI). A validated 14-item questionnaire PREDIMED (Prevención con dieta Mediterránea) was used for the assessment of adherence to the MD. The 25OHD levels were determined by a direct competitive chemiluminescence immunoassay. Females have a higher PREDIMED score than males (7.4 ± 2.8 vs. 6.7 ± 3.1 score, p = 0.001), and according to PREDIMED categories, a greater percentage of males had low adherence to the MD compared to their female counterparts (40.2% vs. 37.1%; χ2 = 8.94, p = 0.003). The 25OHD levels were higher in males than in females (18.3 ± 7.3 vs. 16.8 ± 7.8 ng/mL, p = 0.01), and a higher percentage of males had sufficient 25OHD levels (>30 ng/mL) than their female counterparts (10.5% vs. 3.4%, χ2 = 10.96, p < 0.001). Stratifying the sample population according to 25OHD categories, BMI decreased and PREDIMED score increased significantly along with the increased 25OHD levels, in both males and females, respectively (p < 0.001). Looking at the bivariate correlations, PREDIMED score was positively correlated with 25OHD levels after adjusting for age and BMI, in both males (r = 0.21, p < 0.001) and females (r = 0.30, p < 0.001). At the bivariate proportional odds ratio (OR) model, 25OHD levels presented the highest OR values in the category low adherence vs. high adherence to the MD, in both genders (OR 1.21 and OR 1.31, in males and females, respectively). Receiver operator characteristic (ROC) analysis was performed to determine the cut-off values of PREDIMED scores predictive of 25OHD levels: PREDIMED score >5 in males (p < 0.001) and >7 in females (p < 0.001) could serve as thresholds for 25OHD levels above the median. The results of our study highlighted a novel positive association between adherence to the MD and 25OHD levels in both genders. Although 25OHD levels were higher in males than females, 69.7% were deficient. To the best of our knowledge, this is the first study to show that high adherence to the MD is associated with low BMI and high 25OHD levels in both genders, probably through the anti-inflammatory and anti-oxidant effects that are synergistically exerted by either MD or vitamin D on body weight.

Luteinizing hormone and ovarian steroids affect in vitro prostaglandin production in the equine myometrium and endometrium

Prostaglandins (PGs) play crucial roles in the regulation of the oestrus cycle and establishment of pregnancy in animals. Luteinizing hormone (LH) and ovarian steroids are involved in regulating endometrial PG production in many species. Their effects on PG production and associated pathways in the mare myometrium and endometrium are the subjects of our interest. This study aimed to evaluate the specific effects of LH and ovarian steroids on equine myometrial and endometrial tissues on (i) PGE2 and PGF2α secretion and (ii) transcription of genes encoding specific enzymes responsible for PG synthesis, such as prostaglandin–endoperoxide synthase (PTGS2), PGE2 synthases (PGES), PGF2α synthases (PGFS), and PGI2 synthases (PGIS), using equine myometrial and endometrial explants. Equine myometrial and endometrial tissues were collected at the mid-luteal (n = 6) and follicular (n = 6) phases of the oestrus cycle and were exposed to: (1) vehicle (control), (2) arachidonic acid (AA, 50 ng/mL, positive control), (3) LH (10 ng/mL), (4) progesterone (P4, 10−7M) and (5) 17-β oestradiol (E2, 10−9M) for 24 h. After exposure, PGF2α and PGE2 concentrations were determined using direct enzyme immunoassays. Alterations in PG synthase mRNA expression were determined using RT-qPCR. After 24 h, LH and P4 increased PGE2 and PGF2α secretion by myometrial tissues at the mid-luteal phase (P < 0.05), whereas PG secretion was augmented by LH and E2 during the follicular phase (P < 0.01). In contrast, LH and E2 increased PGE2 and PGF2α secretion by endometrial tissues during the mid-luteal phase (P < 0.05), while E2 enhanced PGE2 secretion during the follicular phase of the oestrus cycle (P < 0.01). These results indicate that LH and ovarian steroids modulate PG production in equine myometrial and endometrial tissues and affect PG synthase expression at the mRNA level. We conclude that the equine myometrium is an alternative source of PG production and participates in the regulation of uterus function during the oestrus cycle.

