Enzyme-linked Immunosorbent Assay Immunoassay Research Articles

Comparison of ECL, ELISA and ELFA immunoassays

The aim of these study was the evaluation of electrochemiluminescence assay (ECL), enzyme-linked immunosorbent assay (ELISA) and enzyme-linked fluorescent assay (ELFA), for an early diagnosis of Cytomegalovirus infections in pregnant women. In medical diagnostics, it is necessary to determine the most sensitive techniques for the detection of this pathogen, due to it’s multiple fetal infections during pregnancy. The ECL technique resulted with the highest sensitivity and specificity values. However, for diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history and other clinical examinations.

Anti-DFS70 antibodies in systemic lupus erythematosus: Prevalence in a large Chinese cohort and an unexpected association with anti-dsDNA antibodies by a long-term follow-up.

ObjectiveMonospecific autoantibodies to dense fine speckles 70 (DFS70) antigen are purported to aid in excluding systemic autoimmune rheumatic diseases (SARD) such as systemic lupus erythematosus (SLE). However, the non-isolated anti-DFS70 still has a certain prevalence in SLE patients, and the clinical significance remains unclear. We aimed to investigate the prevalence, clinical relevance, and value of long-term monitoring of anti-DFS70 antibodies in SLE patients.MethodsAnti-DFS70 antibodies were measured by enzyme-linked immunosorbent assay (ELISA) in 851 SLE patients, 211 healthy individuals, and 194 patients with other SARD (except SLE). Demographic, serological, and clinical associations of anti-DFS70 antibodies were analyzed by a stepwise multivariable logistic regression model. The correlation of anti-DFS70 with anti-dsDNA, anti-C1q, and SLE Disease Activity Index 2000 (SLEDAI-2K) was analyzed. Sixty-one SLE patients with follow-up time ranging from 2 to 57 months were measured anti-DFS70 antibodies using both ELISA and line immunoassay. The dynamic variations of anti-DFS70 antibodies were evaluated with anti-dsDNA, anti-C1q, and SLEDAI-2K during the follow-up.ResultsThe prevalence of anti-DFS70 was significantly higher in SLE (20.7% (176/851)) than in healthy individuals (9.5% (20/211), p = 0.0002) and other SARD (10.8% (21/194), p = 0.002). Multivariable analysis revealed that anti-DFS70-positive SLE patients were associated with younger age (odds ratio (OR) = 0.982; 95% confidence interval (CI) = 0.969, 0.995), higher frequencies of anti-dsDNA (OR 1.598; 95% CI 1.107, 2.306) and anti-PCNA (OR 6.101; 95% CI 2.534, 14.688), and higher levels of serum IgG (OR 1.097; 95% CI 1.067, 1.129) and were more likely to be accompanied by mucosal ulcers (OR 5.921; 95% CI 1.652, 21.215). The O.D. value of anti-DFS70 positively correlated with levels of anti-dsDNA (r = 0.183, p < 0.0001) and anti-C1q (r = 0.181, p < 0.0001), respectively, but not with SLEDAI-2K (p = 0.920). During the follow-up, 49 (42 negative and 7 positive) patients remained stable with anti-DFS70 levels. The other 12 patients experienced significant changes in anti-DFS70, and 83.3% (10/12) of them showed similar trends between anti-DFS70 and anti-dsDNA by evaluation of dynamic variations.ConclusionAnti-DFS70 antibodies seem to be prevalent in Chinese SLE patients. The positive association of anti-DFS70 with anti-dsDNA and consistent dynamic variation between anti-DFS70 and anti-dsDNA during the follow-up suggested a potential relationship between anti-DFS70 and anti-dsDNA in patients with SLE.

Diagnosing Human Fascioliasis Using ELISA Immunoassays at a Tertiary Referral Hospital in Hanoi: A Cross-Sectional Study.

