In-house Enzyme-linked Immunosorbent Assay Research Articles

Effect of collection tube type and freeze-thaw cycles on myeloperoxidase concentrations in blood samples of acute coronary syndrome patients.

Background Myeloperoxidase has shown potential as a marker for prognosis of coronary artery disease, but presently little is known about preanalytical handling of samples for quantifying myeloperoxidase. The present study was conducted to evaluate the effect of collection tube and freeze-thaw cycles on myeloperoxidase concentrations. Methods Acute coronary syndrome patients ( n = 88) were enrolled after obtaining written informed consent from coronary care unit of a tertiary care hospital (January 2012-June 2014). About 5 mL venous blood was collected from patients and divided into serum, lithium heparin, ethylenediaminetetraacetic acid and sodium citrate tubes. Except serum, all tubes were kept on ice immediately after collection. Samples were centrifuged at -4℃, separated immediately after centrifugation and stored at -40℃ until analysis. Myeloperoxidase was quantified by in-house and commercial assays and re-quantified after five freeze-thaw cycles. Results Myeloperoxidase concentrations, (serum samples) determined by commercial and in-house assays correlated well (r = 0.946) ( P < 0.001) and were higher in serum samples. Within plasma, myeloperoxidase concentrations were slightly higher in ethylenediaminetetraacetic acid (307.7 ± 52.1) and lower in lithium heparin (290.3 ± 49.2) and sodium citrate (221.4 ± 40.3) but not statistically significant. Correlation between myeloperoxidase concentrations (in-house enzyme-linked immunosorbent assay) after first cycle and fifth freeze-thaw cycle dropped to r = 0.448 ( P < 0.001). Conclusion Myeloperoxidase concentrations are comparable in three types of plasma tubes when samples are placed on ice immediately, centrifuged at low temperatures and separated immediately after centrifugation. Multiple freeze-thaw cycles have an effect on myeloperoxidase and should be avoided for quantifying myeloperoxidase.

Redefining Gold Standard Testing for Diagnosing Leptospirosis: Further Evidence from a Well-Characterized, Flood-Related Outbreak in Sri Lanka.

A gap in the leptospirosis field remains the lack of well-characterized sample collections that allow for comparison of new methods to standard ones. In the context of a flood-related outbreak of leptospirosis evaluated in Anuradhapura, Sri Lanka, a specimen bank was obtained with detailed metadata accompanied by gold standard diagnostic test results. Blood samples collected on admission and 14 days later from suspected cases of leptospirosis were tested using microscopic agglutination test (MAT) (17 serovars), an in-house enzyme-linked immunosorbent assay (ELISA) using a locally obtained strain of Leptospira kirschneri as sonicated antigen, a commercially available ELISA based on sonicated Leptospira biflexa, and a quantitative polymerase chain reaction (qPCR) assay targeting the pathogenic Leptospira-specific 16S rRNA gene. Of 62 patients presenting within the first 2 days of illness, 31 had confirmed leptospirosis based either on paired-sample MAT or qPCR. During the acute phase, qPCR was most sensitive, detecting 74% of definitively diagnosed cases; immunoglobulin G (IgG) ELISA (in-house), IgG ELISA (commercial), and MAT had sensitivities of 35.5%, 12.0%, and 22.6%, respectively, in detecting definitively diagnosed cases using acute phase serum. Of 40 patients with paired sera, 10 were qPCR positive. Of these, five samples were negative by paired-sample MAT. Of the 11 MAT-positive samples, only five were detected using qPCR confirming that both tests are needed for maximal sensitivity. Regional leptospiral serovar-specific IgG ELISA was superior to MAT. Knowing the regionally dominant serovars improves serological sensitivity in the analysis of acute specimens by ELISA, but qPCR was most sensitive in this patient population.

Development and use of specific ELISA methods for quantifying the biological activity of bevacizumab, cetuximab and trastuzumab in stability studies.