The Sun's Vitamin in Adult Patients Affected by Prader-Willi Syndrome.

Prader–Willi syndrome (PWS) is a genetic disorder characterized by hyperphagia with progressive, severe obesity, and an increased risk of obesity-related comorbidities in adult life. Although low dietary vitamin D intake and low 25-hydroxy vitamin D (25OHD) levels are commonly reported in PWS in the context of bone metabolism, the association of low 25OHD levels with fat mass has not been extensively evaluated in PWS adults. The aims of this study were to investigate the following in PWS adults: (1) 25OHD levels and the dietary vitamin D intake; (2) associations among 25OHD levels with anthropometric measurements and fat mass; (3) specific cut-off values for body mass index (BMI) and fat mass predictive of the 25OHD levels. In this cross-sectional, single-center study we enrolled 30 participants, 15 PWS adults (age 19–41 years and 40% males) and 15 control subjects matched by age, sex, and BMI from the same geographical area (latitude 40° 49’ N; elevation 17 m). Fat mass was assessed using a bioelectrical impedance analysis (BIA) phase-sensitive system. The 25OHD levels were determined by a direct competitive chemiluminescence immunoassay. Dietary vitamin D intake data was collected by three-day food records. The 25OHD levels in the PWS adults were constantly lower across all categories of BMI and fat mass compared with their obese counterpart. The 25OHD levels were negatively associated with BMI (p = 0.04), waist circumference (p = 0.03), fat mass (p = 0.04), and dietary vitamin D intake (p < 0.001). During multiple regression analysis, dietary vitamin D intake was entered at the first step (p < 0.001), thus explaining 84% of 25OHD level variability. The threshold values of BMI and fat mass predicting the lowest decrease in the 25OHD levels were found at BMI ≥ 42 kg/m2 (p = 0.01) and fat mass ≥ 42 Kg (p = 0.003). In conclusion, our data indicate that: (i) 25OHD levels and dietary vitamin D intake were lower in PWS adults than in the control, independent of body fat differences; (ii) 25OHD levels were inversely associated with BMI, waist circumference, and fat mass, but low dietary vitamin D intake was the major determinant of low vitamin D status in these patients; (iii) sample-specific cut-off values of BMI and fat mass might help to predict risks of the lowest 25OHD level decreases in PWS adults. The presence of trained nutritionists in the integrated care teams of PWS adults is strongly suggested in order to provide an accurate nutritional assessment and tailored vitamin D supplementations.

Detection and Quantification of HspX Antigen in Sputum Samples Using Plasmonic Biosensing: Toward a Real Point-of-Care (POC) for Tuberculosis Diagnosis.

Advancements that occurred during the last years in the diagnosis of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis infection, have prompted increased survival rates of patients. However, limitations related to the inefficiency of an early detection still remain; some techniques and laboratory methods do not have enough specificity and most instruments are expensive and require handling by trained staff. In order to contribute to a prompt and effective diagnosis of tuberculosis, we report the development of a portable, user-friendly, and low-cost biosensor device for its early detection. By using a label-free surface plasmon resonance (SPR) biosensor, we have established a direct immunoassay for the direct detection and quantification of the heat shock protein X (HspX) of Mtb, a well-established biomarker of this pathogen, directly in pretreated sputum samples. The method relies on highly specific monoclonal antibodies that are previously immobilized on the plasmonic sensor surface. This technology allows for the direct detection of the biomarker without amplification steps, showing a limit of detection (LOD) of 0.63 ng mL-1 and a limit of quantification (LOQ) of 2.12 ng mL-1. The direct analysis in pretreated sputum shows significant differences in the HspX concentration in patients with tuberculosis (with concentration levels in the order of 116-175 ng mL-1) compared with non-tuberculosis infected patients (values below the LOQ of the assay).