Infections with the zoonotic liver flukes Fasciola gigantica and Fasciola hepatica may result in severe disease in humans. In Vietnam, an emergence of fascioliasis cases has been observed from the late 1990s onwards. Various diagnostic tools are used in the country, but the agreement between these tools has not been critically evaluated. We aimed to describe the clinical presentation and diagnostic outcomes in fascioliasis patients in Vietnam. A retrospective, cross-sectional study was conducted on the medical records of a group of 145 patients diagnosed with fascioliasis at a tertiary referral hospital in Hanoi. Based on the review, sociodemographic background and clinical presentation were recorded. These patients all received standard routine serologic tests, including internal antibody (Ab)-ELISA, an enzyme-linked immunosorbent assay (ELISA), and commercial coproantigen (cAg)-ELISA. The majority of cases were between 30 and 59 years old (68.3%), and about half of them were male (51.0%). Upper quadrant and epigastric pain were the most commonly reported symptoms (61.4% and 35.2%, respectively). All but one patient had liver lesions upon ultrasound examination (99.3%), and eosinophilia was present in most of the patients (89.7%). A high number of patients were positive in the in-house and the commercial Ab-ELISA (95.9% and 87.4%, respectively), yet only a slight agreement was observed between the two tests (kappa coefficient, 0.06). A further 47.4% of cases were positive for the commercial cAg-ELISA, whereas stool microscopy indicated the presence of Fasciola spp. eggs in 25.7% of patients. The current study emphasizes the challenges related to the diagnosis of human fascioliasis in Vietnam.

Ability of matrix metalloproteinase-8 biosensor, IFMA, and ELISA immunoassays to differentiate between periodontal health, gingivitis, and periodontitis.

ObjectiveThe aim of this study was to determine the diagnostic utility of an MMP‐8 biosensor assay in differentiating periodontal health from gingivitis and periodontitis and compare it with an established time‐resolved immunofluorescence assay (IFMA) and enzyme‐linked immunosorbent assay (ELISA).BackgroundCurrently available antibody‐based assays display a wide variability in their ability to accurately measure matrix metalloproteinase‐8 (MMP‐8) levels in saliva.MethodsSalivary MMP‐8 levels were analyzed in 189 systemically healthy participants using an antibody‐based biosensor prototype that operates using a surface acoustic wave technology and compared with IFMA and ELISA antibody assays. Participants were categorized into 3 groups: periodontal health (59), gingivitis (63), and periodontitis (67). A sub‐population of participants (n = 20) with periodontitis received periodontal treatment and were monitored for 6 months.ResultsAll the assays demonstrated significantly higher salivary MMP‐8 concentrations in participants with periodontitis versus gingivitis, periodontitis versus health, and gingivitis versus health (all p < .05). The biosensor data demonstrated significant correlations with IFMA (r = .354, p < .001) and ELISA (r = .681, p < .001). Significant reductions in salivary MMP‐8 concentrations were detected by the biosensor (p = .030) and IFMA (p = .002) in participants with periodontitis 6 months after non‐surgical periodontal treatment. IFMA had the best sensitivity (89.2%) for detecting periodontitis and gingivitis versus health and 96.6% for detecting periodontitis versus health and gingivitis. The biosensor had an AUC value of 0.81 and diagnostic accuracy of 74.2% for differentiating periodontitis and gingivitis from health; an AUC value of 0.86 and diagnostic accuracy of 82.8% for periodontitis versus health and gingivitis.ConclusionsThe biosensor, IFMA, and ELISA assays differentiated between periodontal health, gingivitis, and periodontitis based on salivary MMP‐8 levels. Only the biosensor and, particularly, IFMA identified an effect of periodontal treatment in the participants with periodontitis. Our findings support the potential utility of salivary oral fluid aMMP‐8‐based point‐of‐care technology in the future of periodontal diagnostics.

Internal quality control for HIV testing of blood donors - Dried tube specimen as a cost-effective alternative.

