Use Of Enzyme-linked Immunosorbent Assay Research Articles

Clinical implications of carcinoembryonic antigen distribution in serum exosomal fraction-Measurement by ELISA.

BackgroundSerum exosomal proteins have great potential as indicators of disease status in cancer, inflammatory or metabolic diseases. The association of a fraction of various serum proteins such as carcinoembryonic antigen (CEA) with circulating exosomes has been debated. The establishment of a method to measure the exosomal fraction of such proteins might help resolve this controversy. The use of enzyme-linked immunosorbent assays (ELISAs) to measure serum exosomal molecules, for example CEA, is rare in research laboratories and totally absent in clinical biology. In this study, we optimized a method for assessment of serum exosomal molecules combining a treatment by volume-excluding polymers to isolate the exosomes, their subsequent solubilization in an assay buffer and ELISA.MethodsOne hundred sixteen consecutive patients with colorectal cancer were enrolled for this study between June 2015 and June 2016 at Wakayama Medical University Hospital (WMUH). Whole blood samples were collected from patients during surgery. Exosomes were isolated using the ExoQuick reagent, solubilized in an assay buffer and subjected to CEA detection by ELISA. The procedure of serum exosome isolation and the formulation of the assay buffer used for the ELISA were optimized in order to improve the sensitivity and specificity of the assay.ResultsA five-fold increase in the concentration of the exosomes in the assay buffer (using initial serum volume as a reference) and the addition of bovine serum albumin (BSA) resulted in more accurate measurements of the serum exosomal CEA. The thawing temperature of frozen serum samples before exosome extraction was also optimized. A validation study that included one hundred sixteen patients with colorectal cancer demonstrated that serum exosomal CEA from samples thawed at 25°C exhibited a better AUC value, sensitivity, and specificity as well as a more correct classification than serum CEA.ConclusionsWe optimized an easy and rapid detection method for assessment of serum exosomal CEA. The thawing temperature of frozen serum prior to exosome extraction, the formulation of the assay buffer used for exosome solubilization and the concentration of the exosomes in this buffer were fine-tuned to enable the appropriate and accurate measurement of serum exosomal CEA.

A novel strategy with combined assays for detection of anti-neutrophil cytoplasmic antibody (ANCA) in clinically ANCA-negative granulomatosis with polyangiitis patients

ObjectiveGranulomatosis with polyangiitis (GPA) that is localized to the upper airway presents a diagnostic challenge because of a tendency towards anti-neutrophil cytoplasmic antibody (ANCA)-negativity. The purpose of this study was to investigate whether positivity of ANCA detection might be elicited with combined use of enzyme-linked immunosorbent assay (ELISA) kits. MethodsTwenty-nine serum samples obtained from GPA patients were used in this study. In addition to routine biochemical investigation for ANCA, tests for detecting PR3-, MPO-ANCAs, and minor ANCAs were performed with commercially available ELISA kits. Cytoplasmic (C)-ANCA and perinuclear (P)-ANCA were evaluated using the indirect immunofluorescence (IIF) technique. ResultsTwelve patients were positive for PR3- or MPO-ANCA in the clinical laboratory test, and 17 patients were negative for both ANCAs. Of the 17 ANCA-negative patients, four were positive for PR3- or MPO-ANCA, and three were positive for minor ANCA according to results obtained from six different ELISA kits. These findings indicated that performing detection tests with six different ELISA kits might improve the positivity of ANCA and might contribute to establishing the diagnosis of ANCA-associated vasculitis. Together with results from IIF, the samples of eight patients with clinically ANCA-negative results (8/17, 47.1%) were converted to ANCA-positive results, and the ANCA detection rate was significantly improved from 12/29 (41.4%) to 20/29 (69.0%, p=0.03). ConclusionsAdditional detection techniques should be used to confirm the results of clinically ANCA-negative samples, particularly when vasculitis is suspected. Minor ANCAs should also be evaluated with detection tests when PR3- and MPO-ANCA are negative.

