Antibody Enzyme-linked Immunosorbent Assay Research Articles

Correlation between Virus Replication and Antibody Responses in Macaques following Infection with Pandemic Influenza A Virus.

Influenza virus infection of nonhuman primates is a well-established animal model for studying pathogenesis and for evaluating prophylactic and therapeutic intervention strategies. However, usually a standard dose is used for the infection, and there is no information on the relation between challenge dose and virus replication or the induction of immune responses. Such information is also very scarce for humans and largely confined to evaluation of attenuated virus strains. Here, we have compared the effect of a commonly used dose (4 × 10(6) 50% tissue culture infective doses) versus a 100-fold-higher dose, administered by intrabronchial installation, to two groups of 6 cynomolgus macaques. Animals infected with the high virus dose showed more fever and had higher peak levels of gamma interferon in the blood. However, virus replication in the trachea was not significantly different between the groups, although in 2 out of 6 animals from the high-dose group it was present at higher levels and for a longer duration. The virus-specific antibody response was not significantly different between the groups. However, antibody enzyme-linked immunosorbent assay, virus neutralization, and hemagglutination inhibition antibody titers correlated with cumulative virus production in the trachea. In conclusion, using influenza virus infection in cynomolgus macaques as a model, we demonstrated a relationship between the level of virus production upon infection and induction of functional antibody responses against the virus. There is only very limited information on the effect of virus inoculation dose on the level of virus production and the induction of adaptive immune responses in humans or nonhuman primates. We found only a marginal and variable effect of virus dose on virus production in the trachea but a significant effect on body temperature. The induction of functional antibody responses, including virus neutralization titer, hemagglutination inhibition titer, and antibody-dependent cell-mediated cytotoxicity, correlated with the level of virus replication measured in the trachea. The study reveals a relationship between virus production and functional antibody formation, which could be relevant in defining appropriate criteria for new influenza virus vaccine candidates.

Correlation between serum cytokeratin-18 and the progression or regression of non-alcoholic fatty liver disease

Background. Diagnosis of non-alcoholic fatty liver disease (NAFLD) is limited by the need for liver biopsies. Serum cytokeratin 18 (CK-18) levels have been investigated as potential biomarkers for the presence of NAFLD and non-alcoholic steatohepatitis (NASH). Herein, we assessed the correlation between CK-18 levels and NAFLD progression.Material and methods. Serum CK-18 levels were estimated using the M30 antibody enzyme-linked immunosorbent assay in 147 patients diagnosed with NAFLD. In 72 patients, disease progression was evaluated by repeated liver biopsy, which was conducted after 4.3 ± 2.6 years. The relationship between the CK-18 levels and liver histological findings was assessed.Results. The CK-18 levels were useful for identifying NAFLD patients with NAFLD activity scores (NAS) ≥ 5 (NAS ≥ 5 vs. < 4: 675.1 U/L vs. 348.7 U/L; p < 0.0001). A cut-off value of 375 U/L was calculated using the receiver operating characteristic curve approach, with a specificity and sensitivity of 81.5 and 65%, respectively, for the diagnosis of NASH. Among the 72 patients who underwent repeated liver biopsy, 11 patients with a progressed NAS also had significantly increased serum CK-18 levels (p < 0.01); in 30 patients with an improved NAS, there was a significant improvement in the mean CK-18 levels (p < 0.0001). The 31 patients with static NAS had static CK-18 levels.Conclusions. In conclusion, serum CK-18 levels can predict NAS ≥ 5 in NAFLD patients. In NAFLD patients, serum CK-18 levels reflect NAS values and correlate with histological changes, and they appear to be useful indicators of progression and improvement.

Séroprévalence et facteurs de risque des hémoparasitoses (theilériose, babésiose et anaplasmose) chez les bovins dans quatre grandes régions d’élevage du Maroc

Les hémoparasitoses (theilériose, babésiose et anaplasmose) sont des maladies vectorielles graves dont l’impact économique et sur la santé des élevages bovins au Maroc est considérable. L’objectif de cette étude, réalisée dans quatre régions d’élevage bovin laitier, a été de déterminer la séroprévalence et les facteurs de risque de ces infections. Au total 1 040 bovins ont été examinés dans 96 exploitations. La technique competitive enzyme-linked immunosorbent assay (cELISA) et la réaction d’immunofluorescence indirecte ont été utilisées pour la détermination de la séroprévalence, et le test statistique V de Cramer a permis d’analyser les facteurs de risque d’infection des bovins par les hémoparasites. Sur 1 040 sérums analysés, 689 (66,3 p. 100) ont été séropositifs ; la séroprévalence a varié selon le parasite considéré : Theileria annulata (35,4 p. 100), Anaplasma marginale (20,5 p. 100), Babesia bigemina (13,6 p. 100) et B. bovis (12,0 p. 100). Par ailleurs, 155 sérums (14,9 p. 100) ont présenté des infections mixtes (deux ou trois hémoparasites simultané- ment). L’analyse statistique a montré que l’âge (C = 0,62) et le type d’élevage (C = 0,60) ont eu une forte influence sur la séroprévalence, tandis que la race (C = 0,46) et le sexe (C = 0,29) ont eu une influence moindre. Le climat (C = 0,06) n’a pas semblé avoir d’impact. Ces résultats aideront à comprendre la situation épizootique des hémoparasitoses vectorielles chez les bovins afin d’établir une stratégie adéquate pour leur contrôle.