An important aspect of ensuring blood safety is the performance of mandatory serological testing for transfusion transmissible infections. The practice of internal quality control (IQC) in blood banks in India is nonuniform, especially the use of third-party materials. Cited reasons are cost, lack of access to control materials, and need for deep-freezers for storage, if prepared in-house. Validation of dried tube specimen (DTS) from HIV-positive plasma as a low-cost, stable material for use as IQC material in blood banks. Fresh-frozen plasma (FFP) prepared from four HIV-positive blood-donors were pooled. Equal numbers of seronegative FFPs were pooled. Twenty microlitre aliquots of plasma were made in micro-centrifuge tubes and air-dried overnight at room-temperature. These were stored in 2-8°C refrigerators and tested once weekly for 6 months on multiple platforms with different detection principles: Rapid tests, second-generation enzyme-linked immunosorbent assay (ELISA), fourth-generation ELISA, and fourth-generation Chemiluminescence immunoassay. The protocol was sustained over the next 6 months with decreased testing frequency to study the extended stability of DTS. A total of 139 positive-DTS and 139 negative-DTS were tested with 100% samples showing consistent results on all platforms over 1 year. There was mild deterioration in reaction strengths, which did not interfere in result interpretations. Plasma in form of DTS maintained stability when stored at 2-8°C for 1 year. This provides evidence that DTS can be a modality for the production of cost-effective, stable, in-house control material for resource-restricted countries.

Life Sciences Discovery and Technology Highlights

Life Sciences Discovery and Technology Highlights

The Evolution of the Enzyme Immunoassay/Enzyme-Linked Immunosorbent Assay

50 years ago the Enzyme Immunoassay Enzyme-Linked Immunosorbent Assay, mostly known as ELISA was developed. This is a powerful but simple method that is very widely used in the diagnostic practice, as well as in biomedical research. During this time a number of ELISA modification were developed that significantly increased its properties, especially the senstivity, such as avidin-biotin assay, immuno-PCR, nano-ELISA and finally, the digital ELISA. This short review describes the principles of ELISA and the evolution from a conventional assay to the modern ultra-sensitive method. Most of the immunological methods have two components: antigen and antibody. The high specificity of their interaction gives a possibility to detect one of them if other one is included in the reaction as a specific partner. The simplest method for antigen detection in the presence of the antibody is immune diffusion (radial immune diffusion in that case), which practically the formation of precipitate of the “antigen-antibody” complex, when the target antigen diffuses from well into agarose containing the specific antibody. Unfortunately, this assay, as well as other traditional methods, like hemagglutination or complement fixation, have a low sensitivity and are unwieldy.

A Brief Review on Cell Biology

Cell Sciences is a supplier of premium biochemical reagents including antibodies, proteins, peptides, and receptors as well as immunoassay Enzyme-Linked Immunosorbent Assay (ELISA) packs, tissues, liquids, and lysates. We give items to humans, non-human primates, and little and huge animal model exploration projects across the globe. Cell science (likewise cell science or cytology) is a part of science that reviews the construction, capacity, and conduct of cells. Cell science envelops both prokaryotic and eukaryotic cells and can be separated into many sub-themes which may incorporate the investigation of cell digestion, cell correspondence, cell cycle, organic chemistry, and cell organization. The investigation of cells is performed a few strategies, for example, cell culture, different sorts of microscopy, and cell fractionation. These have been considered and are presently being utilized for revelations and exploration relating to how cells work, eventually giving knowledge into understanding bigger creatures. Knowing the parts of cells and how cells work is principal to all-natural sciences while likewise being fundamental for research in biomedical fields like malignancy, and different illnesses. Exploration in cell science is interconnected to different fields like hereditary qualities, atomic hereditary qualities, natural chemistry, sub-atomic science, clinical microbiology, immunology, and cytochemistry.

Comparison of ELISA and chemiluminescence immunoassay methods for quantification of human Placental growth factor in serum

Placental growth factor (PlGF) is crucial during placental development in early pregnancy. Several studies in pregnancies with complications such as preeclampsia or small for gestational age neonates find that PlGF levels are significantly lower in the first trimester, which implies that the concentration of PlGF could be used as an early screening biomarker for these conditions. This study aimed to compare the performance of chemiluminescence immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) for the quantification of human PlGF in serum. This is a comparative study on 88 pregnant women in the first trimester subjected to measurement of PlGF in serum using two commercially available kits: Human PlGF Quantikine HS ELISA (R&amp;amp;D Systems) and PlGF CLIA (Snibe). The overall coefficient of correlation between the tests was 0.93. When the cut-off value of 40 pg/mL was applied, it dropped significantly to 0.50 towards the lower values, while remaining an excellent 0.91 in the group with higher concentrations of PlGF. While R&amp;amp;D Systems’s ELISA seems to have better sensitivity, it is not very convenient to use for a small number of samples. Snibe’s CLIA automated method is user-friendly, fast and powerful. Both tests show excellent performance when indicating risk-free pregnancies.

