Sandwich-type Enzyme-linked Immunosorbent Assay Research Articles

РАСПРЕДЕЛЕНИЕ ГЕНОТИПОВ ГЕНА SLC2A9, УРОВЕНЬ МОЧЕВОЙ КИСЛОТЫ И МЕТАБОЛИТЫ ПУРИНОВОГО ОБМЕНА У ПАЦИЕНТОВ С АРТЕРИАЛЬНОЙ ГИПЕРТЕНЗИЕЙ И ФИБРИЛЛЯЦИЕЙ ПРЕДСЕРДИЙ

The aim is to investigate the frequency of genotypes and alleles of the SLC2A9 gene rs734553 polymorphism, the level of serum uric acid (sUA) and metabolites of purine metabolism in patients with arterial hypertension (AH) and atrial fibrillation (AF), as well as in healthy individuals. Material and methods. The study included 154 patients: 50 were healthy individuals (group 0), of which 22 (44%) were men and 28 (56%) were women aged 50 [45;53] years and 104 were patients with AH and AF (main group), of which 94 (90.4%) were men and 10 (9.6%) were women aged 55 [45; 61] years. The main group was divided into subgroups: subgroup I – patients with AF without a history of AH and other rhythm disorders (n=13); subgroup II – patients with AH in combination with AF (n=68); subgroup III – patients with AH without a history of AF or other rhythm disturbances (n=23). Hyperuricemia was detected in 34 (22.1%) patients, normal uric acid levels were revealed in 120 (77.9%) patients. All patients were studied using clinical, laboratory, instrumental and molecular genetic research methods. The level of sUA was determined by the enzymatic colorimetric method. Serum xanthine oxidase was measured using a method based on a solid phase sandwich-type enzyme-linked immunosorbent assay. Metabolites of purine metabolism in blood plasma were measured using the method of high-performance liquid chromatography. The SLC2A9 gene rs734553 polymorphism was determined using the polymerase chain reaction with real-time detection of results. Results. The patients with AH and AF, as compared to healthy individuals, had more severe disturbances of purine metabolism, characterized by higher concentration of sUA (330 [283; 412] µmol/l and 197 [161; 229] µmol/l (p<0.001), respectively). Also, in contrast to the group of healthy individuals, the group of patients with AH and AF demonstrated an increase in the level of adenosine (p=0.001), and a decrease in the levels of hypoxanthine and xanthine (p<0.001). There were no statistically significant differences in xanthine oxidase activity level (p>0.05), however, in 54% of patients in the main group it was higher than normal values. The dominant allele A and the dominant genotype A/A of the SLC2A9 gene rs734553 polymorphism (75%, p=0.005; 64%, p=0.001, respectively) occurred significantly more often in healthy individuals, while the recessive allele C and the heterozygous genotype A/C were found significantly more often in the group of patients with AH and AF (41.3%, p=0.005; 48.1%, p=0.003, respectively). The C/C genotype (41.7%, p=0.001) was significantly more common in patients with AH in combination with AF and hyperuricemia, compared to patients with AH combined with AF without hyperuricemia as well as healthy individuals (6.8%; 14%, p=0.001, respectively). Patients with AH in combination with AF and the C/C genotype were characterized by a significantly higher level of sUA (p=0.003) compared to patients with the A/A genotype. Conclusions. A statistically significant predominance of the recessive allele C of the SLC2A9 gene rs734553 polymorphism was established in patients with AH and AF compared with healthy individuals (p=0.005). In patients with AH in combination with AF and hyperuricemia, the C/C genotype was significantly more common (41.7%, p=0.001). Patients with AH in combination with AF and the C/C genotype were characterized by a significantly higher level of sUA (p=0.003) compared to patients with the A/A genotype.