Highly sensitive detection of salbutamol by ALP-mediated plasmonic ELISA based on controlled growth of AgNPs

Salbutamol (SAL), a short-acting β-2 adrenergic receptor agonist, is an undesirable addition to livestock farming and used to improve the growth rates. A direct competitive colorimetric immunoassay for SAL was developed. The alkaline phosphatase (ALP)-mediated controlled growth of the prereduction silver nanoparticles (AgNPs) was used to achieve the highly sensitive, colorimetric detection of SAL through the plasmonic ELISA. Click chemistry reaction was used to synthesize salbutamol biotin label (SAL-Bio) to achieve signal amplification and specific binding with antibody. ALP can efficiently hydrolyze sodium L-ascorbyl-2-phosphate and generate L-ascorbic acid, thereby reducing Ag+ and forming AgNPs. The plasmon resonance absorption signals of AgNPs were enhanced along with a distinct color change. The limit of detection of SAL can be decreased to 26.14 pg/mL. The method offers a good technique for the detection of many small molecular contaminants, such as pesticide residues and veterinary drug residues in food and edible water.

A competitive immunoassay for electrochemical impedimetric determination of chlorpyrifos using a nanogold-modified glassy carbon electrode based on enzymatic biocatalytic precipitation.

A direct competitive impedimetric immunoassay for chlorpyrifos (CPF) was developed that is based on the specific affinity of immunoassay and the enzymatic biocatalytic precipitation amplification strategy. The CPF antibody (anti-CPF) was anchored onto an electro-deposited nanogold modified glassy carbon electrode surface by adsorption of the Au-NH2 bond and Au-SH bond. This improved the electrode reactivity and the loading amount of anti-CPF. Abundant horseradish peroxidase (HRP) and bovine serum albumin-CPF (BSA-CPF) were anchored onto spherical gold nanoparticles (AuNPs, 16 ± 2nm) to form HRP-AuNP-BSA-CPF (analyte competitor). CPF determination was achieved when the competitive immunoassay occurred between CPF and analyte competitor with anti-CPF. In the presence of H2O2 and 4-chloro-1-naphthol, anenzyme-mediated biocatalytic precipitation process was triggered and produced an insoluble 4-chloro-1(4H)-naphthalenone. This insoluble substance increased the Faradaic impedance of the base electrode. The impedimetric signal was determined at the formal potential of 220mV and alternating voltage of 10mV. This signal decreased with increasing concentration of CPF over a linear range of 1.0 × 10-3ngmL-1~10ngmL-1 with a detection limit of 0.070pgmL-1. The immunoassay has been tested for determination of chlorpyrifos in complex matrices such as artificially spiked vegetables with recoveriesin the range 85 to 110%. The relative standard deviations were less than 7.5%. Graphical abstractSchematic representation of electrochemical impedimetric immunoassay for chlorpyrifos determination before enzymatic biocatalytic precipitation (BCP, red line) process and after BCP process (blue line).

Kвантитативно одредување на калпротектин во асцит кај пациенти со спонтан бактериски перитонитис