Development of ELISA and chemiluminescence enzyme immunoassay for quantification of histamine in drug products and food samples.

Histamine (HA) is a biogenic amine associated with allergies and food poisoning. It is an important indicator of food freshness and quality. In recent years, a series of medical negligence cases have been reported to be related to the intravenous injection of antibiotics produced via fermentation with fish peptone due to HA contamination. To detect HA efficiently, mouse monoclonal antibody was developed. An enzyme-linked immunosorbent assay (ELISA) and a chemiluminescence enzyme immunoassay (CLEIA) were developed and compared with conventional HPLC analysis. Both immunoassays showed low cross-reactivity, low 50% inhibitive concentration (IC50; 1.2μg/mL and 1.1μg/mL), low limits of detection (LODs, IC10; 89.0ng/mL and 73.4ng/mL), and appreciable recoveries in spiked foods and drugs (from 73.4 to 131.0% and from 77.0 to 119.0%, espectively), demonstrating that the developed methods are sensitive, specific, fast, and reliable for HA detection in complicated real samples. Graphical abstract.

Comparative Analysis of ELISA Immunoassay and LC-QTOF for Opiate Screening

A comparative analysis of enzyme-linked immunosorbent assay (ELISA) and quadropole time-of-flight mass spectrometry (LC-QTOF) for the detection of opioids in blood samples is presented. The Orange County Crime Lab (OCCL) was concerned that the opioid drug class was not accurately detected at low concentrations due to the use of LC-QTOF as a non-targeted screening method for multiple classes of drugs. In order to investigate this issue, 968 ante-mortem and postmortem blood samples were analyzed by ELISA for the presence of the following opioids: morphine, morphine-glucuronide, codeine, codeine-glucuronide, hydrocodone, hydromorphone, hydromorphone-glucuronide, oxycodone, oxymorphone, and oxymorphone-glucuronide. All samples had been previously analyzed by LC-QTOF. Overall, 84 samples tested positive for opioids. Discrepant samples between ELISA and LC-QTOF were analyzed by a liquid chromatography tandem mass spectrometry confirmation method in order to determine the true composition of the sample. Upon review of the discrepant samples, no forensically relevant concentration of opioids was missed by LC-QTOF. Thus, the ability of the Orange County Crime Lab's LC-QTOF screening method was verified to detect opioids at low concentrations.

Analysis of anti-RNA polymerase III antibodies in Chinese Han systemic sclerosis patients

ObjectivesThis study aimed to assess the prevalence and clinical correlation of anti-RNA polymerase III antibodies (anti-RNAP III) in Chinese Han systemic sclerosis (SSc) patients.MethodsSerum samples from 236 patients with SSc, 125 patients with connective tissue diseases (CTD), and 166 healthy controls (HCs), recruited from Peking Union Medical College Hospital and 21 other medical centers in China, were tested for antibodies to RNA polymerase III by means of a line immunoassay (LIA) or an enzyme-linked immunosorbent assay (ELISA) kit.ResultsAnti-RNAP III antibodies were found in 14/236 SSc patients (5.93%), 1/125 (0.80%) CTD patients, and 0/166 (0.00%) HCs. The prevalence of anti-RNAP III was higher in SSc patients than in the CTD and HC groups (p = 0.02, p = 0.001, respectively). Renal crisis was significantly more common in patients with anti-RNAP III than patients without anti-RNAP III (42.9 vs. 4.1%, p < 0.0001). Gastrointestinal involvement was significantly more common in patients without anti-RNAP III than patients with anti-RNAP III (53.6 vs. 21.4%, p = 0.039). There was good agreement between the ELISA and line immunoassay (LIA) detection capabilities for anti-RNAP III.ConclusionsThe anti-RNAP III antibody, which was detected by ELISA, has diagnostic value for SSc and predictive value for SSc-related renal crisis. Both ELISA and LIA are very reliable methods for anti-RNAP III.Key Points• The prevalence of anti-RNAP III antibody was determined in Chinese SSc patients and performed ethnic differences.• The clinical association between anti-RNAP III antibody and Chinese SSc patients was evaluated in this research.• Methodological consistency of detection of anti-RNAP III antibody using commercial ELISA and LIA methods was evaluated in this research.