Bevacizumab (BVZ), cetuximab (CTX) and trastuzumab (TTZ) are monoclonal antibodies (mAbs) used worldwide for the treatment of several widespread kinds of cancer. They are marketed as medicines under their respective tradenames: Avastin(®), Erbitux(®) and Herceptin(®). The aim of this research was to develop in-house specific enzyme-linked immunosorbent assays (ELISA) to assess the long-term stability of these three mabs. These assays assess the biological functionality of the mAbs by quantifying their biological activity. For this purpose, we developed an indirect ELISA procedure whereby the specific antigens against which the mAbs are directed are used as specific "capturing" antibodies on the ELISA plates. We therefore used vascular endothelial growth factor (VEGF) in the ELISA for BVZ; human epidermal growth factor receptor (hEGFR) in the ELISA for CTX and human receptor HER2 (hHER2) in the ELISA for TTZ. After the mAbs had attached to their antigen, we used an anti-human IgG (whole molecule) peroxidase-conjugate and o-phenylenediaminedihydrochloride substrate. The reaction was stopped using sulphuric acid and absorbance was recorded at a wavelength of 450nm. The three ELISA methods were validated in terms of calibration models, range of the assay, limits of detection and quantitation, intra and interday precision and accuracy, and specificity by cross reactions. Forced degradation studies were also conducted on the medicines, providing useful information. Finally, the proposed ELISA were successfully used in a long-term stability study to quantify the remaining biological activity in medicines that had been opened and then stored under two different storage conditions, i.e. refrigerated at 4°C and frozen at -20°C. Results indicated that BVZ (Avastin(®)) is the most stable of the three in terms of its biological functionality.

Absolute quantification of HTLV-1 basic leucine zipper factor (HBZ) protein and its plasma antibody in HTLV-1 infected individuals with different clinical status.

BackgroundHuman T cell leukemia virus type 1 (HTLV-1) basic leucine zipper factor (HBZ), which is encoded by a minus strand mRNA, is thought to play important roles in the development of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, a comprehensive analysis of HBZ, including mRNA and protein expression, humoral immunoreactivity against HBZ, and HTLV-1 proviral load (PVL), in HTLV-1-infected individuals with different clinical status has not been reported previously.ResultsIn this study, using novel monoclonal antibody-based in-house enzyme-linked immunosorbent assay systems, we report the absolute quantification of HBZ protein and its plasma antibody in clinical samples from HTLV-1-infected individuals with different clinical status. The data were compared to both HBZ mRNA levels and PVL. The results showed that plasma anti-HBZ antibody was detectable only in 10.4 % (5/48) of asymptomatic carriers (ACs), 10.8 % (13/120) of HAM/TSP patients, and 16.7 % (7/42) of ATL patients. HBZ protein was detected in three out of five patients with acute ATL, but was not detected in patients with HAM/TSP (0/10) or ACs (0/4). Thus, an antibody response to HBZ was not associated with the PVL or the expression of HBZ (both at the mRNA and protein levels) or the clinical status of the infection.ConclusionsThe present results emphasize the extremely low expression and immunogenicity of HBZ in natural HTLV-1 infection. However, there is a possibility that the low but distinct expression of HBZ protein in PBMCs is associated with the survival of HTLV-1-infected cells and the development of ATL.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0263-z) contains supplementary material, which is available to authorized users.

Development of a Luminex-Based DIVA Assay for Serological Detection of African Horse Sickness Virus in Horses.

African horse sickness (AHS) is considered a fatal re-emergent vector-borne disease of horses. In the absence of any effective treatment for AHS, vaccination remains the most effective form of disease control. The new generation of vaccines, such as one based on purified, inactivated AHS virus (AHSV, serotype 4), which does not induce antibodies against non-structural protein 3 (NS3), enables the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA assays). As detecting AHS in AHSV-free countries may lead to restrictions on international animal movements and thereby cause significant economic damage, these DIVA assays are crucial for reducing movement restrictions. In this article, we describe a Luminex-based multiplex assay for DIVA diagnosis of AHS, and we validate it in a duplex format to detect antibodies against structural protein 7 (VP7) and NS3 in serum samples from horses vaccinated with inactivated AHSV4 vaccine or infected with a live virus of the same serotype. Results of the Luminex-based assay for detecting anti-NS3 antibodies showed good positive correlation with results from an in-house enzyme-linked immunosorbent assay (ELISA). Thus, the Luminex-based technique described here may allow multiplex DIVA antibody detection in a single sample in less than 2h, and it may prove adaptable for the development of robust, multiplex serological assays.