IgG4 anti-neurofascin155 antibodies in chronic inflammatory demyelinating polyradiculoneuropathy: Clinical significance and diagnostic utility of a conventional assay

We aimed to validate the diagnostic utility of enzyme-linked immunosorbent assay (ELISA) for the detection of anti-neurofascin (NF) 155 antibody in 191 patients with chronic inflammatory demyelinating polyneuropathy (CIDP). Human NF155-based ELISA clearly distinguished between anti-NF155 antibody-positive and -negative sera. Fifteen CIDP patients (8%) were IgG4 anti-human NF155 antibody-positive, which were confirmed by western blot, cell-based assay and immunohistochemical study. None of disease controls or healthy subjects had positive results. Clinical presentation of IgG4 anti-NF155 antibody-positive patients was consistent with those in previous reports. This ELISA combined with determination of the IgG4 subclass is useful in screening for anti-NF155 antibodies.

Development of a Quasi-Steady Flow Electrochemical Paper-Based Analytical Device.

An electrochemical paper-based analytical device (ePAD) was developed for quasi-steady flow detection at microwire electrodes, for the first time. The device implements a fan shaped geometry connected to an analysis channel whereby solution is pulled from an inlet, through a channel, and into the steadily increasing capillary network of the fan. The network counteracts the decrease in solution flow rate associated with increasing viscosity within the channel, generating quasi-steady flow within the analysis channel. Microwire electrodes were embedded between two paper layers within the analysis channel, such that solution flow occurred on both sides of the wire electrodes. The quasi-steady flow ePAD increased the current by 2.5 times and 0.7 times from a saturated channel with no flow and from a single-layer paper device with flow, respectively. Amperometric detection was used for flow injection analysis (FIA) of multiple analytes at both Au and Pt microwire working electrodes, both of which provided similar sensitivity (ca. 0.2 mM-1) when normalized to the same standard. The two-layer paper devices provided a detection limit of 31 μM for p-aminophenol (PAP) using Pt electrodes and was also used to detect enzyme activity for the reaction of β-galactosidase with p-aminophenyl-galactopyranoside (PAPG). Measured enzyme kinetics provided similar Vmax (0.079 mM/min) and Km (0.36 mM) values as those found in the literature. This device shows great promise toward use in enzyme-linked immunosorbent assays or other analytical techniques where flow or washing steps are necessary. The developed sensor provides a simple and inexpensive device capable of performing multiple injection analysis with steady-flow and online detection that would normally require an external pump to perform.

High-Throughput Production of Chromium(III) Complexes for Antibody Immobilization.

A robot-assisted high-throughput methodology was employed to produce chromium(III) complexes suitable for the surface modification of the commercially available PerkinElmer Optiplate96 well plate for use in enzyme-linked immunosorbent assays (ELISAs). The complexes were immobilized to the native functionality of the well plate and first screened using a horseradish-peroxidase-tagged (HRP) mouse antibody to quantify binding. The top "hits" were further assessed for their ability to present the antibody in a functional state using an ELISA. "Hits" from the second screen yielded four complexes capable of improving the signal intensity of the ELISA by greater than 500%. The metal/base ratio of these complexes was also investigated, and we isolated the most stable and reproducible candidate, [Cr(OH)6]3-, which was formed from chromium(III) perchlorate and pH adjusted with ethylenediamine. This chromium solution was employed in a clinically relevant setting for the detection of bovine TNFα producing up to a 200% increase in signal intensity.

Prevalence of anti-hepatitis C virus antibody among pregnant women and blood donors at Bowen University Teaching Hospital, Ogbomoso, Oyo State, Nigeria

ABSTRACTHepatitis C virus is one of the emerging infectious diseases that can be transmitted through blood-to-blood contact. This study was carried out to determine the prevalence of anti-HCV antibodies among potential blood donors and pregnant women attending Bowen University Teaching Hospital (BUTH), Ogbomoso, Oyo State. This hospital-based study was conducted from December 2014 to September 2015. The study group (N = 279) included potential blood donors and pregnant women. Data on socio-demographic characteristics and potential risk factors were collected using a structured questionnaire. The presence of anti-HCV antibodies in serum samples of the studied subjects were detected using third-generation Enzyme Linked Immunosorbent Assay (ELISA) (WKEA Med Supplies Corp, China). Chisquare test was utilized to assess the association between the socio-demographic variables and HCV status. Logistic regression was done to determine the strength of association between risk factors and HCV status. Statistical significance was set at P ˂ 0.05. Overall seroprevalence of hepatitis C virus infection was found to be 1.79% consisting 0.36% of pregnant women and 1.43% of blood donors. None of the socio-demographic characteristics and potential risk factors among the study groups were significantly associated with hepatitis C virus infection. This study found a seroprevalence of anti-HCV antibody to be 1.79%, thus, screening of pregnant women and blood donors for HCV infections with the use of ELISA is recommended because of its important role in detecting the presence of anti-HCV antibody with utmost specificity and sensitivity.