Association of Fok1 VDR polymorphism with Vitamin D and its associated molecules in pulmonary tuberculosis patients and their household contacts

Status of Fok I VDR polymorphism along with vitamin D, Vitamin D receptor (VDR), and cathelicidin levels in Tuberculosis (TB) patients compared to household contacts and implication of these findings in susceptibility to TB is not known. 150 active TB patients, 150 household contacts and 150 healthy controls were recruited from North Indian population. Fok1 VDR polymorphism was studied by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP).VDR mRNA and protein levels were studied using quantitative real time PCR (q rt PCR) and enzyme linked immunosorbent assay (ELISA) respectively. Cathelicidin and Vitamin D levels were measured using ELISA and chemiluminescence immunoassay (CLIA) respectively. Significant association was found between Fok1 polymorphism and susceptibility to TB (P < 0.0005). VDR mRNA, VDR protein and vitamin D levels were significantly lower in active TB group when compared to household contacts and healthy controls (P < 0.0001, 0.0001 and 0.0005 respectively). Cathelicidin levels were higher in active TB patients compared to other groups (P < 0.0001). Expression of VDR and cathelicidin was significantly higher among ‘FF’ genotypes of VDR (more active form of VDR) compared to ‘ff’ genotype (less active form of VDR). ‘f’ allele was associated with increased susceptibility to TB. Higher frequency of ‘F’ allele, increased VDR expression along with increased vitamin D levels in household contacts compared to active TB group might be responsible for protection against active TB.

Comparison of Immunochromatography test (ICT) with real time polymerase chain reaction (RT – PCR) in detection of HCV.

Objectives: To compare ICT rapid test with RT- PCR.&#x0D; Methodology: This study conducted at Pathological Laboratory Muhammad Medical College Hospital during July-August 2016 with collaboration of The Health Foundation District Sanghar at Goth Haji Khair Muhammad Junejo Kandiari Dist: Sanghar Sindh. 400 subjects from whole the population approximately 674 persons of Goth Haji Khair Mohammad Junejo taulka kandiari district Sanghar were selected in between the age of 10 years to 70 years. The purpose was to treat them if they are HCV positive. All the subjects were first screened for Anti – HCV on ICT method, and then all positive cases were confirmed on RT – PCR.&#x0D; Results: This study conducted during the month of July and August 2016. Total 400 asymptomatic subjects from both male and female were screened by ICT method for Anti – HCV. Out of 400 subjects, 142 (35.5%) were positive. The serum of all 142 subjects positive on ICT, sent to Rawalpindi for HCV qualitative test on RT – PCR, HCV detected in 84 (59.16%) cases and 58 (40.84%) cases were negative. The ratio of variation (ICT: RT-PCR) was 1.44.&#x0D; Conclusion: Rapid ICT method is less sensitive compare to RT – PCR. There is variation of results in between two methods. All positive cases on ICT must be confirmed on RT – PCR. There is false positivity in ICT method.&#x0D; &#x0D; Key word: ICT (Immunochromatography test) RT – PCR (Real time polymerase chain reaction) RIA&#x0D; (Radio immunoassay) ELISA (Enzyme linked immunosorbent assay) EIA (Enzyme immunoassay).

Immunological measurement of aspartate/alanine aminotransferase in predicting liver fibrosis and inflammation.