Phenotypic variants of occupational bronchial asthma (immune profile, cytokines, diagnosis and prognosis)

Currently, occupational bronchial asthma is considered as a phenotypically and genotypically heterogeneous disease. The assessment of immunological characteristics opens up new opportunities in assessing the disease development, predicting the characteristics of the course, and establishing a personalized approach to pharmacotherapy of occupational asthma, as well as in developing an individual strategy for its prevention. The purpose of the study is to determine immunological markers of the risk of developing occupational bronchial asthma under conditions of exposure to sensitizing substances for various phenotypes of this disease. Materials and methods. The study included 170 patients with various phenotypes of occupational asthma and 50 participants in the control group. The levels of immunoglobulins A, M, and G in serum were determined by the Mancini technique (radial immunodiffusion). The determination of the levels of cytokines IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17, TNF-α, MCP- 1, and IFN-γ, total IgE, C-reactive protein, and fibronectin in blood serum was carried out by enzyme-linked immunosorbent assay using kits and reagents (OOO Proteinovy Kontur, Vektor-Best, Diateks-E, DIA-plus, Pharmacia diagnostika). To study the content of vascular endothelial growth factor (VEGF) in blood serum, we used a sandwich-type enzyme-linked immunosorbent assay with a set of reagents from eBioscience. Genotyping was performed by real-time polymerase chain reaction using primers and probes developed with the PrimerQuest program (Integrated DNA Technologies, Inc.). Results. For the first time, the study has established the features of immunological manifestations in allergic and non-allergic phenotypes of occupational bronchial asthma, as well as the phenotypes of the combination of occupational asthma with occupational chronic obstructive pulmonary disease and metabolic syndrome. The features of the formation, pathogenesis, and prognosis of various phenotypes of occupational bronchial asthma have been identified. This allows recommending the determination of these immunological parameters during in-depth periodic medical examinations of workers exposed to sensitizing and irritating substances for differential diagnosis of various occupational asthma phenotypes in a specialized inpatient examination. Limitations of the study. The study has regional (Samara Region) and professional (in terms of detailing working conditions in the studied comparison groups) limitations. Conclusion. The identified immunological features in various phenotypes of occupational bronchial asthma and the established profiles of occupational asthma genotypes can optimize the approach to early diagnosis, prognosis, prevention, and pharmacotherapy of this disease, as well as expand the list of immunological research indicators used during preliminary and periodic medical examinations, in-depth examination of patients with occupational asthma in occupational disease clinics and occupational pathology centers, and the use of new reliable criteria for predicting the course of the disease.

Evaluating the influence of cold plasma bubbling on protein structure and allergenicity in sesame milk.

Sesame is a traditional oilseed comprising essential amino acids. However, the presence of allergens in sesame is a significant problem in its consumption; thus, this study attempted to reduce these allergens in sesame oilseeds. The present study aimed to evaluate the effect of cold plasma processing on structural changes in proteins, and thereby the alteration of allergenicity in sesame milk. Method: Sesame milk (300 mL) was processed using atmospheric pressure plasma bubbling unit (dielectric barrier discharge, power: 200 V, and airflow rate: 16.6 mL/min) at different exposure times (10, 20, and 30 min). The efficiency of plasma-bubbling unit as measured by electron paramagnetic resonance in terms of producing reactive hydroxyl (OH) radicals proved that generation of reactive species increased with exposure time. Further, the plasma-processed sesame milk subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and differential scanning calorimetery analysis revealed that plasma bubbling increased the oxidation of proteins with respect to bubbling time. The structural analysis by Fourier transform infrared spectroscopy and circular dichroism revealed that the secondary structure of proteins was altered after plasma application. This change in the protein structure helped in changing the immunoglobulin E (IgE)-binding epitopes of the protein, which in turn reduced the allergen-binding capacity by 23% at 20-min plasma bubbling as determined by the sandwich-type enzyme-linked immunosorbent assay. However, 30-min plasma bubbling intended to increase allergenicity, possibly because of increase in IgE binding due to the generation of neo epitopes. These changes proved that plasma bubbling is a promising technology in oxidizing protein structure, and thereby reducing the allergenicity of sesame milk. However, increase in binding at 30-min bubbling is to be studied to facilitate further reduction of the binding capacity of IgE antibodies.

Quercetin Attenuates the Production of Pro-Inflammatory Cytokines in H292 Human Lung Epithelial Cells Infected with Pseudomonas aeruginosa by Modulating ExoS Production.

