Microplate Enzyme-linked Immunosorbent Assay Research Articles

Rapid magneto-enzyme-linked immunosorbent assay for ultrasensitive protein detection

Protein-based diagnostics are the standard of care for screening and diagnosing a broad range of diseases and medical conditions. The current gold standard method for quantifying proteins in clinical specimens is the enzyme-linked immunosorbent assay (ELISA), which offers high analytical sensitivity, can process many samples at once, and is widely available in many diagnostic laboratories worldwide. However, running an ELISA is cumbersome, requiring multiple liquid handling and washing steps, and time-intensive (∼2 − 4 h per test). Here, we demonstrate a unique magneto-ELISA that utilizes dually labeled magnetic nanoparticles (DMPs) coated with horseradish peroxidase (HRP) and an HRP-conjugated detection antibody, enabling rapid immunomagnetic enrichment and signal amplification. For proof of concept, this assay was used to detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a malaria parasite biomarker, which exhibited a lower limit of detection of 2 pg mL−1 (33 fM) in human serum. Measurements of PfHRP2 in clinical blood samples from individuals with and without P. falciparum infection revealed that this magneto-ELISA offers a superior diagnostic accuracy compared to a commercial PfHRP2 ELISA kit. We also demonstrate the versatility of this platform by adapting it for the detection of SARS-CoV-2 nucleocapsid protein, which could be detected at concentrations as low as 8 pg mL−1 (174 fM) in human serum. In addition to its high analytical performance, this assay can be completed in 30 min, requires no specialized equipment, and is compatible with standard microplate readers and ELISA protocols, allowing it to integrate readily into current clinical practice.

Changes in the organism of patients with chronic inflammatory diseases of the upper respiratory tract at different periods after administration of influenza vaccine. Message 1. Status of congenital immune parameters after a single anti-influenza vaccination

Introduction: Studies of the clinical status, production of antibodies to hemagglutinins of influenza A and B viruses and the content of immune complexes in patients with chronic inflammatory diseases (CID) of the upper respiratory tract (URT) before and within 36 weeks of vaccination after parenteral vaccination Vaxigrip leads to a short-term (~ 6 months) increase in anti-influenza hemagglutinin t itres. According to modern ideas about the mechanisms of antiviral protection, in addition to humoral, mainly due to antibodies, antiviral protection is largely due to the activation of innate immune factors, both systemic and local. Aim: to evaluate in the dynamics of observations the influence of a single parenteral influenza vaccination on the factors of innate immunity in patients with chronic inflammatory diseases of the upper respiratory tract. Material and methods: Among the factors of innate immunity a significant role is played by interferons, the activity of natural cytolytic cells (NK) of various histogenesis, phagocytic cells and non-specific proteins such as defensins, elafins. Therefore, quantitative and functional characteristics of NK-cells, phagocytic absorption activity of polymorphonuclear leukocytes (PMN), systemic content of γ-interferon (IFN-γ) were studied in patients at the systemic level. At the local level, the concentrations of defensin-β and α-interferon were studied in mucosa secretes (MS) according to modern ideas about the state of the mechanisms of innate antiviral immunity. From the autumn-winter period of 2019 to November 2020, 32 donors (control group) and 32 patients with CID of both sexes were examined, 11 of whom were diagnosed with chronic rhinosinusitis, 9 with chronic tonsillitis, and 12 with chronic pharyngitis. No vaccinations were given 1 year before the study. Samples of venous serum and oropharyngeal secretes of all clinically healthy patients, as well as persons with CID were obtained at the initial examination. After parenteral vaccination with Vaxigrip (SANOFI PASTEUR, S.A., France), blood and MS were tested twice more in vaccinated patients: 12 and 36 weeks after vaccination. The obtained samples of experimental material were stored at a temperature of -20 C◦ until the simultaneous determination of innate immune factors (interferons), in particular with the help of reagents from Cytokine, Hema Medica (RF), defensin-β (Hucalt-bio, the Netherlands). Measurements were performed on a microplate enzyme-linked immunosorbent assay "STAT FAX 2100 (USA). Statistical processing was performed using the non-parametric criterion "U" - Mann-Whitney or Fisher's exact method. Differences were considered statistically significant at p <0.05. The results were given in the form of arithmetic mean (M), median (Me) and percentiles (P25-P75). Results: In determining the role of innate immunity factors in vaccination with influenza vaccine, we conducted clinical and immunological examination of patients with chronic inflammatory diseases of the upper respiratory tract, which included systemic determination of γ-interferon, quantitative and functional activity of natural cytotoxic cells, phagocytosis activity at the stage of attraction and capture. Levels of α-interferon and defensin-β in oropharyngeal secretion (MS) were studied. It was found that before influenza vaccination in patients with chronic inflammatory diseases of the upper respiratory tract found an increased content of γ-interferon and decreased phagocytic activity of polymorphonuclear leukocytes. Decreased α-interferon and defensin-β concentrations were detected in MS patients before vaccination. After vaccination, there were positive changes in the direction of increasing the concentration of nonspecific local immunity factor – defensing 1β and partly in the observation period of 3 months after vaccination- α-interferon. Сonclusion: Among the factors of innate immunity in patients with CІD at the systemic level, decreased cytolytic activity of NK, increased levels of γ-interferon and decreased phagocytic function of neutrophils. Decreased concentration of α-interferon and defensin-1β is determined in the saliva of patients with CID. Vaccination of patients with chronic inflammatory diseases of the upper respiratory tract with influenza vaccine has a positive effect on the state of innate immune factors of local and systemic level: concentration of γ-interferon, phagocytic activity of neutrophils and enhancing the functional activity of natural killer cells, increase in saliva of defensin-1β.