Background/AimsEnzymatic analysis of aspartate/alanine aminotransferase (AST/ALT) does not exactly represent the progression of liver fibrosis or inflammation. Immunoassay for AST (cytoplasmic [c] AST/mitochondrial [m] AST) and ALT (ALT1/ALT2) has been suggested as one alternatives for enzymatic analysis. The objective of this study was to evaluate the efficacy of immunoassay in predicting liver fibrosis and inflammation.MethodsA total of 219 patients with chronic hepatitis B (CHB) who underwent hepatic venous pressure gradient (HVPG) and liver biopsy before antiviral therapy were recruited. Serum samples were prepared from blood during HVPG. Results of biochemical parameters including enzymatic AST/ALT and immunological assays of cAST, mAST, ALT1, and ALT2 through sandwich enzyme-linked immunosorbent assay (ELISA) immunoassay with fluorescence labeled monoclonal antibodies were compared with the results of METAVIR stage of live fibrosis and the Knodell grade of inflammation.ResultsMETAVIR fibrosis stages were as follows: F0, six (3%); F1, 52 (24%); F2, 88 (40%); F3, 45 (20%); and F4, 28 patients (13%). Mean levels of AST and ALT were 121 ± 157 and 210 ± 279 IU/L, respectively. Mean HVPG score of all patients was 4.7 ± 2.5 mmHg. According to the stage of liver fibrosis, HVPG score (p < 0.001, r = 0.439) and ALT1 level (p < 0.001, r = 0.283) were significantly increased in all samples from patients with CHB. ALT (p < 0.001, r = 0.310), ALT1 (p < 0.001, r = 0.369), and AST (p < 0.001, r = 0.374) levels were positively correlated with Knodell grade of inflammation.ConclusionsALT1 measurement by utilizing sandwich ELISA immunoassay can be useful method for predicting inf lammation grade and fibrosis stage in patients with CHB.

Prevalence and Overtesting of True Heparin-Induced Thrombocytopenia in a 591-Bed Tertiary Care, Teaching Hospital.

Heparin-induced thrombocytopenia type II (HIT) is a rare but potentially fatal antibody-mediated reaction to all forms of heparin (unfractionated heparin, low-molecular weight heparin, heparin flushes, and heparin-coated catheters), which can lead to HIT with thrombosis. Two tests commonly used to screen for HIT include the enzyme-linked immunosorbent assay (ELISA) and serotonin release assay (SRA). This is a retrospective chart review study conducted from January 1, 2013, through December 31, 2014, to estimate the rate of true HIT in critical care patients at Winthrop-University Hospital, located in Mineola, New York. Patients are classified as positive for HIT if both ELISA and SRA immunoassays are positive. We reviewed 507 heparin immunoassays, excluding 64 who had an inappropriate ELISA test sent due to no administration of heparin, enoxaparin, or heparin lock flush at this or previous hospital stays at Winthrop. Of the 443 heparin immunoassays, ELISA results were positive for 66 patients (15.1%), and only 11 (2.5%) patients had true cases of HIT with a 95% confidence interval of 1.3% to 4.4%. The 4T score for those with true HIT (median: 5.0) was statistically higher compared to those without true HIT (median: 2.0; P < .001). Despite guidelines in place, overtesting for HIT is still a prevalent issue.

An ELISA immunoassay employing a conserved Leishmania hypothetical protein for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans

In the present study, a conserved Leishmania hypothetical protein, namely LiHypA, was evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans. This protein showed a high amino acid sequence homology between viscerotropic and cutaneotropic Leishmania species. An enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant antigen (rLiHypA), in addition to the A2 protein and two parasite antigenic preparations, which were used as controls. Regarding human diagnosis, results showed that rLiHypA was more sensitive and specific than ELISA-L. braziliensis SLA in detecting both cutaneous or mucosal leishmaniasis patients, but not those from Chagas disease patients or healthy subjects. Regarding canine diagnosis, this recombinant antigen showed higher sensitivity and specificity values, as well as a perfect accuracy to identify asymptomatic and symptomatic visceral leishmaniasis (VL) in dogs, but not those from vaccinated animals or those developing babesiosis, ehrlichiosis, or Chagas disease. However, using the rA2 protein or L. braziliensis SLA as controls, significant cross-reactivity was found when these samples were used, hampering their sensitivity and specificity values for the diagnosis. In this context, LiHypA could be considered a candidate to be evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans.

Clinical effectiveness and cost-effectiveness of use of therapeutic monitoring of tumour necrosis factor alpha (TNF-α) inhibitors [LISA-TRACKER® enzyme-linked immunosorbent assay (ELISA) kits, TNF-α-Blocker ELISA kits and Promonitor® ELISA kits] versus standard care in patients with Crohn's disease: systematic reviews and economic modelling.