The type three secretion system (T3SS) is a major virulence system of Pseudomonas aeruginosa (P. aeruginosa). The effector protein Exotoxin S (ExoS) produced by P. aeruginosa is secreted into the host cells via the T3SS. For the purpose of an experiment on inhibitors with regard to ExoS secretion, we developed a sandwich-type enzyme-linked immunosorbent assay (ELISA) system. Quercetin was selected because it has a prominent ExoS inhibition effect and also is known to have anti-inflammatory and antioxidant effects on mammalian cells. In this study, we investigated the effects of quercetin on the expression and secretion of ExoS using ELISA and Western blot analysis methods. The results showed that the secretion of ExoS was significantly decreased by 10 and 20 μM of quercetin. Also, popB, popD, pscF, and pcrV which are composed of the T3SS needle, are reduced by quercetin at the mRNA level. We also confirmed the inhibitory effect of quercetin on cytokines (IL-6, IL-1β, and IL-18) in P. aeruginosa-infected H292 cells by real-time polymerase chain reaction (PCR) and ELISA. Collectively, quercetin inhibits the secretion of ExoS by reducing both ExoS production and the expression of the needle protein of T3SS. Furthermore, these results suggest that quercetin has the potential to be used as an anti-toxic treatment for the inflammatory disease caused by P. aeruginosa infection.

Chronic musculoskeletal pain in degenerative changes of the spine in young and middle-aged patients

To identify clinical and laboratory indicators associated with the development of chronic musculoskeletal pain syndrome in young and middle-aged patients with degenerative changes of the spine. The study included 103 patients (59 women and 44 men) with a mean age 42 [36; 47] years with chronic musculoskeletal back pain. To verify degenerative changes of the spine, MRI was performed using Magnetom Symphony (Siemens; Germany), Magnetom Verio (Siemens; Germany) devices. The intensity of the pain syndrome, the neuropathic component of pain sensations, and affective disorders were assessed. Blood levels of osteocalcin, parathyroid hormone, thyroid stimulating hormone were determined using an immunochemiluminescent analyzer Immulite 2000 (Siemens, USA), vitamin D - using an immunochemical analyzer Architect i2000SR (Abbott, USA), vitamin PP and 17-hydroxyprogesterone by sandwich-type ELISA on a plate reader VICTOR 2 (Perken Elmer, USA). The levels of interleukin-1β, interleukin-6 and interleukin-8 and tumor necrosis factor alpha (TNF-α) in the blood were determined by enzyme-linked immunosorbent assay (ELISA) on a Real-best plate ELISA analyzer (Russia) using Cloud Clone Corparation kits (USA, China). The main causes of musculoskeletal pain in the main group (83 patients) with a mean age of 42 [38; 46] years, were degenerative changes of the spine without signs of compression of the spinal nerves and spinal canal stenosis. In the control group (20 patients) with a mean age of 41 [34; 47] years, main causes were static and functional disorders. Patients with degenerative changes of the spine often led a sedentary lifestyle and had an increased body mass index. Tobacco smoking was noted in 48.2% of patients of the main group and 25% of patients in the control group (p=0.080). The intensity of pain in patients in both groups had a moderate degree, the neuropathic component of pain was not detected. Asthenia, situational and personal anxiety were mild or elevated in both groups In the main group, there was an increase in the levels of pro-inflammatory factors (C-reactive protein, TNF-α, IL-1β, IL-6 and IL-8), as well as a decrease in the level of osteocalcin. The revealed increase in the levels of pro-inflammatory factors (C-reactive protein, TNF-α, IL-1β, IL-6 and IL-8) in the blood confirms the role of inflammatory reactions in the pathogenesis of degenerative changes of the spine and the associated musculoskeletal pain syndrome. A decrease in the level of osteocalcin with a normal content of parathyroid hormone in the blood in patients of main group may be associated with desorganized bone remodeling and increased bone resorbtion.