The protective mechanism of Grx2 in ultraviolet-B (UVB)-induced cataract formation

ObjectiveWe established a mouse cataract model by irradiating Grx2 knockout (KO) and knock-in (KI) genetically modified mice with UVB to explore the protective mechanism of Grx2 against UVB lens damage. MethodsAfter irradiating Grx2 KO and Grx2 KI mice with UVB lamps, we observed and recorded the general physiological conditions and lens opacity of the mice. The crystalline grading system of the University of Oxford was used to classify the opacity of the lens. Lens reactive oxygen species (ROS) contents were detected using a microplate reader, western blot, and enzyme-linked immunosorbent assay (ELISA) to detect antioxidant and antioxidant enzyme contents. Statistical analysis of the recorded data was performed by using SPSS 19.0 software. ResultsAfter UVB irradiation, the weight of Grx2 KO mice was slightly lower than that of wild-type (WT) mice of the same age. Compared to WT mice, the lens opacity of Grx2 KO mice appeared earlier, the nucleus density of the lens increased, and the opacity increased in the first week after UVB irradiation. Meanwhile, the lenses of Grx2 KI mice remained transparent. The experiment showed that the content of ROS increased, the level of glutathione (GSH) decreased, the content of 8-OHdG increased, and the expression of BCL2 decreased after UVB irradiation. Compared to WT mice, these changes were more significant in Grx2 KO mice. ConclusionThis experiment found that knocking out the Grx2 gene accelerated the occurrence and development of UVB-induced cataracts in mice and that Grx2 plays an important role in the oxidative damage caused by UVB radiation by repairing the antioxidant enzymes of the lens. This study provides a new animal model and research ideas for the study of cataract pathogenesis.

Gravity-Driven Microfluidic Siphons: Fluidic Characterization and Application to Quantitative Immunoassays.

A range of biosensing techniques including immunoassays are routinely used for quantitation of analytes in biological samples and available in a range of formats, from centralized lab testing (e.g., microplate enzyme-linked immunosorbent assay (ELISA)) to automated point-of-care (POC) and lateral flow immunochromatographic tests. High analytical performance is intrinsically linked to the use of a sequence of reagent and washing steps, yet this is extremely challenging to deliver at the POC without a high level of fluidic control involving, e.g., automation, fluidic pumping, or manual fluid handling/pipetting. Here we introduce a microfluidic siphon concept that conceptualizes a multistep ″dipstick″ for quantitative, enzymatically amplified immunoassays using a strip of microporous or microbored material. We demonstrated that gravity-driven siphon flow can be realized in single-bore glass capillaries, a multibored microcapillary film, and a glass fiber porous membrane. In contrast to other POC devices proposed to date, the operation of the siphon is only dependent on the hydrostatic liquid pressure (gravity) and not capillary forces, and the unique stepwise approach to the delivery of the sample and immunoassay reagents results in zero dead volume in the device, no reagent overlap or carryover, and full start/stop fluid control. We demonstrated applications of a 10-bore microfluidic siphon as a portable ELISA system without compromised quantitative capabilities in two global diagnostic applications: (1) a four-plex sandwich ELISA for rapid smartphone dengue serotype identification by serotype-specific dengue virus NS1 antigen detection, relevant for acute dengue fever diagnosis, and (2) quantitation of anti-SARS-CoV-2 IgG and IgM titers in spiked serum samples. Diagnostic siphons provide the opportunity for high-performance immunoassay testing outside sophisticated laboratories, meeting the rapidly changing global clinical and public health needs.