Immunoassay for determination of trilobolide

Trilobolide (Tb) is a pharmacologically interesting sesquiterpene lactone isolated from Laser trilobum (L.) Borkh. Structural relation to a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin bring promising prospects for Tb to be used in the development of new anti-cancer drugs. As long as there are still unanswered questions regarding its investigation, a need for novel analytical tools emerge. Since immunoassays serve as one of powerful tools within the investigation of natural products, the development of indirect competitive enzyme-linked immunosorbent assay (ELISA) utilizing coating based on avidin-biotin technology is described. In our set-up of ELISA, newly synthesized biotinylated Tb served as immobilized competitor. Tb-carboxymethyloxime-bovine serum albumin (BSA) and Tb-succinoyl-BSA conjugates were used separately for immunization of rabbits. Two sets of polyclonal antibodies (RAbs) were obtained. Antibodies against Tb-succinoyl-BSA conjugate (RAb No. 206) were chosen as the best. Under optimized conditions, limit of detection and 50% intercept of our ELISA were 849pg/mL and 8.89ng/mL, respectively. The cross-reactivity (CR) was tested on 10 structurally related compounds and CR did not exceed 6.1%. The reproducibility of the system is expressed as intra- and inter-assay coefficients of variation (9.7% and 11.4%, respectively). Based on conducted experiments, we proposed the use of ELISA for quantification of Tb in complex biological matrices such as plant extracts. A method was applied to analyze three extracts obtained from different parts of L. trilobum. Data obtained were compared to those acquired by UHPLC-MS/MS. The concordance between the methods (103–87%) showed the ability of ELISA to quantify Tb.

Platelet-derived CXCL4 regulates neutrophil infiltration and tissue damage in severe acute pancreatitis

Platelets are known to play an important role in acute pancreatitis (AP) via promotion of neutrophil accumulation, although mechanisms behind platelet-dependent accumulation of neutrophils in the pancreas remain elusive. Platelets contain a wide spectrum of different pro-inflammatory compounds, such as chemokines. CXCL4 (platelet factor 4) is one of the most abundant chemokine in platelets, and we hypothesized that CXCL4 might be involved in platelet-dependent accumulation of neutrophils in the inflamed pancreas. The aim of this study was to examine the role of CXCL4 in severe AP. Pancreatitis was provoked by infusion of taurocholate into the pancreatic duct or by intraperitoneal administration of L-arginine in C57BL/6 mice. Animals were treated with an antibody against platelets or CXCL4 before induction of pancreatitis. Plasma and lung levels of CXCL2, CXCL4, and interleukin (IL)-6 were determined by use of enzyme-linked immunosorbent assay. Flow cytometry was used to examine surface expression of macrophage-1 (Mac-1) on neutrophils. Plasma was obtained from healthy individuals (controls) and patients with AP. Challenge with taurocholate increased plasma levels of CXCL4, and depletion of platelets markedly reduced plasma levels of CXCL4 indicating that circulating levels of CXCL4 are mainly derived from platelets in AP. Inhibition of CXCL4 reduced taurocholate-induced neutrophil recruitment, IL-6 secretion, edema formation, amylase release, and tissue damage in the pancreas. However, immunoneutralization of CXCL4 had no effect on CXCL2-evoked neutrophil expression of Mac-1 or chemotaxis invitro, suggesting an indirect effect of CXCL4 on neutrophil recruitment in AP. Targeting CXCL4 significantly attenuated plasma and lung levels of CXCL2, which is a potent neutrophil chemoattractant, and inhibition of the CXCL2 receptor attenuated neutrophil infiltration and tissue damage in the inflamed pancreas. A significant role of CXCL4 was confirmed in an alternate model of AP induced by L-arginine challenge. Moreover, patients with AP had significantly increased plasma levels of CXCL4 compared with healthy controls. These findings' results suggest that platelet-derived CXCL4 is a potent stimulator of neutrophil accumulation in AP and that this is mediated via generation of CXCL2 in the inflamedpancreas. We conclude that CXCL4 plays an important role in pancreatic inflammation and that targeting CXCL4 might be a useful way to ameliorate tissue damage in AP.