Development of a sensitive sandwich-ELISA assay for reliable detection of fish residues in foods

A new sandwich-type Enzyme-Linked Immunosorbent Assay (ELISA) method was developed based on goat IgG as capturing antibody and rabbit IgG as detecting antibody targeting soluble antigenic fish proteins in foods as detection targets. The assay has provided a relatively lower limit of quantitation (LoQ) for fish proteins with LoQ 0.5 ng/ml and appears highly sensitive. The analysis of 24 different substances, both raw and boiled, revealed no cross-reactivity above the cut-off point of the limit of quantitation. Recoveries of the SB spiked food matrixes were in the range of 83–131%. Assay precision testing proved that repeatability (<5%) and reproducibility (<11%) had an acceptable level of variation. The sandwich ELISA was capable of detecting all tested commercially important fish. As a potential analytical tool, the newly developed immunoenzymatic method is suitable for detecting undeclared fish residues in real food samples available in the market, thereby will help to reduce the incidents of fish allergies.

Serum Biomarkers in Differential Diagnosis of Idiopathic Pulmonary Fibrosis and Connective Tissue Disease-Associated Interstitial Lung Disease.

Introduction: The goal of this study is to determine whether Advanced glycosylated end-products (AGE), Advanced oxidation protein products (AOPP) and Matrix metalloproteinase 7 (MMP7) could be used as differential biomarkers for idiopathic pulmonary fibrosis (IPF) and connective tissue disease-associated interstitial lung disease (CTD-ILD). Method: Seventy-three patients were enrolled: 29 with IPF, 14 with CTD-ILD, and 30 healthy controls. The study included a single visit by participants. A blood sample was drawn and serum was analysed for AGE using spectrofluorimetry, AOPP by spectrophotometry, and MMP7 using sandwich-type enzyme-linked immunosorbent assay. Results: AGE, AOPP and MMP7 serum levels were significantly higher in both IPF and CTD-ILD patients versus healthy controls; and AGE was also significantly elevated in CTD-ILD compared to the IPF group. AGE plasma levels clearly distinguished CTD-ILD patients from healthy participants (AUC = 0.95; 95% IC 0.86–1), whereas in IPF patients, the distinction was moderate (AUC = 0.78; 95% IC 0.60–0.97). Conclusion: In summary, our results provide support for the potential value of serum AGE, AOPP and MMP7 concentrations as diagnostic biomarkers in IPF and CTD-ILD to differentiate between ILD patients and healthy controls. Furthermore, this study provides evidence, for the first time, for the possible use of AGE as a differential diagnostic biomarker to distinguish between IPF and CTD-ILD. The value of these biomarkers as additional tools in a multidisciplinary approach to IPF and CTD-ILD diagnosis needs to be considered and further explored. Multicentre studies are necessary to understand the role of AGE in differential diagnosis.

Quantitation of venom Antigens from Moroccan vipers in serum by using an Enzyme-Linked Immunosorbent Assay (ELISA) toward improving health vigilance systems

In the present study an ELISA assay was developed and validated for detection and determination of the concentration of snakes venom in biological samples. Individual component of each venom (Cerastes cerastes and Macrovipera mauretanica) used as immunogen to raise specific rabbit IgGs in order to set up a sandwich-type ELISA. Lower detection limit, linearity, accuracy, precision, reproducibility, and reference intervals were determined. The method proved to be simple, specific, reproducible, sensitive (detection limit = 0.5 ng/ml) and the calibration plot was based on linear regression analysis (r = 0.980) between 0.9 and 1000 ng/mL of venom concentration, with a lower limit of quantification of 1.58 ng/mL. The intra- and interassay coefficient of variation ranged from 2,02 to 4.62% and 5.29 to 7.40%, respectively. The specificity of the assay was tested using vipers, cobra and scorpion venom. This method detected venom from all viper species tested without significant cross reactivity with other venoms in the concentration range of 0.9–1000 ng/mL. This ELISA described is sufficiently validated for clinical evaluation. The method is adaptable to other venoms. This is potentially useful for clinical diagnosis of snakebite, to monitor antivenom dose, and consequently to improve the national health monitoring systems.

Molecular affinity rulers: systematic evaluation of DNA aptamers for their applicabilities in ELISA.