Directional immobilization of antibody onto magnetic nanoparticles by Fc-binding protein-assisted photo-conjugation for high sensitivity detection of antigen

Immobilized antibodies with site-specific, oriented, and covalent pattern are of great significance to improve the sensitivity of solid-phase immunoassay. Here, we developed a novel antibody conjugation strategy that can immobilize antibodies in a directional and covalent manner. In this study, an IgG-Fc binding protein (Z domain) carrying a site-specific photo-crosslinker, p-benzoyl-L-phenylalanine, and a single C-terminal cysteine (Cys) handle was genetically engineered. Upon UV irradiation, the chimeric protein enables the Cys handle to couple with the native antibody in Fc-specific and covalent conjugation pattern, resulting in a novel thiolated antibody. Thus, an approach for the covalent, directional immobilization of antibodies to maleimide-modified magnetic nanoparticles (MNPs) was developed on the basis of the crosslinking between sulfhydryl and maleimide groups. The antibody-conjugated MNPs were applied in MNP-based enzyme-linked immunosorbent assay (ELISA) for the detection of carcinoembryonic antigen. The MNP-based ELISA presented a quantification linear range of 0.1–100 ng mL−1 and detection limit of 0.02 ng mL−1, which was approximately 100 times more sensitive than the traditional microplate ELISA (2.0 ng mL−1). Thus, the proposed antibody immobilization approach can be used in surface functionalization for the sensitive detection of various biomarkers.

Pixel-Based Machine Learning and Image Reconstitution for Dot-ELISA Pathogen Diagnosis in Biological Samples.

Serological methods serve as a direct or indirect means of pathogen infection diagnosis in plant and animal species, including humans. Dot-ELISA (DE) is an inexpensive and sensitive, solid-state version of the microplate enzyme-linked immunosorbent assay, with a broad range of applications in epidemiology. Yet, its applicability is limited by uncertainties in the qualitative output of the assay due to overlapping dot colorations of positive and negative samples, stemming mainly from the inherent color discrimination thresholds of the human eye. Here, we report a novel approach for unambiguous DE output evaluation by applying machine learning-based pattern recognition of image pixels of the blot using an impartial predictive model rather than human judgment. Supervised machine learning was used to train a classifier algorithm through a built multivariate logistic regression model based on the RGB (“Red,” “Green,” “Blue”) pixel attributes of a scanned DE output of samples of known infection status to a model pathogen (Lettuce big-vein associated virus). Based on the trained and cross-validated algorithm, pixel probabilities of unknown samples could be predicted in scanned DE output images, which would then be reconstituted by pixels having probabilities above a cutoff. The cutoff may be selected at will to yield desirable false positive and false negative rates depending on the question at hand, thus allowing for proper dot classification of positive and negative samples and, hence, accurate diagnosis. Potential improvements and diagnostic applications of the proposed versatile method that translates unique pathogen antigens to the universal basic color language are discussed.

Integration of Acoustic Liquid Handling into Quantitative Analysis of Biological Matrix Samples.

Acoustic liquid handlers deliver small volumes (nL-µL) of multiple fluid types with accuracy and dynamic viscosity profiling. They are widely used in the pharmaceutical industry with applications extending from high-throughput screening in compound management to gene expression sequencing, genomic and epigenetic assays, and cell-based assays. The capability of the Echo to transfer small volumes of multiple types of fluids could benefit bioanalysis assays by minimization of sample volume and by simplifying dilution procedures by direct dilution. In this study, we evaluated the Labcyte Echo 525 liquid handler for its ability to deliver small volumes of sample preparations in biological matrix (plasma and serum) and to assess the feasibility of integration of the Echo with three types of bioanalytical assay platforms: microplate enzyme-linked immunosorbent assay, Gyrolab immunoassay, and liquid chromatography with tandem mass spectrometry. The results demonstrated acceptable consistency of dispensed plasma samples from multiple lots and species by the Echo. Equivalent assay performance demonstrated between the Echo and manual liquid procedures indicated great potential for the integration of the Echo with the bioanalytical assay, which allows the successful implementation of microsampling strategies in drug discovery and development.