The Expression of TNFα, IL-6, IL-2 and IL-8 in the Serum of Patients with Idiopathic Sudden Sensorineural Hearing Loss: Possible Prognostic Factors of Response to Corticosteroid Treatment

Introduction: Idiopathic sudden sensorineural hearing loss (ISSNHL) remains one of the major unsolved otologic emergencies. A viral infection, a systemic inflammatory disorder, as well as physical, mental and metabolic stress can trigger an innate immune response in the inner ear resulting in ISSNHL. Proinflammatory cytokines play a central role in this cochlear immunological cascade. Objective: To examine the expression of proinflammatory cytokines in the serum of patients with ISSNHL in correlation with the therapeutic outcome of intravenous administration of corticosteroids. Method: Forty-three patients primarily diagnosed with ISSNHL underwent intravenous corticosteroid treatment for 8 days. The expression of tumor necrosis factor-α (TNFα), interleukin-6 (IL-6), interleukin-2 (IL-2) and interleukin-8 (IL-8) was detected with the use of enzyme-linked immunosorbent assay in serum specimens on the 1st and 8th day of treatment and it was correlated with the treatment outcome. Results: TNFα reduction and IL-6 increase strongly correlate with a good therapeutic result [χ²(2) = 13.12, p = 0.001 and χ²(2) = 16.78, p = 0.0001]. IL-8 increase reflects negatively on the outcome, however, not in a statistically significant way. No association was established between IL-2 variations and the therapeutic outcome. Conclusions: TNFα and IL-6 can be used as prognostic factors for the treatment outcome, whereas the prognostic value of IL-8 requires further statistical confirmation.

A Review of Cry Protein Detection with Enzyme-Linked Immunosorbent Assays.

The widespread use of Cry proteins in insecticide formulations and transgenic crops for insect control has led to an increased interest in the environmental fate of these proteins. Although several detection methods are available to monitor the fate of Cry proteins in the environment, enzyme-linked immunosorbent assays (ELISAs) have emerged as the preferred detection method, due to their cost-effectiveness, ease of use, and rapid results. Validation of ELISAs is necessary to ensure accurate measurements of Cry protein concentrations in the environment. Validation methodology has been extensively researched and published for the areas of sensitivity, specificity, accuracy, and precision; however, cross validation of ELISA results has been studied to a lesser extent. This review discusses the use of ELISAs for detection of Cry proteins in environmental samples and validation of ELISAs and introduces cross validation. The state of Cry protein environmental fate research is considered through a critical review of published literature to identify areas where the use of validation protocols can be improved.

Pregnancy-induced adaptations in intramuscular extracellular matrix of rat pelvic floor muscles

Birth trauma to pelvic floor muscles is a major risk factor for pelvic floor disorders. Intramuscular extracellular matrix determines muscle stiffness, supports contractile component, and shields myofibers from mechanical strain. Our goal was to determine whether pregnancy alters extracellular matrix mechanical and biochemical properties in a rat model, which may provide insights into the pathogenesis of pelvic floor muscle birth injury. To examine whether pregnancy effects were unique to pelvic floor muscles, we also studied a hind limb muscle. Passive mechanical properties of coccygeus, iliocaudalis, pubocaudalis, and tibialis anterior were compared among 3-month old Sprague-Dawley virgin, late-pregnant, and postpartum rats. Muscle tangent stiffness was calculated as the slope of the stress-sarcomere length curve between 2.5 and 4.0 μm, obtained from a stress-relaxation protocol at a bundle level. Elastin and collagen isoform concentrations were quantified by the use of enzyme-linked immunosorbent assay. Enzymatic and glycosylated collagen crosslinks were determined by high-performance liquid chromatography. Data were compared by the use of repeated-measures, 2-way analysis of variance with Tukey post-hoc testing. Correlations between mechanical and biochemical parameters were assessed by linear regressions. Significance was set to P < .05. Results are reported as mean ± SEM. Pregnancy significantly increased stiffness in coccygeus (P< .05) and pubocaudalis (P< .0001) relative to virgin controls, with no change in iliocaudalis. Postpartum, pelvic floor muscle stiffness did not differ from virgins (P> .3). A substantial increase in collagen V in coccygeus and pubocaudalis was observed in late-pregnant, compared with virgin, animals, (P< .001). Enzymatic crosslinks decreased in coccygeus (P< .0001) and pubocaudalis (P< .02) in pregnancy, whereas glycosylated crosslinks were significantly elevated in late-pregnant rats in all pelvic floor muscles (P< .05). Correlations between muscle stiffness and biochemical parameters were inconsistent. In contrast to the changes observed in pelvic floor muscles, the tibialis anterior was unaltered by pregnancy. In contrast to other pelvic tissues, pelvic floor muscle stiffness increased in pregnancy, returning to prepregnancy state postpartum. This adaptation may shield myofibers from excessive mechanical strain during parturition. Biochemical alterations in pelvic floor muscle extracellular matrix due to pregnancy include increase in collagen V and a differential response in enzymatic vs glycosylated collagen crosslinks. The relationships between pelvic floor muscle biochemical and mechanical parameters remain unclear.