Many nucleic acid aptamers that bind to target molecules have been reported as antibody alternatives. However, while the affinities of aptamers vary widely, little is known about the relationship between the affinities and their applicabilities for practical use. Here, we developed molecular affinity rulers: a series of DNA aptamers with different affinities that bind to the same area of target molecules, to measure the aptamer and its device applicabilities. For the ruler preparation, we used high-affinity DNA aptamers containing a hydrophobic unnatural base (Ds) as the fifth base. By replacing Ds bases with A bases in Ds-DNA aptamers targeting VEGF165 and interferon-γ, we prepared two sets of DNA aptamers with dissociation constants (KD) ranging from 10−12 to 10−8 M. Using these molecular affinity rulers, we evaluated the sensitivity of DNA aptamers in ELISA (enzyme-linked immunosorbent assay), which showed the clear relationship between aptamer affinities and their detection sensitivities. In sandwich-type ELISA using combinations of aptamers and antibodies, aptamers with KD values lower than ∼10−9 M were required for sufficient sensitivities (limit of detection (LOD) < 10 pM) and signal intensities, but optimizations improved the lower-affinity aptamers’ applicabilities. These aptamer affinity rulers could be useful for evaluating and improving aptamer applicabilities.

Validation of Enzyme Immunoassay for Preclinical Pharmacokinetic Trials of Rituximab

An integral part of preclinical pharmacokinetic studies is the development of a bioanalytical method for determination of the drug in a biological fluid.The aim of the research was to assess the suitability of the test system based on enzyme-linked immunosorbent assay (ELISA) for quantitative determination of rituximab in the blood serum of laboratory animals after intravenous administration of rituximab at a dose corresponding to the therapeutic dose in humans. Th test system was developed by the Scientific and Production Center Probiotech.Materials and methods: the determination of rituximab in biological samples was carried out using a two-stage sandwich-type ELISA, followed by detection based on horseradish peroxidase. The ELISA results were recorded using a microplate photometer at a wavelength of 450 nm.Results: the experiments helped to establish the detection limit (0.24 ng/mL) and the lower limit of quantitation (1.00 ng/mL) of rituximab in rabbit blood serum, they also demonstrated high selectivity of analyte determination in a multicomponent biological matrix. The mean rituximab concentration was within 14 % of the nominal value in the entire working range of the method. The within-run and between-run precision of the assay did not exceed 7.4 %, the total error of the method did not exceed 20.1 %. The linearity of dilution makes it possible to use the assay for the analysis of biological samples with a wide range of rituximab concentrations. The stability of the analyte in the rabbit blood serum was confirmed by storing samples for 6 hours at room temperature, for 50 days at —35 °C, and after 3 freeze-thaw cycles. The validated immunoassay was successfully used to determine the rituximab concentration in biological samples obtained in the rituximab pharmacokinetic trial in rabbits. The accuracy of the results was confirmed for the entire range of the determined concentrations; parallelism was demonstrated between the calibration curve and the results of analysis of serially diluted rabbit serum samples with the maximum concentration of rituximab.Conclusions: the proposed enzyme immunoassay test system can be used for quantitative determination of rituximab in the blood serum of laboratory animals, as it meets acceptance criteria for all validation parameters described in the international guidelines on validation of bioanalytical methods.

High level of serum Cripto-1 in hepatocellular carcinoma, especially with hepatitis B virus infection.

Background:Human Cripto-1 (CR-1), a member of the epidermal growth factor-Cripto-1/FRL-1/Cryptic protein family (EGF-CFC), is highly expressed in a variety of human cancers. We aimed to detect serum CR-1 level in liver diseases especially in hepatocellular carcinoma (HCC) patients.Methods:Serum CR-1 level was Sandwich-type enzyme-linked immuno sorbent assay (ELISA) detected in 330 patients with liver diseases including HCC, cirrhosis, and chronic hepatitis and 50 volunteers without hepatitis B virus (HBV) or hepatitis C virus (HCV) infection as control.Results:The serum CR-1 level was significantly higher in HCC patients than volunteer controls and it was also significantly higher in HBV-related HCC than HCV-related HCC. In addition, serum CR-1 level was correlated with serum alpha-feto-protein (AFP) in HBV-related HCC patients. The serum CR-1 was also higher in cirrhosis and chronic hepatitis than volunteer controls. The serum CR-1 in HBV-related cirrhosis was higher than chronic hepatitis B, but there was no significant difference between HCV-related cirrhosis and chronic hepatitis C.Conclusions:Serum CR-1 was higher in HCC patients and might serve as a complementary biomarker to clinical diagnosis of HBV-related HCC. The high level of serum CR-1 in HBV-related liver disease might be partly attributed to HBV infection.