Development of Multispecies Recombinant Nucleoprotein-Based Indirect ELISA for High-Throughput Screening of Crimean-Congo Hemorrhagic Fever Virus-Specific Antibodies.

Crimean-Congo hemorrhagic fever (CCHF) is a re-emerging zoonotic viral disease prevalent in many parts of Asia, Europe, and Africa. The causative agent, Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV), is transmitted through hard ticks. Tick vectors especially belonging to the Hyalomma species serve as the reservoir and amplifying host. The vertebrate animals including sheep, goat, and bovine act as a short-lasting bridge linking the virus and ticks. CCHFV causes fatal hemorrhagic fever in humans. Humans are usually infected with CCHFV either through the bite of infected ticks or by close contact with infected animals. Immunological assays, primarily enzyme-linked immunosorbent assay (ELISA) using whole viral antigen, are widely used for serosurveillance in animals. However, the whole virus antigen poses a high biohazard risk and can only be produced in biosafety level 4 laboratories. The present study focuses on the development and evaluation of safe, sensitive, and specific IgG indirect enzyme-linked immunosorbent assay (iELISA) using recombinant nucleoprotein (NP) of CCHF virus as an antigen. The codon-optimized NP gene sequence was synthesized, cloned, and expressed in pET28a+ vector. The recombinant NP was purified to homogeneity by affinity chromatography and characterized through Western blot and MALDI-TOF/MS analysis. The characterized protein was used to develop an indirect IgG microplate ELISA using a panel of animal sera. The in-house ELISA was comparatively evaluated vis-à-vis a commercially available ELISA kit (Vector-Best, Russia) with 76 suspected samples that revealed a concordance of 90% with a sensitivity and specificity of 79.4 and 100%, respectively. The precision analysis revealed that the assay is robust and reproducible in different sets of conditions. Further, the assay was used for serosurveillance in ruminants from different regions of India that revealed 18% seropositivity in ruminants, indicating continued circulation of virus in the region. The findings suggest that the developed IgG iELISA employing recombinant NP is a safe and valuable tool for scalable high-throughput screening of CCHFV-specific antibodies in multiple species.

Evaluation of commercially available diagnostic tests for the detection of dengue virus NS1 antigen and anti-dengue virus IgM antibody.

Commercially available diagnostic test kits for detection of dengue virus (DENV) non-structural protein 1 (NS1) and anti-DENV IgM were evaluated for their sensitivity and specificity and other performance characteristics by a diagnostic laboratory network developed by World Health Organization (WHO), the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Pediatric Dengue Vaccine Initiative (PDVI). Each network laboratory contributed characterized serum specimens for the panels used in the evaluation. Microplate enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT formats) were represented by the kits. Each ELISA was evaluated by 2 laboratories and RDTs were evaluated by at least 3 laboratories. The reference tests for IgM anti-DENV were laboratory developed assays produced by the Armed Forces Research Institute for Medical Science (AFRIMS) and the Centers for Disease Control and Prevention (CDC), and the NS1 reference test was reverse transcriptase polymerase chain reaction (RT-PCR). Results were analyzed to determine sensitivity, specificity, inter-laboratory and inter-reader agreement, lot-to-lot variation and ease-of-use. NS1 ELISA sensitivity was 60–75% and specificity 71–80%; NS1 RDT sensitivity was 38–71% and specificity 76–80%; the IgM anti-DENV RDTs sensitivity was 30–96%, with a specificity of 86–92%, and IgM anti-DENV ELISA sensitivity was 96–98% and specificity 78–91%. NS1 tests were generally more sensitive in specimens from the acute phase of dengue and in primary DENV infection, whereas IgM anti-DENV tests were less sensitive in secondary DENV infections. The reproducibility of the NS1 RDTs ranged from 92-99% and the IgM anti-DENV RDTs from 88–94%.