Usefulness of enzyme-linked immunosorbent assay in the diagnosis of pemphigus and bullous pemphigoid in upper Egypt: correlation with intensity score

Background The availability of complementary DNA clones for pemphigus and pemphigoid antigens has led to the production of recombinant proteins representing epitopes of native antigens Dsg1, Dsg3, BP180 and BP230, and to the development of related enzyme-linked immunosorbent assay (ELISA) systems. Objective The aim of this study was to evaluate the diagnostic value of Dsg1, Dsg3, BP180 and BP230 ELISAs in upper Egypt patients, and to assess its correlation with the severity of diseases. Patients and methods Forty-six patients with pemphigus and bullous pemphigoid were enrolled. The diagnosis was confirmed by means of histopathology and direct immunofluorescence. Autoantibodies against Dsg1, Dsg3, BP180 and BP230 were assayed using ELISA in the serum of pemphigus and bullous pemphigoid patients. In pemphigus vulgaris, anti-Dsg1 and anti-Dsg3 antibody titre were raised above the cutoff value in 88.5 and 96.1% of patients, respectively. A strong positive correlation between the titre of anti-Dsg1 and Dsg3 and the total Autoimmune Bullous Skin Disorder Intensity Score (ABSIS) in (mucocutaneous) pemphigus patients (P Conclusion ELISA can be considered an important step in the laboratory diagnosis of pemphigus and bullous pemphigoid, especially as it is a simple, noninvasive and fast manoeuvre. Anti-Dsg1 Ab and anti-Dsg3 Ab ELISA values are correlated with the disease activity of pemphigus. Anti-BP180 and anti-BP230 Ab ELISA values are correlated with the disease activity of bullous pemphigoid.

Leukocyte-Reduced Platelet-Rich Plasma Normalizes Matrix Metabolism in Torn Human Rotator Cuff Tendons

Background: The optimal platelet-rich plasma (PRP) for treatment of supraspinatus tendinopathy has not been determined. Purpose: To evaluate the effect of low- versus high-leukocyte concentrated PRP products on catabolic and anabolic mediators of matrix metabolism in diseased rotator cuff tendons. Study Design: Controlled laboratory study. Methods: Diseased supraspinatus tendons were treated with PRP made by use of 2 commercial systems: Arthrex Autologous Conditioned Plasma Double Syringe System (Llo PRP) and Biomet GPS III Mini Platelet Concentrate System (Lhi PRP). Tendon explants were placed in 6-well plates and cultured in Llo PRP, Lhi PRP, or control media (Dulbecco’s Modified Eagle Medium + 10% fetal bovine serum) for 96 hours. Tendons were processed for hematoxylin-eosin histologic results and were scored with the modified Bonar scale. Group 1 tendons were defined as moderate tendinopathy (Bonar score <3); group 2 tendons were assessed as severely affected (Bonar score = 3). Transforming growth factor β–1 (TGFβ-1), interleukin-1β (IL-1β), interleukin-1 receptor antagonist (IL-1Ra), interleukin-6 (IL-6), interleukin-8 (IL-8), and matrix metalloproteinase–9 (MMP-9) concentrations in PRP media were measured by use of enzyme-linked immunosorbent assay after 96 hours of culture with diseased tendon. Tendon messenger RNA expression of collagen type I (COL1A1), collagen type III (COL3A1), cartilage oligomeric matrix protein (COMP), MMP-9, MMP-13, and IL-1β was measured with real-time quantitative polymerase chain reaction. Results: Leukocytes and platelets were significantly more concentrated in Lhi PRP compared with Llo PRP. Increased IL-1β was present in Lhi PRP after culture with group 1 tendons. IL-6 was increased in Lhi PRP after culture with group 2 tendons. Both TGFβ-1 and MMP-9 were increased in Lhi PRP after culture with either tendon group. In Llo PRP cultures, IL-1Ra:IL-1β in PRP used as media and COL1A1:COL3A1 gene expression were increased for group 1 tendon cultures. Gene expression of MMP-9 and IL-1β was increased in group 2 tendons cultured in Llo PRP. There was no significant difference in the expression of MMP-13 or COMP in either group of tendons cultured in Llo PRP or Lhi PRP. Conclusion: Llo PRP promotes normal collagen matrix synthesis and decreases cytokines associated with matrix degradation and inflammation to a greater extent than does Lhi PRP in moderately degenerative tendons. In severely degenerative tendons, neither PRP preparation enhanced matrix synthesis. Clinical Relevance: Llo PRP may promote healing in moderately degenerative rotator cuff tendons.