A panel of autoantibodies as potential early diagnostic serum biomarkers in patients with cervical cancer.

The study was designed to test whether circulating autoantibodies against associated antigens (TAAs) were altered in early cervical cancer and benign cervical tumors. A total of 111 cervical cancer patients, 137 cervical benign tumor patients, and 160 healthy volunteers matched in age were recruited in this study. The expression of autoantibodies was tested using in-house developed enzyme-linked immunosorbent assay (ELISA) with linear peptide envelope antigens derived from TAAs. One-way ANOVA test showed that there was no difference in the CD25 autoantibody expression among the cervical cancer group, benign tumor group, and healthy control group (P = 0.063; P = 0.191). The expression of autoantibodies against survivin and TP53 in the cervical cancer group was significantly higher than that in the benign tumor group (P < 0.001; P < 0.001). The levels of autoantibodies against cyclinB-1 and ANXA-1 were higher in the cervical cancer group than in the healthy control group (P = 0.010; P = 0.001), while autoantibodies in the cervical cancer group showed no difference in expression compared with that in the benign tumor group. The panel of five TAAs showed a sensitivity of 37.8% and a specificity of 90%, which was much higher than the sensitivity of the single-TAA testing group. The data from this study further support our previous hypothesis that the detection of autoantibodies for the diagnosis of a specific cancer type can be enhanced using a panel of several selected TAAs as target antigens.

Leishmania infantum INFECTION IN DOGS FROM THE SOUTHERN REGION OF MINAS GERAIS STATE, BRAZIL.

SUMMARYVisceral leishmaniasis is a systemic and chronic disease and dogs are the main reservoir of the etiologic agent, Leishmania infantum (syn L. chagasi). A serological and molecular investigation of canine visceral leishmaniasis (CVL) was performed in the municipality of Alfenas, located in the southern region of Minas Gerais, where the disease is not endemic. Samples from 87 dogs were submitted to serological tests including the Dual Path Platform (DPP(r) ) CVL Bio-Manguinhos rapid test, an in-house enzyme-linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT), as well as molecular techniques such as a conventional polymerase chain reaction (PCR) with the RV1/RV2 primers and a quantitative PCR (qPCR) with the LinJ31, Ldon and DNApol primers. Of the 87 serum samples, eight (9.2%) were positive for Leishmania using the DPP rapid test, but only four (4.6%) were confirmed by ELISA and two (2.3%) by IFAT. In these two serologically confirmed cases, spleen and liver samples were positive by all the employed molecular and parasitological procedures performed on spleen samples. When whole blood samples were used in the molecular assays, two samples (2.3%) were positive only by qPCR. DNA extracted and amplified from the spleens of seropositive dogs was sequenced, showing 100% of similarity with the Leishmania infantum (syn L. chagasi) sequence. Thus, the first cases of CVL have been confirmed in the Alfenas region, suggesting the importance of canine surveys in non-endemic municipalities for CVL to monitor disease progression and to prevent outbreaks.

First report of seroprevalence of Toxoplasma gondii infection in sheep in Pomerania, northern Poland.