Cloning and expression of an envelope gene of West Nile virus and evaluation of the protein for use in an IgM ELISA

West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis. The early confirmatory diagnosis of WNV infections is important for timely clinical management and epidemiologic control in areas where multiple flaviviruses are endemic. The coexistence of WNV along with other members of flaviviruses like dengue and Japanese encephalitis in India has complicated the serodiagnosis due to cross-reactive antigens. In the present study, the development and evaluation of a highly sensitive and specific IgM enzyme-linked immunosorbent assay (ELISA) using the recombinant envelope protein (rWNV-Env) for rapid, early, and accurate diagnosis of WNV are reported. The gene coding for the envelope protein of WNV was cloned and expressed in pET 28a vector followed by purification of recombinant protein by affinity chromatography. An indirect IgM microplate ELISA using purified rWNV-Env protein was optimized having no cross reactivity with healthy human serum. Furthermore, the specificity of this assay was confirmed by cross checking with serum samples obtained from patients with dengue and Japanese encephalitis viruses. The comparative evaluation of this rWNV-Env protein–specific IgM ELISA with plaque reduction neutralization test assay using 105 acute phase of clinical samples revealed 95% concordance with sensitivity and specificity of 92% and 97%, respectively. The positive and negative predictive values of recombinant-based Env ELISA were 94% and 96%, respectively. The recombinant envelope protein–based WNV-specific ELISA reported in this study will be useful for rapid screening of large numbers of clinical samples in endemic areas during outbreaks.

Preparation of Antibodies and Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Determination of Doxycycline Antibiotic in Milk Samples

This paper reports the development of an immunoassay for the specific analysis of doxycycline (DC), a congener of the tetracycline antibiotic family (TCs), in milk samples. This is the first time that DC antibody production is reported, based on a rationally designed and well-characterized immunizing hapten. The chemical structure of the immunizing hapten (13-[(2-carboxyethyl)thiol]-5-hydroxy-6-α-deoxytetracycline, TC1) was designed to maximize recognition of the tetracycline characteristic moiety defined as lower periphery of the TCs plus the region of the upper periphery composed by the hydroxyl group at position C(5) (B ring) and the dimethylamino group in ring A. Polyclonal antibodies raised against TC1 coupled to horseshoe crab hemocianyn (HCH) were used to develop a homologous indirect competitive enzyme-linked immunosorbent assay (ELISA). The microplate ELISA can detect DC in buffer down to 0.1 μg L(-1). The ELISA has been proven to tolerate a wide range of ionic strengths and pH values. The assay is very selective for DC with a minor recognition of methacycline (32% of cross-reactivity). Experiments performed with whole milk samples demonstrate that samples can be directly analyzed after a simple treatment method, reaching detectability values below 5 μg L(-1).

Urinary estrogen levels in women on contraceptives in Enugu, South-East Nigeria

Background:Substantial evidence supports a causal relationship between the risk of human breast cancer and levels of endogenous estrogens.Aim:To evaluate the urinary estrogen of women on contraceptives and also compare the levels in two different classes of contraceptives; hence, the possible predisposition of such women to the risk of breast cancer.Setting and Design:Urinary estrogen level was evaluated in 84 women attending family planning clinic in University of Nigeria Teaching Hospital Enugu, Nigeria, who have been on contraceptive device for 10 years or less (≤10 years). They were aged between 21 and 50 years and were divide into three groups (21-30 years, 31-40 years, and >40 years). The control group consisted of 30 age-matched apparently-healthy women who were not on any contraceptive device.Materials and Methods:Estrogen was analyzed using Ecologenia; Estrogen (E1/E2/E3) microplate enzyme-linked immunosorbent assay (ELISA) kit, Batch No. T2GR4, from Japan Envirochemicals Ltd, Japan.Statistical Analysis Used:Significant differences between means were determined by two-tailed Student's t-test using graph pad prism computer software program.Result:There was a statistically significant increase (P=0.0462), in the mean urinary estrogen level of women on contraceptives when compared with the control. The highest amount of estrogen was excreted by the women in the 21-30 years age group. When the contraceptive devices were divided into two classes of intra-uterine device and oral/injectables, there was no statistical difference (P=0.8112) in the mean urinary estrogen output of the women.Conclusion:The synthetic estrogen content of contraceptive device most probably contributed to the level excreted in the urine. The increased estrogen output observed in women on contraceptive device was not dependent on the class of contraceptive device used.