Altered Levels of Fatty Acids and Inflammatory and Metabolic Mediators in Epicardial Adipose Tissue in Patients With Systolic Heart Failure

BackgroundAdipose tissue has endocrine properties, secreting a wide range of mediators into the circulation, including factors involved in cardiovascular disease. However, little is known about the potential role of adipose tissue in heart failure (HF), and the aim of this study was to investigate epicardial (EAT) and subcutaneous (SAT) adipose tissue in HF patients. Methods and ResultsThirty patients with systolic HF and 30 patients with normal systolic function undergoing thoracic surgery were included in the study. Plasma was sampled and examined with the use of enzyme-linked immunosorbent assays, whereas SAT and EAT biopsies were collected and examined by means of reverse-transcription polymerase chain reaction and gas chromatography. Significantly higher expressions of mRNA encoding interleukin-6, adrenomedullin, peroxisome proliferator–activated receptor α, and fatty acid (FA)–binding protein 3, as well as higher levels of monounsaturated FA and palmitoleic acid, were seen in the EAT of HF patients, whereas the levels of docosahexaenoic acid were lower. Palmitoleic acid levels in EAT were correlated with 2 parameters of cardiac remodeling: increasing left ventricular end-diastolic diameter and N-terminal pro–B-type natriuretic peptide. ConclusionsOur results demonstrate adipose tissue depot–specific alterations of synthesis of FA and inflammatory and metabolic mediators in systolic HF patients. EAT may be a source of increased circulatory and myocardial levels of these mediators through endocrine actions.

Aggregated Silver Nanoparticles Based Surface-Enhanced Raman Scattering Enzyme-Linked Immunosorbent Assay for Ultrasensitive Detection of Protein Biomarkers and Small Molecules

Lowering the detection limit is critical to the design of bioassays required for medical diagnostics, environmental monitoring, and food safety regulations. The current sensitivity of standard color-based analyte detection limits the further use of enzyme-linked immunosorbent assays (ELISAs) in research and clinical diagnoses. Here, we demonstrate a novel method that uses the Raman signal as the signal-generating system of an ELISA and combines surface-enhanced Raman scattering (SERS) with silver nanoparticles aggregation for ultrasensitive analyte detection. The enzyme label of the ELISA controls the dissolution of Raman reporter-labeled silver nanoparticles through hydrogen peroxide and generates a strong Raman signal when the analyte is present. Using this assay, prostate-specific antigen (PSA) and the adrenal stimulant ractopamine (Rac) were detected in whole serum and urine at the ultralow concentrations of 10(-9) and 10(-6) ng/mL, respectively. The methodology proposed here could potentially be applied to other molecules detection as well as PSA and Rac.

P016: Utility of enzyme-linked immunosorbent assay for detectsHistoplasma capsulatumantigenuria-like screening in patients with HIV/AIDS

P016: Utility of enzyme-linked immunosorbent assay for detectsHistoplasma capsulatumantigenuria-like screening in patients with HIV/AIDS

Remarks on an Enzymatic Amplification System for Use in Point of Care Diagnostics

Detection methods to quantify specific protein levels in biological samples for diagnostic purposes typically rely on the use of enzyme linked immunosorbent assays (ELISA). With an ELISA, total protein, rather than the bioactive amount may be quantified, as it is often complexed to naturally occurring aggregates or inhibitors. Thus, in cases where the protein is an enzyme, an activity assay may be more informative and correlative with disease status.