Toxoplasmosis is parasitic disease which has economic relevance for both veterinary and human medicine. In sheep, toxoplasmosis is a major cause of abortion and can thus cause reproductive problems. The current study aimed to determine the occurrence of anti-Toxoplasma gondii IgG antibodies in sheep from 13 districts of northern Poland and thereby obtain actual data about T. gondii seroprevalence in this population of animals. Blood samples from 1,646 animals from 99 herds were collected, and an in-house enzyme-linked immunosorbent assay (ELISA) based on native Toxoplasma lysate antigen (TLA) was used for serological testing. The diagnostic sensitivity of diagnostic test used in this study was 98.6%, and specificity 94.9% for the group of 113 sheep sera (74 seropositive and 39 seronegative) previously characterized by using an commercial agglutination test. Antibodies against T. gondii were found in 921 (55.9%) of all tested animals. The percentage of infected sheep was the highest (67.6%) for older animals (>6 years), whereas for younger ones it was significantly lower (50.1% - 57.2% for 1-5-year-old animals, respectively). Furthermore, a higher percentage of seropositive animals was noted among males (63%) than females (55.5%). The results also showed that the size of the herd is not a factor which may affect the seroprevalence of T. gondii infection in the examined population of sheep. The results of this study indicate that T. gondii infection in sheep from region of northern Poland is relatively high, and consumption of ovine meat and milk can be regarded as a significant source of infection for humans.

Immunoglobulin G response in patients with Campylobacter concisus diarrhea

Limited information is available on the systemic immunoglobulin response in patients infected with the emerging pathogen Campylobacter concisus. The aim of the present study was to detect anti–C. concisus antibodies in serum of 88 patients with C. concisus gastroenteritis. Specific IgG antibodies to C. concisus were measured in serum using an in-house enzyme-linked immunosorbent assay, and pooled donor serum was used as a control. The mean optical density was 0.135 (SEM: 0.020) for the 88 adult patients and 0.100 (SEM: 0.011) in controls. When using an optical density value equal to the mean +3 SEM for the control serum, 22 (25%) C. concisus–positive patients had increased IgG antibodies. Patients with high IgG levels more often reported headache, and they had a trend toward more mucus in stools, whereas IgG levels were unrelated to age, duration of diarrhea, number of stools per day, and weight loss.

Increased Sensitivity of HIV-1 p24 ELISA Using a Photochemical Signal Amplification System.

In this study we describe a photochemical signal amplification method (PSAM) for increasing of the sensitivity of enzyme-linked immunosorbent assay (ELISA) for determination of HIV-1 p24 antigen. The photochemical signal amplification method is based on an autocatalytic photochemical reaction of a horseradish peroxidase (HRP) substrate, orthophenylenediamine (OPD). To compare the performance of PSAM-boosted ELISA with a conventional colorimetric ELISA for determination of HIV-1 p24 antigen we employed a PerkinElmer HIV-1 p24 ELISA kit, using conventional ELISA alongside ELISA + PSAM. In the present study, we show that PSAM technology allows one to increase the analytical sensitivity and dynamic range of a commercial HIV-1 p24 ELISA kit, with and without immune-complex disruption, by a factor of approximately 40-fold. ELISA + PSAM is compatible with commercially available microtiter plate readers, requires only an inexpensive illumination device, and the PSAM amplification step takes no longer than 15 min. This method can be used for both commercially available and in-house ELISA tests, and has the advantage of being considerably simpler and less costly than alternative signal amplification methods. This method can be used for both commercially available and in-house ELISA tests, and has the advantage of being considerably simpler and less costly than alternative signal amplification methods.

Development and validation of an Enzyme Linked Immunosorbent Assay (ELISA) test for the diagnosis of toxoplasmosis in Sri Lanka.

ELISA is the most widely used form of diagnosis for toxoplasmosis. Several commercial kits are currently used in Sri Lanka. However, these kits are not affordable in resource-limited settings. Objectives Aim of this study was to develop a cost effective in-house ELISA for the detection of Toxoplasma antibody and to estimate the diagnostic accuracy compared to a commercial kit. Vero cell lines were inoculated with tachyzoites and harvested after 2-6 days and sonicated to obtain somatic antigen. The antigen was used as coating material in ELISA to detect antibodies against T. gondii in patient sera. Hundred and three patients' sera were analysed by in-house ELISA and kit ELISA. Optical density (OD) values were analysed statistically. Toxoplasma IgG avidity test was used to determine the chronic and acute phase of infection. The optimum working dilutions for antigen was 0.846 μg/ml and for serum 1 in 100. The optimal cut-off values for the in-house ELISA within the range 0.85 to 0.98 at which the sensitivity was 95.3% and specificity was 98.3. The OD values of in-house ELISA were compared with OD values of kit ELISA and the results showed strong correlation between the two tests. The results of our study demonstrated that our in-house ELISA for detection of T. gondii antibody was as sensitive and specific as the commercial kit used in this study. Thus, the in-house ELISA is a useful, costeffective tool for diagnostic and screening purposes.