Systematic reviews and economic modelling of clinical effectiveness and cost-effectiveness of therapeutic monitoring of tumour necrosis factor alpha (TNF-α) inhibitors [using LISA-TRACKER® enzyme-linked immunosorbent assay (ELISA) kits (Theradiag, Marne La Vallee, France, or Alpha Laboratories, Heriot, UK), TNF-α-Blocker ELISA kits (Immundiagnostik AG, Bensheim, Germany) and Promonitor® ELISA kits (Proteomika, Progenika Biopharma, Bizkaia, Spain)] versus standard care for Crohn's disease (CD). Multiple electronic databases were searched from inception to December 2014 in order to identify primary studies and meta-analyses. Patients with moderate to severe active CD treated with infliximab (IFX) (Remicade®, Merck Sharp & Dohme Ltd, Kenilworth, NJ, USA) or adalimumab (ADA) (Humira®, AbbVie Inc., North Chicago, IL, USA). Monitoring of serum anti-TNF-α (IFX or ADA) and/or of anti-drug antibody levels using test assays with a test-treatment algorithm. Standard care. Any patient-related outcome, test agreement and cost-effectiveness estimates. The quality assessments used recognised checklists (Quality Assessment of Diagnostic Accuracy Studies-2, Cochrane, Philips and Consolidated Health Economic Evaluation Reporting Standards). Evidence was synthesised using narrative review and meta-analysis. A Markov model was built in TreeAge Pro 2013 (TreeAge Software, Inc., Williamstown, MA, USA). The model had a 4-week cycle and a 10-year time horizon, adopted a NHS and Personal Social Services perspective and used a linked evidence approach. Costs were adjusted to 2013/14 prices and discounted at 3.5%. We included 68 out of 2434 and 4 out of 2466 studies for the clinical effectiveness and cost-effectiveness reviews, respectively. Twenty-three studies comparing test methods were identified. Evidence on test concordance was sparse and contradictory, offering scant data for a linked evidence approach. Three studies [two randomised controlled trials (RCTs) and one retrospective observational study] investigated outcomes following implementation of a test algorithm. None used the specified commercial ELISA immunoassay test kits. Neither of the two RCTs demonstrated clinical benefit of a test-treatment regimen. A meta-analysis of 31 studies to estimate test accuracy for predicting clinical status indicated that 20-30% of test results are likely to be inaccurate. The four cost-effectiveness studies suggested that testing results in small cost reductions. In the economic analysis the base-case analysis showed that standard practice (no testing/therapeutic monitoring with the intervention tests) was more costly and more effective than testing for IFX. Sensitivity and scenario analyses gave similar results. The probabilistic sensitivity analysis indicated a 92% likelihood that the 'no-testing' strategy was cost-effective at a willingness to pay of £20,000 per quality-adjusted life-year. Rigorous systematic reviews were undertaken; however, the underlying evidence base was poor or lacking. There was uncertainty about a linked evidence approach and a lack of gold standard for assay comparison. The only comparative evidence available for economic evaluation was for assays other than the intervention assays. Our finding that testing is not cost-effective for IFX should be viewed cautiously in view of the limited evidence. Clinicians should be mindful of variation in performance of different assays and of the absence of standardised approaches to patient assessment and treatment algorithms. There is substantial variation in the underlying treatment pathways and uncertainty in the relative effectiveness of assay- and test-based treatment algorithms, which requires further investigation. There is very little research evidence on ADA or on drug monitoring in children with CD, and conclusions on cost-effectiveness could not be reached for these. This study is registered as PROSPERO CRD42014015278. The National Institute for Health Research Health Technology Assessment programme.

Development of monoclonal antibodies and serological assays including indirect ELISA and fluorescent microsphere immunoassays for diagnosis of porcine deltacoronavirus.