Спонтаниот бактериски перитонитис (СБП) кај пациентите со црнодробна цироза е новонастаната, спонтана бактериска инфекција на стерилна асцитна течност, во отсуство на интраабдоминални извори на инфекција или малигнитет. Најсензитивен показател за поставување на дијагнозата е бројот на полиморфонуклеарни клетки (ПМНК) ≥250 во 1 мл асцитна течност (рачно микроскопско или автоматизирано пребројување) и/или кога во микробиолошката култура биде изолиран еден бактериски вид. Цел на трудот е да се одреди концентрацијата на калпротектин во асцит кај пациентите со СБП и не-СБП, да се споредат просечните вредности на Turcotte-Pugh II и MELD скорот кај пациентите со СБП и не-СБП и просечните вредности на C-реактивниот протеин во серум и во асцит во испитуваните групи. Материјали и методи. Во оваа проспективно-аналитичко-опсервациска пилот студија беа вклучени 30 пациенти со црнодробна цироза и асцит, поделени во две групи, СБП и не-СБП. Квантитативното мерење на калпротектин во асцит се вршеше со тестот Quantum Blue Calprotectin Ascites (LF-ASC25). Тестот е дизајниран за селективно мерење на антигенот на калпротектинот (MRP8/14) со директен сендвич имуноесеј. Примероците од асцитот се разредуваа со Chase Buffer во однос 1:5 и по 12 минути инкубација на собна температура, интензитетот на сигналот на линијата за тестирање и контролната линија се мереа квантитативно со BÜHLMANN Quantum Blue®Reader. Собраните податоци се обработија со помош на статистичкиот програм SPSS 23 за Windows. Резултати. Во нашата студија просечната вредност на калпротектин кај пациентите со СБП изнесуваше 1,4 μg/mL. Најниската вредност на калпротектин во испитуваната група беше регистрирана кај еден пациент со вредност од 0,61 μg/mL, додека највисока вредност од 1,81μg/mL кај четири пациенти. Резултатите покажаа повисоки вредности на калпротектин во асцит кај пациентите со алкохолна болест на црниот дроб во споредба со останатите етиологии. Рефракторен асцит се регистрира кај 60,0% од испитаниците, а само кај еден пациент (6,7%) се регистрира Klepsiella pneumoniae во микробиолошката анализа на асцитот. Според Child-Turcotte-PughII класификација сите пациенти од испитуваната група беа класа C, додека просечната вредност на MELD скорот изнесуваше 29,8±6,14. Разликата помеѓу просечните вредности наC-реактивниот протеин во серум и во асцит кај пациентите со СБП беше статистички сигнификантна во споредба со не-СБП. Заклучок. Kвантитативното одредување на калпротектинот во асцит со тестот Quantum Blue Calprotectin Ascites(LF-ASC25) може да се користи како алтернатива наполиморфонуклеарните клетки (ПМНК) во асцит. СБП се јавува кај пациенти со сериозна црнодробна дисфункција пресметана според Child-Pugh II скорот и MELD скорот. Вредноста на C-реaктивниот протеин во серум и во асцит кај пациентите со СБП немаше високи вредности ,но сепак беше утврдена сигнификантна разлика во споредба со пациентите со не-СБП.&#x0D;

Acoustic Immunosensing of Exosomes Using a Quartz Crystal Microbalance with Dissipation Monitoring.

Exosomes are endocytic lipid-membrane bound bodies with the potential to be used as biomarkers in cancer and neurodegenerative disease. The limitations and scarcity of current exosome characterization approaches have led to a growing demand for translational techniques, capable of determining their molecular composition and physical properties in physiological fluids. Here, we investigate label-free immunosensing, using a quartz crystal microbalance with dissipation monitoring (QCM-D), to detect exosomes by exploiting their surface protein profile. Exosomes expressing the transmembrane protein CD63 were isolated by size-exclusion chromatography from cell culture media. QCM-D sensors functionalized with anti-CD63 antibodies formed a direct immunoassay toward CD63-positive exosomes in 75% v/v serum, exhibiting a limit-of-detection of 2.9 × 108 and 1.4 × 108 exosome sized particles (ESPs)/mL for frequency and dissipation response, respectively, i.e., clinically relevant concentrations. Our proof-of-concept findings support the adoption of dual-mode acoustic analysis of exosomes, leveraging both frequency and dissipation monitoring for use in bioanalytical characterization.

A novel biomimetic immunoassay method based on Pt nanozyme and molecularly imprinted polymer for the detection of histamine in foods

ABSTRACT This study used surface imprinting polymerization to synthesize molecularly imprinted polymer on the surface of PAF-45 material (PAF-45@MIP). SEM, FT-IR, static and kinetic adsorption experiments were adopted to investigate the morphology and properties of the MIP. The results showed that the PAF-45@MIP could rapidly absorb histamine with a high adsorption capacity. Using PAF-45@MIP as a biomimetic antibody and Pt nanozyme as a marker, a novel direct competitive biomimetic immunoassay (BIA) method for rapidly detecting histamine in foods was developed. Under optimal conditions, the limit of detection and sensitivity of the BIA method were 0.128 and 8.306 mg/L, respectively. Vinegar spiked with histamine was determined using the BIA method with recoveries ranging from 81.50% to 90.38%. The BIA method was also applied to determine histamine in dried shrimp and soy sauce samples, the results correlating well with those obtained using high-performance liquid chromatography.