A sandwich-type ELISA for the detection of Listeria monocytogenes using the well-oriented single chain Fv antibody fragment

Listeria monocytogenes (LM) poses a serious threat to public health. The present study details the development of a sandwich-type enzyme-linked immunosorbent assay (S-ELISA) for the detection of LM using well-oriented single chain Fv antibody fragment (scFv) and a polyclonal antibody. The anti-LM scFv, used as capture antibody, was gene-optimized and expressed with a C-terminal PS-tag in Escherichia coli BL21(DE3) for oriented-immobilization on hydrophilic polystyrene microtiter plates. The anti-LM rabbit polyclonal antibody, used as the detection antibody, was prepared using heat-killed LM cells as an immunogen. The developed S-ELISA showed great specificity to LM and had a detection limit of lg 6.5 CFU/mL, which was better than the control S-ELISA using non-oriented scFv as the capture antibody. When the S-ELISA was applied to pork and pasteurized milk samples spiked with LM, strong responses were observed. In conclusion, the developed S-ELISA shows great potential for the rapid detection of LM.

A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9

BackgroundKallikrein 9 (KLK9) is a member of the human kallikrein-related peptidases family, whose physiological role and implications in disease processes remain unclear. The active form of the enzyme is predicted to have chymotryptic activity. In the present study, we produced for the first time the active recombinant protein and monoclonal antibodies, and developed novel immunoassays for the quantification of free and bound KLK9 in biological samples.MethodsThe coding sequence of mature KLK9 isoform (mat-KLK9) was expressed in an Expi293F mammalian system and the synthesized polypeptide was purified through a two-step protocol. The purified protein was used as an immunogen for production of monoclonal antibodies in mice. Hybridomas were further expanded and antibodies were purified. Newly-produced monoclonal antibodies were screened for reaction with the KLK9 recombinant protein by a state-of-the-art immunocapture/parallel reaction monitoring mass spectrometry-based methodology.ResultsAnti-KLK9 antibodies were combined in pairs, resulting in the development of a highly sensitive (limit of detection: 15 pg/mL) and specific (no cross-reactivity with other KLKs) sandwich-type ELISA. Highest KLK9 protein levels were found in tonsil and sweat and lower levels in the heart, kidney and liver. Hybrid immunoassays using an anti-KLK9 antibody for antigen capture and various anti-serine protease inhibitor polyclonal antibodies, revealed the presence of an a1-antichymotrypsin-bound KLK9 isoform in biological samples.ConclusionsThe ELISAs for free and bound forms of KLK9 may be highly useful for the detection of KLK9 in a broad range of biological samples, thus enabling the clarification of KLK9 function and use as a potential disease biomarker.

Sensitivity improvement of a sandwich-type ELISA immunosensor for the detection of different prostate-specific antigen isoforms in human serum using electrochemical impedance spectroscopy and an ordered and hierarchically organized interfacial supramolecular architecture

A gold millielectrode (GME) functionalized with a mixed (16-MHA + EG3SH) self-assembled monolayer (SAM) was used to fabricate an indirect enzyme-linked immunosorbent assay (ELISA) immunosensor for the sensitive detection of prostate-specific antigen (PSA), a prostate cancer (PCa) biomarker, in human serum samples. To address and minimize the issue of non-specific protein adsorption, an organic matrix (amine-PEG3-biotin/avidin) was assembled on the previously functionalized electrode surface to build up an ordered and hierarchically organized interfacial supramolecular architecture: Au/16-MHA/EG3SH/amine-PEG3-biotin/avidin. The electrode was then exposed to serum samples at different concentrations of a sandwich-type immunocomplex molecule (BtnAb-AgPSA-HRPAb), and its interfacial properties were characterized using electrochemical impedance spectroscopy (EIS). Calibration curves for polarization resistance (RP) and capacitance (1/C) vs. total and free PSA concentrations were obtained and their analytical quality parameters were determined. This approach was compared with results obtained from a commercially available ELISA immunosensor. The results obtained in this work showed that the proposed immunosensor can be successfully applied to analyze serum samples of patients representative of the Mexican population.