BackgroundA novel porcine deltacoronavirus (PDCoV), also known as porcine coronavirus HKU15, was reported in China in 2012 and identified in the U.S. in early 2014. Since then, PDCoV has been identified in a number of U.S. states and linked with clinical disease including acute diarrhea and vomiting in the absence of other identifiable pathogens. Since PDCoV was just recently linked with clinical disease, few specific antibody-based reagents were available to assist in diagnosis of PDCoV and limited serological capabilities were available to detect an antibody response to this virus. Therefore, the overall objective of this project was to develop and validate selected diagnostic reagents and assays for PDCoV antigen and antibody detection.ResultsThe nucleoprotein of PDCoV was expressed as a recombinant protein and purified for use as an antigen to immunize mice for polyclonal, hyperimmune sera and monoclonal antibody (mAb) production. The resulting mAbs were evaluated for use in fluorescent antibody staining methods to detect PDCoV infected cells following virus isolation attempts and for immunohistochemistry staining of intestinal tissues of infected pigs. The same antigen was used to develop serological tests to detect the antibody response to PDCoV in pigs following infection. Serum samples from swine herds with recent documentation of PDCoV infection and samples from expected naïve herds were used for initial assay optimization. The tests were optimized in a checkerboard fashion to reduce signal to noise ratios using samples of known status. Statistical analysis was performed to establish assay cutoff values and assess diagnostic sensitivities and specificities. At least 629 known negative serum samples and 311 known positive samples were evaluated for each assay. The enzyme linked immunosorbent assay (ELISA) showed diagnostic sensitivity (DSe) of 96.1 % and diagnostic specificity (DSp) of 96.2 %. The fluorescent microsphere immunoassay (FMIA) showed a DSe of 95.8 % and DSp of 98.1 %. Both ELISA and FMIA detected seroconversion of challenged pigs between 8–14 days post-infection (DPI). An indirect fluorescent antibody (IFA) test was also developed using cell culture adapted PDCoV for comparative purposes.ConclusionThese new, specific reagents and serological assays will allow for improved diagnosis of PDCoV. Since many aspects of PDCoV infection and transmission are still not fully understood, the reagents and assays developed in this project should provide valuable tools to help understand this disease and to aid in the control and surveillance of porcine deltacoronavirus outbreaks.

Versatile and Amplified Biosensing through Enzymatic Cascade Reaction by Coupling Alkaline Phosphatase in Situ Generation of Photoresponsive Nanozyme.

The alkaline phosphatase (ALP) biocatalysis followed by the in situ enzymatic generation of a visible light responsive nanozyme is coupled to elucidate a novel amplification strategy by enzymatic cascade reaction for versatile biosensing. The enzymatic hydrolysis of o-phosphonoxyphenol (OPP) to catechol (CA) by ALP is allowed to coordinate on the surface of TiO2 nanoparticles (NPs) due to the specificity and high affinity of enediol ligands to Ti(IV). Upon the stimuli by CA generated from ALP, the inert TiO2 NPs is activated, which demonstrates highly efficient oxidase mimicking activity for catalyzing the oxidation of the typical substrate of 3,3',5,5'-tetramethylbenzidine (TMB) under visible light (λ ≥ 400 nm) irradiation utilizing dissolved oxygen as an electron acceptor. On the basis of the cascade reaction of ALP and the nanozyme of CA coordinated TiO2 (TiO2-CA) NPs, we design exquisitely colorimetric biosensors for probing ALP activity and its inhibitor of 2, 4-dichlorophenoxyacetic acid (2,4-DA). Quantitative probing of ALP activity in a wide linear range from 0.01 to 150 U/L with the detection limit of 0.002 U/L is realized, which endows the methodology with sufficiently high sensitivity for potentially practical applications in real samples of human serum (ALP level of 40-190 U/L for adults). In addition, a novel immunoassay protocol by taking mouse IgG as an example is validated using the ALP/nanozyme cascade amplification reaction as the signal transducer. A low detection limit of 2.0 pg/mL is attained for mouse IgG, which is 4500-fold lower than that of the standard enzyme-linked immuno-sorbent assay (ELISA) kit. Although only mouse IgG is used as a proof-of-concept in our experiment, we believe that this approach is generalizable to be readily extended to other ELISA systems. This methodology opens a new horizon for amplified and versatile biosensing including probing ALP activity and following ALP-based ELISA immunoassays.