Validation of the ADVIA Centaur® XP system for the determination of insulin and its application

In this study, a direct chemiluminescent immunoassay for the determination of human serum insulin levels using the ADVIA Centaur® XP system was validated. Dilution recovery, linearity, precision, sensitivity, between analyzer variation, reference interval and stability were analyzed. The linear range of the insulin assay was from 0.64 to 277.27 mU/L. Intra- and inter-assay coefficients of variation were 3.67–7.96% and 4.66–8.69%, respectively. The lower and upper limits of quantification were 0.61 mU/L and 8872.64 mU/L, respectively. In terms of between analyzer variation, our study showed comparable results with a good correlation of r2 = 0.9934. The human serum insulin reference interval was in the range of 3.0–25.0 mU/L. Serum insulin can be kept for 7 days between 2-8 °C and 18–26 °C, and the corresponding results for −20 °C and −70 °C were 1 month and 6 months are reported. We proved that this insulin assay was robust and the analytical performance met the requirements. We successfully applied this insulin assay to a bioequivalence study of miglitol in 48 healthy Chinese subjects. The miglitol bioequivalence study was evaluated based on pharmacokinetic and pharmacodynamic parameter endpoints. The results demonstrated that the test formulation and the reference formulation were bioequivalent.

Female and male serum reference intervals for challenging sex and precursor steroids by liquid chromatography - tandem mass spectrometry

Measuring some sex and precursor steroids is still challenging even by liquid chromatography – tandem mass spectrometry (LC-MS/MS), and few normal values are available. We developed a LC-MS/MS method for estradiol, estrone, dihydrotestosterone and 17-hydroxypregnenolone measurement, compared it with direct immunoassays, and generated sex, age, menopausal and menstrual status specific reference intervals. Liquid-liquid extraction was optimized on 300 μL serum spiked with isotopic internal standards. A 2D-LC system allowed on-line purification and separation in 11 min run. Electrospray ionization was enhanced by ammonium fluoride. MS-detection was obtained by multiple reaction monitoring. Direct ECLIA for estradiol (n = 80) and RIA for estrone (n = 41) were compared with LC-MS/MS. Reference values were estimated in healthy, lean women in reproductive age (n = 118), menopausal women (n = 33) and men (n = 159). The assay showed satisfying imprecision, trueness, recovery and selectivity. Adequate functional sensitivity was achieved for measuring estrone (18.1 pmol/L) and 17-hydroxypregnenolone (117 pmol/L) in all subjects, and estradiol (35.9 pmol/L) and dihydrotestosterone (134 pmol/L) in women in reproductive age and men, but not in menopausal women. Compared with LC-MS/MS, immunoassays showed good agreement for estradiol but severe disagreement for estrone. Estrogens exhibited sex, menopausal and menstrual variations. Dihydrotestosterone and 17-hydroxypregnenolone depended on sex and menopause, the latter also declining with age in men. Strictly defined reference intervals in the adult female and male population were generated for challenging steroids such as estrogens, dihydrotestosterone and 17-hydroxypregnenolone by a novel LC-MS/MS method. Our achievement can be used to deepen the comprehension of several endocrine diseases.