Rapid and Sensitive Detection of Escherichia coli O157:H7 in Milk and Ground Beef Using Magnetic Bead–Based Immunoassay Coupled with Tyramide Signal Amplification

Escherichia coli O157:H7 is a major foodborne pathogen that has posed serious problems for food safety and public health. Recent outbreaks and recalls associated with various foods contaminated by E. coli O157:H7 clearly indicate its deleterious effect on food safety. A rapid and sensitive detection assay is needed for this harmful organism to prevent foodborne illnesses and control outbreaks in a timely manner. We developed a magnetic bead–based immunoassay for detection of E. coli O157:H7 (the most well-known Shiga toxigenic E. coli strain) with a 96-well microplate as an assay platform. Immunomagnetic separation (IMS) and tyramide signal amplification were coupled to the assay to increase its sensitivity and specificity. This immunoassay was able to detect E. coli O157:H7 in pure culture with a detection limit of 50 CFU/ml in less than 3 h without an enrichment step. The detection limit was decreased 10-fold to 5 CFU/ml with addition of a 3-h enrichment step. When this assay was tested with other nontarget foodborne pathogens and common enteric bacteria, no cross-reactivity was found. When tested with artificially contaminated ground beef and milk samples, the assay sensitivity decreased two- to fivefold, with detection limits of 250 and 100 CFU/ml, respectively, probably because of the food matrix effect. The assay results also were compared with those of a sandwich-type enzyme-linked immunosorbent assay (ELISA) and an ELISA coupled with IMS; the developed assay was 25 times and 4 times more sensitive than the standard ELISA and the IMS-ELISA, respectively. Tyramide signal amplification combined with IMS can improve sensitivity and specificity for detection of E. coli O157:H7. The developed assay could be easily adapted for other foodborne pathogens and will contribute to improved food safety and public health.

Combined Cathepsin S and hs-CRP predicting inflammation of Abdominal Aortic Aneurysm

ObjectivesCathepsin S (Cat S) protein expression is increased in human abdominal aortic aneurysm (AAA) lesions and Cat S has been suggested a direct role by promoting inflammatory response partly in experimental AAA. The purpose of this study is to observe the expression of serum Cat S and hs-CRP and its clinical significance in AAA patients. Design and methodsWe collected serum samples from 31 AAA patients and 32 controls. Cat S and hs-CRP levels were measured by a sandwich-type enzyme-linked immunosorbent assay (ELISA) and an enhanced immunoturbidimetric assay respectively. The maximum diameter of the AAA was identified by ultrasonography. ResultsThe patients with AAA had higher serum Cat S and hs-CRP levels than the controls (p<0.05). Furthermore, human serum Cat S levels were strongly correlated with hs-CRP by the nonparametric Spearman correlation tests (B=0.849, p<0.05). Based on Pearson's correlation test, human serum Cat S and hs-CRP levels were positively correlated with AAA diameter size (p<0.05). Cat S was correlated independently with the hs-CRP in all subjects (p<0.01). After adjustment for the maximum diameter of the abdominal aorta-associated variables, Cat S combined hs-CRP (R2=0.801) is better than Cat S (R2=0.740) in predicting the maximum diameter of AAA lesions. ConclusionCombined serum Cat S and hs-CRP levels are better in predicting the inflammatory activity of AAA lesions in the clinical setting.

Development of automated paper-based devices for sequential multistep sandwich enzyme-linked immunosorbent assays using inkjet printing

To the best of our knowledge, this is the first report on paper-based devices for automating the sequential multistep procedures of a sandwich-type enzyme-linked immunosorbent assay (ELISA) that require only a single-step application of the sample solution. The device was based on a piece of nitrocellulose (NC) membrane with specially designed channels, where all the reagents are applied at different locations in order to control the fluid travel to the detection region. The inkjet printing method, a simple and low-cost process, was used to create the flow channel and device barrier patterns. The fabricated barrier was found to be an efficient boundary for the liquid along the printed design in the NC membrane, enabling direct control of the reagent flow time. ELISA results were obtained with a single-step sample application. The developed devices (so-called automated paper-based devices) provided a simple procedure for the sandwich ELISA, while reducing assay time and reagent consumption. Colorimetric results were measured using digital camera imaging with software processing. The capability of the method developed herein was successfully used to determine the levels of human chorionic gonadotropin (hCG) by ELISA.