A High-Throughput Microchromatography Platform for Quantitative Analytical Scale Protein Sample Preparation

Protein analysis (using either specific protein quantitation by methods such as HPLC and immunoassays or structural analysis by methods such as LC-MS) usually requires significant sample preparation, including quantitative purification of the target protein from complex sample matrices and potentially enzymatic treatment or labeling. We have developed platform for high-throughput microchromatography, capable of running 96 or more small volume samples in parallel, producing from 10 pg to 100 μg of purified protein from each sample. The platform is based on disposable cartridge devices with 5 μL packed bed of resin. The cartridges may be operated as spin columns or run on a modified 96-channel liquid handler with ultra-low dead volume syringes that directly connect to the cartridges, providing very precisely controlled positive-displacement flow control. A major application is quantitative purification of target proteins using affinity or physical chromatography. Using large diameter nonporous beads, standard microplate enzyme-linked immunosorbent assay reagents can be used to perform 30-min immunoassays. Enzymatic digestion methods have also been developed on the system for application in glycan profiling.

A high-throughput screening (HTS) immunochemical method for the analysis of stanozolol metabolites in cattle urine samples

A high-throughput immunosorbent solid-phase extraction (HTS-IS-SPE) procedure coupled to enzyme-linked immunosorbent assay (ELISA) has been established for the analysis of stanozolol (St) and its main metabolite in cattle, 16β-hydroxy-stanozolol (16βOH-St), in cow urine samples. The chemical structure of the immunizing hapten 2′H-androst-2-eno[3,2-c]-pyrazol-17-hemiglutarate 5 (hapten A) has been designed to accomplish simultaneous detection of St and 16βOH-St. The antibodies obtained have been used to establish a microplate ELISA method able to detect these metabolites with IC 50 values of 0.57 μg L −1 and 1.46 μg L −1, respectively in PBST. Immunosorbents prepared by covalently attaching the antibodies to Sepharose, efficiently removed the matrix interferences caused by the cattle urine samples. Moreover, St and 16βOH-St were efficiently extracted from urine samples as demonstrated by LC–MS/MS analysis. The immunosorbents are filled on small mini-columns arranges on a 96-SPE-setup compatible with the microplate based ELISA methods. Samples and standards can be run in parallel which increment considerably the speed of the screening method. The recovery values of the whole HTS-IS-SPE-ELISA procedure has found to be 112 ± 10% and St can be detected in hydrolyzed urine samples with LOD of 1.26 ± 0.46 μg L −1 using just 1 mL of sample. As proof-of-concept the urinary excretion profile of St treated animals has been investigated by analyzing individual sampling points. Results from pooled urine samples have also been compared with the results obtained by GC–MS analysis demonstrating the StIR equiv. measured with the HTS-IS-SPE-ELISA protocol are in accordance with the St and 16βOH-St levels found with the chromatographic method. The analytical procedure is rapid, effective and the detectability achieved is below the MPRL (minimum performance required levels) recommended by CRL (Community Reference Laboratory) to the European Community.

Evaluation of Two New Commercial Tests for the Diagnosis of Acute Dengue Virus Infection Using NS1 Antigen Detection in Human Serum