Potential Mechanistic Links between Cancer Treatment and Alzheimer’s Disease: An Insight from the Study of Extracellular Vesicles

In the present work, we designed a microfluidic electrochemical immunosensor with enough sensibility and precision to quantify epithermal growth factor receptor (EGFR) in plasma extracellular vesicles (EVs) of plasma from breast cancer patients. The sensor employs SiNPs coated with chitosan (SiNPs-CH) as reaction's platform, based on the covalently immobilization of monoclonal anti-EGFR on SiNPs-CH retained in the central channel (CC) of the microfluidic device. The synthetized SiNPs-CH were characterized by UV–visible spectroscopy (UV–visible), energy dispersive spectrometry (EDS), Nanoparticle Tracking Analysis (NTA) and transmission electron microscopy (TEM). EGFR was quantified by a direct sandwich immunoassay measuring through a horseradish peroxidase (HRP)-conjugated anti-EGFR. The enzymatic product (benzoquinone) was detected by reduction at − 100 mV on a sputtering gold electrode. The measured current was directly proportional to the level of EGFR in human serum samples. The linear range was from 0 ng mL−1 to 50 ng mL−1. The detection limit was 1.37 pg mL−1, and the within- and between-assay coefficients of variation were below 6.25%. Finally, plasma samples from 30 early breast cancer patients and 20 healthy donor were analyzed by the novel method. EGFR levels in EVs (EVs-EGFR) were significantly higher than in the healthy control group (p = 0.002) and also, more sensitivity and specificity than normal serum markers like CEA and CA15.3 has been observed. EVs-EGFR concentration correlates with EGFR tumor status (p = 0.0003) as well as it correlate with the tumor size and pathological grade. To conclude, plasma EVs are suitable for proteomic characterization of cancer disease, as long as the employed method has sufficient sensitivity, like the case of immune-electrochemical nanosensors with incremented reaction surface.

Application of biological and single-strand conformation polymorphism assays for characterizing potential mild isolates of Citrus tristeza virus for cross protection

Citrus tristeza virus (CTV) by killing millions of citrus cultivars grown on sour orange rootstock worldwide has become one of the most dangerous viral pathogen. Characterization of 12 CTV isolates was analyzed by biological indexing. Infected samples of citrus were collected from citrus growing areas of Pakistan and CTV was detected by symptoms on indicator plants and confirmed by direct tissue blot immunoassay (DTBIA). CTV positive samples were graft inoculated on six biological indicator hosts in the study. A standardized protocol was deployed to study biological characteristics of these isolates. All biological indicators induced mild and from mild to moderate reactions against all of the CTV isolates tested. About two isolates produced stem-pitting symptoms from moderate to severe on Mexican lime. CTV strains were further characterized and confirmed by the analysis of p25 gene of CTV isolates using single-strand conformation polymorphism (SSCP) assay. SSCP analysis revealed that most isolates confined only one predominant sequence variant. SSCP profiles of PCR amplified products from CTV isolates showed bands patterns corresponding to mild and sever strain. Three isolates (4MF, 8KBS and 10GS) from different regions and cultivars were identified as potential source of mild strains for cross protection. These results are the best base for mild strain cross protection (MSCP) in the country.

Quartz Crystal Microbalance Detection of Aflatoxin B1 by Self-Assembled Monolayer Technique

Background: New biosensor techniques allowing detection of low concentrated substances show a great variety nowadays. The construction of a system with modified quartz as a part of a Quartz Crystal Microbalance (QCM) techniques helps the detection and confirmation of low toxin concentrations in a sample. Objective: The study aims to allow the application of methods for preparation and modification of the gold surface of piezoelectric crystal for detection of aflatoxin B1(AFB1) in the concentration range 0.2 - 2.0 µg/L by QCM technique (quartz - crystal microbalance). Methods: The procedure for the preparation of quartz crystal sensors for experimental purposes was performed. The quartz surface was activated and covered with self-assembled monolayer to immobilize antibody (rabbit anti-aflatoxin B1) for the detection of antigen - antibody reaction. Results: The G” corresponds to viscous properties of the material, during applied deformation of the material in the presence of different concentrations, which revealed in the sensitivity of the used resonator. Conclusion: Detection of toxic pollutants may be achieved via QCM methods, ultrasound resonator and piezoelectric quartz techniques for measurement. These techniques allow detection of significantly low concentrations of toxic pollutants, in particular, AFB1, compared to analysis with direct and indirect ELISA immunoassays.