Evaluation and comparison of serological methods for the detection of bovine neosporosis in Argentina

Neospora caninum is a protozoan parasite that causes abortion and important economic losses in cattle worldwide. The accurate diagnosis of neosporosis is essential for management and control measures. The aims of this study were: i) to evaluate the performance of an in-house enzyme-linked immunosorbent assay based on the 38kDa native antigen (p38-ELISA) to diagnose bovine neosporosis in Argentina using a well- characterized local sera panel from experimentally infected and naturally exposed cattle and ii) to compare the diagnostic performance and agreement of three N. caninum serological tests: p38-ELISA, indirect fluorescence antibody test (IFAT) and immunoblotting (IB) using the same sera panel. Serum samples testing either positive or negative by IFAT and IB were considered “Relative Standards of Comparison” (RSC) and used for p38-ELISA evaluation. Receiver operating characteristics analysis revealed that p38-ELISA was highly accurate (area under the curve= 0.982) according to RSC with a cut-off index of 0.0905. Relative sensitivity and specificity of p38-ELISA were 97.8% and 99.5%, respectively and agreement between RSC and p38-ELISA was almost perfect (k= 0.97). The evaluation and performance comparison of serological tests were performed according to the definition of gold standard based on the decision of the “majority of tests”. All tests displayed high sensitivity and specificity values (greater than 95%); and excellent agreement. This study describes the accurate performance of p38-ELISA evaluated locally and the highly accurate diagnostic performance of the studied tests for the detection of anti-N. caninum antibodies in cattle from Argentina.

Abstract 1818: Analytical platform evaluation for quantification of ERG oncoprotein in prostate cancer using protein and mRNA detection methods

Abstract Current molecular tests that aid in the diagnosis of prostate cancer (PCa) are based on the detection of PSA and PSA isoforms (blood), overexpression of PCA3 (urine), and overexpression of AMACR (tissue) as biomarkers. The discovery of cancer specific fusion transcripts, which are detected in over 50% of prostate cancers, revolutionized the field. As such, the cancer specific TMPRSS2-ERG gene fusion drives the overexpression of the ERG oncoprotein. Based on this, we hypothesized that ERG protein may serve as a valuable biomarker and for which quantification methods can be applied for the diagnosis of prostate cancer. An antibody-free assay for ERG protein detection was developed based on PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry. We utilized TMPRSS2-ERG positive VCaP and TMPRSS2-ERG negative LNCaP cells to simulate three different sample types (cells, tissue, and post-DRE urine sediment) for evaluation. Recombinant ERG protein spiked into LNCaP cell lysates could be detected at levels as low as 20 pg by PRISM-SRM analysis. The sensitivity of the PRISM-SRM assay was approximately 10,000 VCaP cells in a mixed cell population model of VCaP and LNCaP cells. Interestingly, ERG protein could be detected in as few as 600 VCaP cells spiked into female urine, presumably due to more effective loading of ERG protein and the reduced sample complexity. In comparison the detection limit of the in-house enzyme-linked immunosorbent assay was 30 pg of recombinant ERG protein and 10,000 VCaP cells, while Western blot analysis demonstrated the detection of 195 pg of recombinant ERG protein and 10,000 VCaP cells in these two sample types, respectively. On the other hand, qRT-PCR exhibited a higher sensitivity, as TMPRSS2-ERG transcripts were detected in as few as 250 VCaP cells spiked into female urine, in comparison to NanoString methodologies which detected 10,000 VCaP cells in the same sample. In summary the data presented here suggest that qRT-PCR and PRISM-SRM platforms are highly sensitive in detecting TMPRSS2-ERG transcripts and proteins, respectively. Compared to other RNA (without preamplifcation step) and protein detection technologies, the PRISM-SRM assay has several significant advantages: it is not affected by RNA stability, it does not rely on a specific antibody, and it is ideal for isoform-specific detection with high multiplexing capability. Therefore, PRISM-SRM assays can be adapted to detect ERG protein in cells present in clinical specimens (e.g., tissue and urine), and cell-free ERG protein present in the blood sera, providing an opportunity for their use in the clinical setting for detection of ERG in prostate cancer patients and for defining appropriate treatment strategies. Citation Format: Jintang He, Athena Schepmoes, Anshu Rastogi, Shyh-Han Tan, Wusheng Yan, Wei Huang, Sreedatta Banerjee, Tujin Shi, Chaochao Wu, Thomas Fillmore, Yuqian Gao, Jacob Kagan, Sudhir Srivastava, Richard Smith, Wei-Jun Qian, David McLeod, Gyorgy Petrovics, Albert Dobi, Alagarsamy Srinivasan, Shiv Srivastava, Karin Rodland, Tao Liu, David Camp. Analytical platform evaluation for quantification of ERG oncoprotein in prostate cancer using protein and mRNA detection methods. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1818. doi:10.1158/1538-7445.AM2015-1818