Detection of proteinaceous binders in ancient Chinese textiles by enzyme-linked immunosorbent assay

The use of binding materials in ancient Chinese textiles was widely practiced to enhance both the bond strength and the cohesion between fibers. An accurate qualitative assessment of such binding materials is of great significance in heritage research and conservation science: first, to understand the historical development of the textile industry and, second, to choose a suitable conservation method. However, this problem has not been fully addressed due to the complexity of the binder composition and the limitations of traditional analytical methods. This study focused on the use of enzyme-linked immunosorbent assay (ELISA) for the specific identification of proteins in archaeological binder materials. Four ancient Chinese textile binder samples were tested by ELISA. The results showed that all the binder samples reacted positive for ovalbumin, while two printing gold samples reacted negative for collagen-I. This is the first time ELISA has been successfully used to detect protein in ancient Chinese textile samples. With its advantages of sensitivity, low cost, and speed, the ELISA technique becomes the perfect tool for protein detection in Chinese heritage research.

Detection of IgM antibrucella antibody in the absence of IgGs: a challenge for the clinical interpretation of brucella serology.

The use of enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM antibodies antibrucella has become widespread in the diagnosis of human brucellosis. IgM anti-Brucella antibodies are indicative of acute infection. Between 2009–2013, 5307 patients were evaluated for serologic diagnosis at the Microbiology Laboratory of the Albacete General Hospital. A ELISA IgM-positive, IgG-negative anti-Brucella antibody serology pattern was detected in 17 of those patients. Epidemiology data, symptoms, laboratory data, treatment and outcome from these patients were reviewed. Sixteen patients presented with musculoskeletal pain, fatigue and/or fever and 1 was asymptomatic. Five patients received treatment with doxycycline combined with rifampin, gentamycin or streptomycin during 6–12 weeks, with no improvement. None of the 17 patients were finally diagnosed with brucellosis. Our results indicate that anti-Brucella IgM positive serology, per se, is not enough to diagnose acute brucellosis and other methods should be used for confirmation. Brucella serology data should be interpreted taking into account the patient's clinical history and epidemiological context.

Is Neutrophil Gelatinase–Associated Lipocalin an Optimal Marker of Renal Function and Injury in Liver Transplant Recipients?

BackgroundRecently, research has focused on the association of neutrophil gelatinase–associated lipocalin (NGAL) with acute and/or active kidney injury. However, it should be remembered that NGAL is involved in iron metabolism and antimicrobial defense mechanisms. MethodsOne hundred seven consecutive liver transplant recipients were included in this study. Plasma and urine NGAL levels were measured with the use of enzyme-linked immunosorbent assay. NGAL levels were studied as plasma concentrations (pNGAL), urine concentrations (uNGAL), urinary NGAL to creatinine ratio (uNGAL/Cr), and fractional NGAL secretion (fNGAL). ResultspNGAL was found to be inversely correlated with estimated glomerular filtration rate (eGFR) and plasma cystatine C (pCysC) (r = −0.26 and P = .007, r = −0.38 and P = .00006, respectively). uNGAL was positively correlated with urinary cystatine C to creatinine ratio (uCysC/Cr) and fractional cystatine C excretion (fCysC) (r = 0.43 and P = .000004; r = 0.4 and P = .1; respectively). uNGAL/Cr was inversely correlated with hematocrit (Htc) and hemoglobin (Hb) (r = −0.35 and P = .0002; r = −0.39 and P = .00004; respectively), and positively correlated with uCysC/Cr (r = 0.5 and P < .0000001). fNGAL was directly correlated with uCysC/Cr and fCysC (r = 0.53 and P < .0000001; r = 0.39 and P = .00005; respectively) and inversely correlated with red blood cell count (RBC; r = −0.31 and P = .001). We observed significant differences of pNGAL, uNGAL/Cr, and fNGAL between sexes, with highest values of uNGAL, uNGAL/Cr, and fNGAL in women and pNGAL in men. In multivariate regression analysis, pNGAL was positively correlated with time elapsed from liver transplantation, neutrophil count, pCysC, and sex (β = 0.36 and P = .00001; β = 0.32 and P = .0001; β = 0.58 and P < .0000001; β = 0.17 and P = .025; respectively) and inversely correlated with patient's age (β = −0.18 and P = .02) with R = 0.67 and R2 = 0.45, independently from blood glucose, eGFR, RBC, white blood cell count, Hb, uCysC, uCysC/Cr, and fCysC. ConclusionsPlasma and urine NGAL levels are strongly correlated not only with kidney function parameters, but also with red and white blood cell parameters and patient's age and sex.