Antibody-responsive signal-off fluorescence of DNA-harbored silver nanoclusters for direct, rapid and sensitive immunoassay

Specific DNA sequence-templated silver nanoclusters (AgNCs) are extremely promising fluorescent emitters in biosensing and bioanalysis. Here, based on the targeted antibody-responsive fluorescent quenching of AgNCs harbored in a unique DNA-based nanoassembly, we propose a specific, sensitive and one-step immunoassay of bivalent anti-digoxigenin (Dig) antibody (antiDA) as a model analyte. For proof-of-concept, this functional nanoassembly (CS1/RS/CS2) consists of a reporter strand tethering Ag-nucleable template (RS) and two capture strands (CS1 and CS2) labeled with hapten Dig, in which two tethered Dig haptens are closely positioned, and brightly emissive AgNCs can be accommodated and stabilized in the unpaired Ag-nucleable scaffold to produce significant fluorescent emission (CS1/RS/CS2-AgNCs). In the presence of antiDA, the specific conjugation of antiDA with two Dig haptens results in the conformation stretch of CS1/RS/CS2, and simultaneously the harbored AgNCs are destabilized to aggregate each other into larger non-fluorescent silver nanoparticles (AgNPs). The remarkable decreasing of AgNCs fluorescence emission is linearly proportional to the antiDA concentration with a high sensitivity down to 4.5 pM. With this regulatory antibody-dependent signal off scheme, the advantageous rapidness and simplification would be attractive and promising for highly specific detection of diverse bivalent antibodies or small molecules.

Signal Enhancement of Silicon Nanowire Field-Effect Transistor Immunosensors by RNA Aptamer

Silicon nanowire field-effect transistors (SiNW-FETs) have been demonstrated as a highly sensitive platform for label-free detection of a variety of biological and chemical entities. However, detecting signal from immunoassays by nano-FETs is severely hindered by the distribution of different charged groups of targeted entities, their binding orientation, and distances to the surface of the FET. Aptamers have been widely applied as a recognition element for plentiful biosensors because of small molecular sizes and moderate to high specific binding affinity with different types of molecules. In this study, we propose an effective approach to enhance the electrical responses of both direct (6×-histidine) and sandwich (amyloid β 1–42) immunoassays in SiNW-FETs with R18, a highly negative charged RNA aptamer against rabbit immunoglobulin G (IgG). Empirical results presented that the immunosensors targeted with R18 expressed a significantly stabilized and amplified signal compared to the ones without this aptamer. The research outcome provides applicability of the highly negative charged aptamer as a bioamplifier for immunoassays by FETs.

Phase Angle: A Possible Biomarker to Quantify Inflammation in Subjects with Obesity and 25(OH)D Deficiency.

Obesity is associated to chronic low-grade metabolic inflammation and hypovitaminosis D. Among extra-skeletal effects, an important role in inflammation has been described for vitamin D (25(OH)D). Phase angle (PhA) is a bioelectrical impedance analysis (BIA) parameter that represents an indicator of cellular health in chronic inflammatory states. However, it is still unknown whether a low 25(OH)D levels might correlate with PhA in obesity. Considering the lack of evidence correlating the 25(OH)D levels with PhA in obesity, the aim of this study was to investigate their possible relationship in a group of patients with obesity stratified according to body mass index (BMI) categories. Four hundred and fifty-five adult subjects (219 males and 236 females; 36 ± 11 years) were enrolled. Body composition, including PhA, was assessed using a BIA phase-sensitive system. Serum levels of 25(OH)D was determined by a direct competitive chemiluminescence immunoassay. Most of the participants were affected by grade III obesity (24%) and had 25(OH)D deficiency (67%). Subjects with 25(OH)D deficiency had highest BMI (p < 0.001). Stratifying the sample population according to the BMI classes, 25(OH)D levels decreased significantly along with the increase in BMI (p < 0.001), with the lowest 25(OH)D levels in the class III obesity. In addition, stratifying the sample population according to 25(OH)D categories, BMI and fat mass (FM) decreased, while PhA increased significantly along with the 25(OH)D categories (p < 0.001). The 25(OH)D levels showed significant positive associations with PhA (r = −0.59, p < 0.001), and this association remained significant also after adjusting for BMI and FM (r = 0.60, p < 0.001). The lowest values of PhA were significantly associated with the severity of obesity (OR 0.3, p < 0.001) and of 25(OH)D deficiency (OR 0.2, p < 0.001). To compare the relative predictive power of body composition parameters associated with the 25(OH)D levels, we performed a multiple linear regression analysis. The most sensitive and specific cut-off for 25(OH)D levels to predict the PhA above the median was >14 ng/mL (p < 0.001). In conclusion, we provided preliminary insights into a novel link between 25(OH)D levels and PhA in the setting of obesity. This association uncovered a new potential usefulness of PhA as expression of cell membrane integrity and predictor of inflammation in low 25(OH)D status that might help in identifying high-risk patients with obesity who could benefit from careful 25(OH)D supplementation.