Severe leptospirosis complicated by Epstein–Barr Virus reactivation

Weil's disease is a severe, potentially fatal illness following Leptospira interrogans infection. The reported case of a patient suffering from acute renal failure, jaundice, thrombocytopenia, rhabdomyolysis and encephalitis syndrome highlights the clinical challenge in reference to Weil syndrome complicated by Epstein-Barr Virus (EBV) reactivation. The diagnosis of leptospirosis was performed using four different diagnostic methods. Sera were analyzed with an in-house IgM and IgG enzyme-linked immunosorbent assay (ELISA) and indirect haemagglutination assay (IHA). Microscopic agglutination test (MAT) was done using 17 reference strains comprising 14 serogroups and 17 serovars. Polyvalent EBV-IgG analysis, EBV-IgG/IgM/IgA western blot analysis as well as quantitative EBV polymerase chain reaction (PCR) were performed. Leptospira IHA showed an initial titer of 1:640 (cut-off 1:320), leptospiral IgG was negative, but IgM was positive. MAT was negative at that time for all 17 strains analyzed. One week later, leptospirosis IHA titer increased to 1:20,480. Leptospiral IgG was now positive, -IgM remained positive and urine was tested negative for leptospiral DNA. The MAT showed positive results for L. interrogans serovar Bataviae, serovar Copenhageni, serovar Pyrogenes and L. borgpetersenii serovar Serjoe. During follow-up examinations, both the leptospiral IgM and IgG remained positive and MAT showed positive results for L. interrogans of different serovars. EBV IgA immunoblot taken at admission was positive for VCA-p18, quantitative EBV-PCR showed an EBV viral load of 2.8E3 copies/ml indicating acute EBV-reactivation. Leptospirosis represents a neglected and re-emerging disease which is difficult to diagnose since Leptospira-PCR from whole blood or urine is frequently negative in the case of early empiric antibiotic treatment. EBV-reactivation might represent a severe complication in Weil's disease which potentially aggravates clinical manifestations of leptospirosis including hepatitis, nephritis, and rhabdomyolysis. Thus, there might be a need for peripheral blood EBV-PCR and EBV blotting in patients suffering from complicated Weil syndrome, also in terms of the choice of antibiotic treatment.

A study of IgG antibodies to the ApoB protein in non-ST segment elevation acute coronary syndrome

Objectives. It has long been noted that there is an association of antibodies against oxidized low-density lipoprotein (oxLDL) with cardiovascular disease, but the anti-oxLDL antibody has not been confirmed as a biomarker for prediction of acute coronary syndrome (ACS). Apolipoprotein B (ApoB) may carry the epitopes for the immune response to oxLDL. The present work was thus undertaken to detect circulating antibodies to ApoB in non-ST segment elevation ACS (NSTE-ACS). Design. A total of 130 patients with NSTE-ACS and 201 control subjects were recruited. Six ApoB-derived peptipe antigens (Ag1–Ag6) were used to develop an in-house enzyme-linked immunosorbent assay to examine circulating anti-ApoB IgG levels. Results. The anti-Ag1 IgG level was significantly higher in the patient group than the control group (P < 0.001) and the non-ST segment elevation myocardial infarction appeared to be the main form of NSTE-ACS contributing to the increased levels of anti-Ag1 IgG (P < 0.001); there was no significant alteration in the levels of IgG to the other 5 antigens in NSTE-ACS. Conclusions. Circulating anti-ApoB IgG test may be useful for prediction of NSTE-ACS although further confirmation is needed in large-scale clinical studies.