Serum sclerostin levels decline in post-menopausal women with osteoporosis following treatment with intermittent parathyroid hormone.

This study was carried out in order to evaluate the effect of 18-month treatment with PTH (1-34) or PTH (1-84) on serum sclerostin levels in humans. We investigated 10 women with severe osteoporosis, previously treated with alendronate and 20 untreated osteoporotic women. Subjects with severe osteoporosis were randomly divided into 2 groups of 5 patients each; the first group was treated with 20 μg of PTH (1-34) and the second one with 100 μg of PTH (1-84) according to an open-label design. Fasting blood samples were collected at baseline and at 2, 4, and 24 h after hormone administration. The same protocol was followed at month 1, 6, 12, 18. Serum sclerostin levels were measured at each time point by a sandwich-type enzyme-linked immunosorbent assay. Basal serum sclerostin levels were not significantly different between patients previously treated with alendronate and those never treated. No significant acute change of serum sclerostin levels was observed after PTH administration. Fitting a mixed effect regression model, we found a significant time effect (p=0.0012) using the sclerostin level as the response variable and the month of drug administration as a single covariate. Treatment with both PTH molecules induced a monthly mean reduction of sclerostin levels of 0.1956 pmol/l. Our results indicate that long-term therapy with PTH (1-34) or PTH (1-84) in women with osteoporosis previously treated with alendronate is associated with a reduction in circulating sclerostin levels. This is a putative mechanism through which PTH performs its anabolic action.

Development of a monoclonal antibody-based sandwich-type enzyme-linked immunosorbent assay (ELISA) for detection of abrin in food samples

Abrin is a plant toxin, which can be easily isolated from the seeds of Abrus precatorius. It may be used as a biological warfare agent. In order to detect abrin in food samples, a two-layer sandwich format enzyme-linked immunosorbent assay based on the monoclonal antibody (mAb) (as capture antibody) and rabbit polyclonal serum (as detecting antibody) was developed and applied for the determination of abrin in some food matrices. The linear range of the mAb was 1–100μgL−1 with a detection limit of 0.5μgL−1 for abrin in phosphate buffered saline (PBS). The recoveries of abrin from sausage, beer and milk samples ranged 97.5–98.6%, 95.8–98.4% and 94.8–9.6%, respectively, with a coefficient of variation (CV) of 3.7% or less. The newly developed sandwich ELISA using the mAb appears to be a reliable and useful method for detection of abrin in sausage, beer and milk.

Vascular Endothelial Growth Factor (VEGF) and Soluble VEGF Receptor 1 (sFlt-1) Levels in Early and Mature Human Milk from Mothers of Preterm versus Term Infants

Vascular endothelial growth factor (VEGF) and its receptors regulate angiogenesis (formation of blood vessels). The soluble VEGF receptor 1 (sFlt-1) binds VEGF as a potent antagonist. The objective of this study was to compare VEGF and sFlt-1 levels in milk from mothers of preterm (n = 50) versus term (n = 49) infants in a longitudinal study. Milk samples were collected on days 3 and 28 of lactation. Vascular endothelial growth factor and sFlt-1 were quantified by sandwich-type enzyme-linked immunosorbent assay. Vascular endothelial growth factor and sFlt-1 were found in high concentrations in early milk (lactation day 3) from mothers of preterm and term infants and were lower in mature milk (lactation day 28). On day 3, median VEGF concentration was lower in preterm than in term milk (37.1 vs 53.9 ng/mL, P < .01). Otherwise, VEGF (day 28) and sFlt-1 (days 3 and 28) did not differ in preterm versus term milk. It was shown for the first time that sFlt-1 is present in human milk. Early human milk contains high concentrations of VEGF and sFlt-1, which decrease over the course of lactation.