BackgroundWe compared the performance of two new commercial tests for the detection of dengue NS1 protein during the clinical phase of dengue virus (DENV) infection—an immunochromatographic test allowing rapid detection of the NS1 antigen, Dengue NS1 Ag STRIP (Bio-Rad Laboratories - Marnes La Coquette, France), and a two-step sandwich-format microplate enzyme-linked immunosorbent assay (ELISA), pan-E Dengue Early ELISA (Panbio - Brisbane, Australia)—with a one-step sandwich-format microplate ELISA, the Platelia Dengue NS1 Ag test (Bio-Rad).MethodsWe tested 272 serum samples from patients with dengue disease. Of these, 222 were from patients with acute infection of one of the four dengue serotypes, detected by RT-PCR and/or virus isolation. Forty-eight acute-phase serum samples from patients not infected with dengue virus were also included.ResultsThe sensitivity of the Platelia Dengue NS1 Ag test on acute serum samples (n = 222) was 87.4% (95% confidence interval: 82.3% to 91.5%); that of Dengue NS1 Ag STRIP was 81.5% (95% CI: 75.8% to 86.4%) after 15 minutes and 82.4% (95% CI: 76.8% to 87.2%) after 30 minutes. Both tests had a specificity of 100% (97.5% CI, one-sided test: 92.6% to 100.0%). The pan-E Dengue Early ELISA had a sensitivity of 60.4% (95% CI: 53.4% to 66.8%) and a specificity of 97.9% (95% CI: 88.9% to 99.9%).ConclusionOur findings support the use of diagnostic tools based on the NS1 antigen detection for the diagnosis of acute DENV infection. The immunochromatographic test, Dengue NS1 Ag STRIP—the first rapid diagnostic test for DENV infection—was highly sensitive and specific, and would therefore be a suitable first-line test in the field. The pan-E Dengue Early ELISA was less sensitive than the Platelia test; this two-step ELISA should be combined with DENV IgM antibody detection for the diagnosis of DENV infection.

Development of a highly sensitive IgG-ELISA based on recombinant arginine kinase of Toxocara canis for serodiagnosis of visceral larva migrans in the murine model

Toxocariasis is a worldwide zoonotic disease caused by the ascarid nematode Toxocara canis. The most common method available for serodiagnosis of toxocariasis is an enzyme-linked immunosorbent assay (ELISA) test using Toxocara excretory-secretory antigen (TES). The present study describes the development of IgG-ELISA based on antiserum prepared against the recombinant arginine kinase of Toxocara canis. Antiserum was prepared against the purified recombinant arginine kinase (AK) using 6-week-old female Japanese white rabbits. Serum samples were collected from experimentally infected BALB/c and C57BL/6 mice at different time periods. The IgG-ELISA was performed using serum samples from mice (infected/uninfected) and TES antigen with antiserum prepared against the recombinant-AK. The optical density (OD450) was measured at 450 nm using a micro-plate ELISA reader. There were significant differences (P<0.01) in the absorbance between infected and control serum samples. Further, we obtained 100% sensitivity for the serum samples from T. canis-infected mice. Therefore, it is suggested that the recombinant-AK based IgG-ELISA could be applied for immunodiagnosis of human toxocariasis. However, it is necessary to evaluate the specificity of this recombinant antigen with similar geohelminth infections.

Immunochemical Analysis of 2,4,6-Tribromophenol for Assessment of Wood Contamination

2,4,6-Tribromophenol (2,4,6-TBP) has been used as a wood preservative and flame retardant and is a synthetic intermediate of the most important brominated flame retardants (BFR) produced. The use of TBP-contaminated wood materials in the food industry poses a risk of significant economical losses due to food contamination. In this work an efficient and reliable immunochemical method for analysis of TBP in wood samples has been established consisting of alkaline wood extraction followed by analysis on a microplate ELISA (enzyme-linked immunosorbent assay). TBP is efficiently extracted from wood samples in 10 min and directly measured after 10-fold buffer dilution to avoid matrix interferences. The analytical procedure has a limit of detection of 45 ng g (-1) of TBP in wood (1.5 microg L (-1) in extracts). The method has been applied to the analysis of contaminated real wood samples, showing that the levels of contamination can reach high TBP concentrations (up to 2000 microg L (-1)). An excellent correlation was observed between TBP levels in wood extracts determined by ELISA and gas chromatography-mass spectrometry (GC-MS) analysis ( R (2) = 0.990, N = 19). The precision found is below 22% CV. The immunoanalytical method developed is fast, reliable, and cost-effective, shows good high-throughput screening capabilities, and can be an excellent tool for assessment of wood contamination at lumber mills or related industries. The TBP ELISA has the potential to be used for environmental, food, and biological monitoring of brominated phenolic compounds considered nowadays as emerging pollutants.

D-dimers in atrial fibrillation: a further step in risk stratification of thrombo-embolism?