Serum Hsp70 Antigen: Early Diagnosis Marker in Perinatal Asphyxia.

Background:Perinatal asphyxia is an important cause of mortality and permanent neurological and developmental deficit. Early and accurate diagnosis would help to establish the likely prognosis and may also help in determining the most appropriate treatment. Studies in experimental animal models suggest that a protein called Hsp70 may be a good and potentially useful marker of cellular stress that may be clinically useful in determining the presence of neonatal asphyxia.Objectives:Regarding the importance of early and accurate diagnosis of asphyxia, we conducted this study, which is the first investigation of the comparison of the serum Hsp70 antigen level between asphyxiated and healthy infants.Patients and Methods:In this observational study, the serum concentrations of Hsp70 antigen were compared between neonates suffering from perinatal asphyxia (n = 50) and normal neonates (n = 51). The inclusion criteria for the cases were neonates who had reached term and had at least two clinical criteria of asphyxia. Exclusion criteria were babies with gestational age < 37 weeks, infants with congenital abnormalities or positive blood culture. Exclusion criteria in this group were the requirement to hospital stay during first week of the life or babies whose mothers had difficulties during pregnancy or delivery. Term neonates without major anomalies who had asphyxia during delivery were enrolled in the first six hours after delivery, and control group consisted of healthy term neonates without problems and normal delivery process in the first week of life. The cord blood was taken during labor to measure Hsp70 antigen level by using an in-house ELISA (The enzyme-linked immunosorbent assay).Results:The median values of serum anti Hsp70 titers were significantly higher in asphyxiated neonates compared with non-asphyxiated neonates (0.36 [0.04 - 1.14] vs 0.24 [0.01 - 0.63]). At cutoff point = 0.3125 ng/mL, sensitivity was 58% and specificity 76% based on ROC curve.Conclusions:A significant difference between the serum concentrations of Hsp70 of the control and patient group was observed in this study. It is inferred serum concentrations of Hsp70 antigen may be a useful marker for the early diagnosis of that prenatal hypoxia.

Spatial analysis and risk mapping of Fasciola hepatica infection in dairy herds in Ireland.

Fasciolosis is generally a subclinical infection of dairy cows and can cause marked economic losses. This study investigated the prevalence and spatial distribution of fasciolosis in dairy cow herds in Ireland using an in-house antibodydetection enzyme-linked immunosorbent assay applied to bulk tank milk (BTM) samples collected during the autumn of 2012. A total of 5,116 BTM samples were collected from 4,602 different herds, with 514 farmers submitting BTM samples in two consecutive months. Analysis of the BTM samples showed that 82% (n = 3,764) of the dairy herds had been exposed to Fasciola hepatica. A total of 108 variables, including averaged climatic data for the period 1981-2010 and contemporary meteorological data for the year 2012, such as soil, subsoil, land cover and habitat maps, were investigated for a possible role as predictor of fasciolosis. Using mainly climatic variables as the major predictors, a model of the predicted risk of fasciolosis was created by Random Forest modelling that had 95% sensitivity and 100% specificity. The most important predictors in descending order of importance were: average of annual total number of rain-days for the period 1981-2010, total rainfall during September, winter and autumn of 2012, average of annual total number of wet-days for the period 1981- 2010 and annual mean temperature of 2012. The findings of this study confirm the high prevalence of fasciolosis in Irish dairy herds and suggest that specific weather and environmental risk factors support a robust and precise distribution model.

Cluster analysis of fasciolosis in dairy cow herds in Munster province of Ireland and detection of major climatic and environmental predictors of the exposure risk.