Highly Specific Chemiluminescence Immunoassay for the Determination of Chloramphenicol in Cosmetics.

A direct and highly specific chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) method for monitoring chloramphenicol (CAP) in cosmetics has been developed. The anti-chloramphenicol antibody (mAb) adopted in this work for direct immunoassay could bind to CAP specifically, with negligible cross-reactivity (CR) (less than 0.01%) with most CAP analogues, including structurally related thiamphenicol (TAP) and florfenicol (FF). The limit of detection (LOD), measured by IC10, was 0.0021 ng mL−1. The detection range (IC20-IC80) was ranged from 0.00979 to 0.12026 ng mL−1. In spiked cosmetics samples, mean recoveries ranged from 82.7% to 99.6%, with intraday and interday variation less than 9.8 and 8.2%, respectively. Moreover, with the help of HRP-labeled anti-CAP mAb, the method could be processed in fast direct immunoreaction mode. This CL-ELISA method could be applied for specific, rapid, semiquantitative, and quantitative detection of CAP in cosmetics, facilitating the precise quality control of CAP contamination.

Lapland extreme 900 km: Biochemical responses during training, competition and restoring period. A case study

This review discusses the state-of-the-art measurement of free and total thyroid hormones in clinical laboratories. We highlight some of the limitations of currently used immunoassays and critically discuss physical separation methods for the measurement of free thyroid hormone. Physical separation methods, such as equilibrium dialysis or ultrafiltration, followed by tandem mass spectrometry for the measurement of free thyroid hormones offer many advantages, which we feel, can deepen our understanding of thyroid hormone metabolism and improve patient diagnosis and care. Problems with direct analogue immunoassay methods for FT4/FT3 as well as immunoassay methods for total T3 at low T3 concentrations and during pregnancy are highlighted. Improved diagnosis and patient management can be achieved utilizing tandem mass spectrometry for these measurements.

Improving direct immunoassay response by layer-by-layer films of gold nanoparticles – Antibody conjugate towards label-free detection

The conjugation of nanoparticles with antibodies has been successfully applied in sandwich immunoassays for detecting cancer biomarkers. However, two antibodies are necessary to perform such experiment, being one of them functionalized with a signal label for optical or electrochemical assay. This approach is time and cost consuming compared to direct label-free immunoassays. In this study, we propose the synthesis of gold nanoparticles conjugated to anti-PSA antibody to produce a label-free impedimetric immunosensors based on nanostructured Layer-by-Layer (LbL) films. Prostate-specific antigen (PSA) detection was performed by electrochemical impedance spectroscopy demonstrating a detection mechanism governed by Langmuir-Freundlich adsorption model. This strategy provided a significant sensitivity using 10-fold less antibody than conventional immunosensors, i.e. decreasing costs using a simple approach, with a limit of detection of 0.17 ng mL−1 and an analytical range of 0.1–20 ng mL−1 indicating that our sensor is potentially useful for clinical applications.