Plasma fibrin d-dimers (hereafter d-dimers) are generated when the endogenous fibrinolytic system degrades fibrin, as in venous thrombo-embolism (VTE) and they consist of two identical subunits derived from two fibrin molecules. Unlike fibrinogen degradation products, which are derived from fibrinogen and fibrin, d-dimers are a specific cross-linked fibrin derivative.1 Because 2–3% of plasma fibrinogen is degraded to fibrin, small amounts are detectable in the plasma of healthy individuals.1 d-dimer levels may be increased in any condition involving the formation and degradation of fibrin, such as VTE, pulmonary embolism, infections, cancer, surgery, cardiac or renal failure, acute coronary syndromes, acute non-lacunar stroke, pregnancy, and sickle cell crises. d-dimer levels increase linearly with age.1 The classic microplate enzyme-linked immunosorbent assay (ELISA) technique is considered the gold standard but it is not used as a routine emergency test. Fully automated techniques have been developed, such as the VIDAS test (ELISA method with a final detection in fluorescence), but also immunofiltration (membrane ELISA) techniques and instantaneous methods which allow a diagnosis within a few minutes at the expense of a semiquantitative approach.1 Levels of coagulative markers are increased in patients with atrial fibrillation (AF), including d-dimers, pro-thrombin fragments 1 + 2, and thrombin–antithrombin complexes, indicating abnormal thrombogenesis.2 d-dimer levels have been shown to be increased in AF, especially in patients having multiple risk factors … *Corresponding author. Tel: +33 1 49 28 28 86; fax: + 33 1 49 28 28 84. E-mail address : ariel.cohen{at}sat.aphp.fr

Diagnostic accuracy of D-dimer test for exclusion of venous thromboembolism: a systematic review.

The reported diagnostic accuracy of the D-dimer test for exclusion of deep vein thrombosis (DVT) and pulmonary embolism (PE) varies. It is unknown to what extent this is due to differences in study design or patient groups, or to genuine differences between D-dimer assays. Studies evaluating the diagnostic accuracy of the D-dimer test in the diagnosis of venous thromboembolism were systematically searched for in the MEDLINE and EMBASE databases up to March 2005. Reference lists of all included studies and of reviews related to the topic of the present meta-analysis were manually searched for other additional potentially eligible studies. Two reviewers independently extracted study characteristics using standardized forms. In total, 217 D-dimer test evaluations for DVT and 111 for PE were analyzed. Several study design characteristics were associated with systematic differences in diagnostic accuracy. After adjustment for these features, the sensitivities of the D-dimer enzyme-linked immunofluorescence assay (ELFA) (DVT 96%; PE 97%), microplate enzyme-linked immunosorbent assay (ELISA) (DVT 94%; PE 95%), and latex quantitative assay (DVT 93%; PE 95%) were superior to those of the whole-blood D-dimer assay (DVT 83%; PE 87%), latex semiquantitative assay (DVT 85%; PE 88%) and latex qualitative assay (DVT 69%; PE 75%). The latex qualitative and whole-blood D-dimer assays had the highest specificities (DVT 99%, 71%; PE 99%, 69%). Compared to other D-dimer assays, the ELFA, microplate ELISA and latex quantitative assays have higher sensitivity but lower specificity, resulting in a more confident exclusion of the disease at the expense of more additional imaging testing. These conclusions are based on the most up-to-date and extensive systematic review of the topic area, including 184 articles, with 328 D-dimer test evaluations.

Detection of Benzodiazepines in Hair Using ELISA and LC-ESI-MS-MS

This study was designed to validate an enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of nine benzodiazepines in hair. Sixteen hair case samples were tested from drug-related deaths where a positive benzodiazepine blood result was obtained. The case samples were decontaminated with 0.1% sodium dodecyl sulfate, distilled water, and dichloromethane. For ELISA analysis, the samples were extracted by incubation in monobasic phosphate buffer for 1 h and then neutralized with dibasic phosphate buffer. They were diluted 1:5 with phosphate buffer saline (PBS) prior to analysis. For LC-MS-MS, the samples were incubated overnight in methanol/25% ammonium hydroxide (20:1). The benzodiazepines were extracted by solid phase. Thirteen samples were confirmed positive by LC-MS-MS. The benzodiazepines detected included diazepam, nordiazepam, temazepam, oxazepam, nitrazepam, and lorazepam. Using a cut-off concentration of 0.1 ng/mg oxazepam, the Immunalysis Benzodiazepine Microplate ELISA demonstrated a sensitivity and specificity of 100% and 81%, respectively, compared with LC-MS-MS results.