Fasciolosis caused by Fasciola hepatica is a widespread parasitic disease in cattle farms. The aim of this study was to detect clusters of fasciolosis in dairy cow herds in Munster Province, Ireland and to identify significant climatic and environmental predictors of the exposure risk. In total, 1,292 dairy herds across Munster was sampled in September 2012 providing a single bulk tank milk (BTM) sample. The analysis of samples by an in-house antibody-detection enzyme-linked immunosorbent assay (ELISA), showed that 65% of the dairy herds (n = 842) had been exposed to F. hepatica. Using the Getis-Ord Gi* statistic, 16 high-risk and 24 low-risk (P &lt;0.01) clusters of fasciolosis were identified. The spatial distribution of high-risk clusters was more dispersed and mainly located in the northern and western regions of Munster compared to the low-risk clusters that were mostly concentrated in the southern and eastern regions. The most significant classes of variables that could reflect the difference between high-risk and low-risk clusters were the total number of wet-days and rain-days, rainfall, the normalized difference vegetation index (NDVI), temperature and soil type. There was a bigger proportion of well-drained soils among the low-risk clusters, whereas poorly drained soils were more common among the high-risk clusters. These results stress the role of precipitation, grazing, temperature and drainage on the life cycle of F. hepatica in the temperate Irish climate. The findings of this study highlight the importance of cluster analysis for identifying significant differences in climatic and environmental variables between high-risk and low-risk clusters of fasciolosis in Irish dairy herds.

Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays

BackgroundEarly diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection. MethodsThe antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA). ResultsThe expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the 30 sera of known genotypes. The antigens did not detect antibodies to genotype-3a, but detected antibodies to all genotypes and did not discriminate them genotype wise. A panel of 175 of HCV-suspected serum samples was subjected to comparative analysis with our in-house ELISA assay and with commercial HCV screening assays. After subjecting the results to the formulas for determining the quality parameters, immunoblot assay had 100% sensitivity and specificity, while the ELISA assay had 100% sensitivity and 98.8% specificity as compared to commercially available assays. ConclusionThis study indicates that a mixture of Core and E2 antigens are potentially valuable antigens and there is the possibility of developing serological assays for monitoring HCV infection.

Amyloid-β oligomers relate to cognitive decline in Alzheimer's disease.

Amyloid-β (Aβ)-oligomers are neurotoxic isoforms of Aβ and are a potential diagnostic biomarker for Alzheimer's disease (AD). 1) Analyze the potential of Aβ-oligomer concentrations in cerebrospinal fluid (CSF) to diagnose and predict progression to AD in a large clinical study sample. 2) Monitor Aβ-oligomer concentrations over-time, both in early and advanced stages of AD. 3) Examine the relation between Aβ-oligomer levels in CSF and cognitive functioning. 24 non-demented, 61 mild cognitive impairment (MCI), and 64 AD patients who underwent lumbar puncture and cognitive testing at baseline and follow-up were selected from the memory clinic based Amsterdam Dementia Cohort. CSF samples were analyzed for standard AD-biomarkers and Aβ-oligomer levels using a validated in-house Aβ-oligomer specific enzyme-linked immunosorbent assay. Aβ-oligomer levels were analyzed as indicators of disease progression (follow-up AD diagnosis) and cognitive decline, respectively. Patient groups did not differ in Aβ-oligomer concentrations at baseline or follow-up. Baseline CSF Aβ-oligomer levels were similar in MCI patients that develop AD as in stable MCI patients. MCI and AD patients showed an annual decrease in Aβ-oligomer levels of 9.4% and 6.8%, respectively. A decrease in Aβ-oligomer levels over time was strongly associated with more severe cognitive decline in AD patients. Despite the limited diagnostic potential of Aβ-oligomer levels in CSF to differentiate between patient groups, and between MCI-AD and MCI-stable patients, changes in CSF Aβ-oligomer levels were related to cognitive decline. Therefore, CSF Aβ-oligomers may aid in the selection of patients with a more aggressive disease course.