Development of ELISA and Colloidal Gold-PAb Conjugate-Based Immunochromatographic Assay for Detection of Abrin-a.

When abrin-a was combined with several polyclonal antibodies (PAb), the detection limit could be increased. In this way, a monoclonal antibody (capture) and polyclonal antibody (detection) sandwich enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-PAb conjugate-based immunochromatographic assay for detection of abrin-a were developed. The ELISA had a detection limit of 3.9 ng/mL for abrin-a in standard solution and 7.8 ng/mL in soybean milk, and was more sensitive than polyclonal antibody (capture) and monoclonal antibody (detection) ELISA, which had a detection limit of 15.6 ng/mL. The test strip had a detection range of 50 to 500 ng/mL for abrin-a and a detection limit in standard solution or soybean milk samples of 50 ng/mL. However, the test strip had a reduced detection capability compared with a colloidal gold-monoclonal antibody conjugate-based immunochromatographic assay test strip, which had a lower detection limit of 10 ng/mL. The developed ELISAs and test strip show the specificity towards abrin-a and have no cross-reactivity towards abrin-b, -c, -d, ricin, or the agglutinins from either castor beans or rosary peas.

Use of a computer-based provider order entry (CPOE) intervention to optimize laboratory testing in patients with suspected heparin-induced thrombocytopenia

IntroductionHeparin-induced thrombocytopenia (HIT) is a rare but frequently considered diagnosis in hospitalized patients. Despite the availability of clinical prediction tools, HIT is often over-diagnosed and patients can be subjected to unnecessary and expensive testing. MethodsA decision-support tool requiring providers to calculate the 4Ts (HIT risk) score prior to ordering laboratory-based tests for anti-PF4/heparin antibody enzyme-linked immunosorbent assay (ELISA) testing was implemented at our institution in January 2014. Charts of adult patients who underwent ELISA or serotonin release assay (SRA) testing during the 8-month time periods prior to and following this intervention were reviewed and 4Ts scores at the time of ELISA or SRA testing were calculated. ResultsA total of 443 ELISA and SRA tests were sent for 411 patients during the time periods studied. We observed a significant decrease from 43tests/month before to 22tests/month (p<0.001) after the intervention. A total of 337 charts were reviewed. We observed a trend toward decrease in the proportion of tested patients with low 4Ts scores (66% vs 56%, p=0.069), as well as an increase in the average 4Ts score of tested patients (3.0 vs 3.4, p=0.010) following our intervention. DiscussionOver-testing and treatment for HIT are frequent and potentially harmful occurrences in hospitalized patients. Our study demonstrates that a clinical decision support tool embedded within the electronic ordering process can decrease unnecessary testing for HIT.

Small intestinal submucosa-derived extracellular matrix bioscaffold significantly enhances angiogenic factor secretion from human mesenchymal stromal cells

IntroductionThe in vivo therapeutic effect of mesenchymal stromal cells (MSCs) is currently believed to be tightly linked to their paracrine secretion ability. However, insufficient or imprecise cell delivery, low cell survival and retention post-transplant, along with harsh donor site microenvironments, are major barriers to the clinical success of MSC therapies. Here we tested a small intestinal submucosa (SIS)-derived extracellular matrix (ECM) bioscaffold augmented with MSCs, with the hypothesis that they will facilitate the precise delivery of increased numbers of MSCs therefore improving cell viability and retention.MethodsIn this study, we evaluated the secretion of angiogenic factors from three human MSC lines cultured on SIS ECM. We used human antibody array and enzyme-linked immunosorbent assay to measure the level of angiogenic factors released from MSCs when cultured on SIS ECM or regular tissue culture plastic. We tested MSCs cultured for three different time points.ResultsWe found that the SIS ECM culture environment can significantly enhance the release of several angiogenic factors when compared to MSCs cultured on standard tissue culture plastic. Specifically, vascular endothelial growth factor and interleukin-8 secretion was significantly increased at 24, 48 and 72 hours postseeding onto SIS ECM whereas vascular endothelial growth factor release for cells cultured on plastic surface remained the same during these time points. We also observed significant donor to donor variation in cytokine production.ConclusionsThis study demonstrates that MSCs transplanted onto a SIS ECM may greatly increase their therapeutic potential through an increase in pro-angiogenic cytokine release.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0165-3) contains supplementary material, which is available to authorized users.

Angiogenic Potential and Secretome of Human Apical Papilla Mesenchymal Stem Cells in Various Stress Microenvironments.

Stem cells from the apical papilla (SCAP) of human adult teeth are considered an accessible source of cells with angiogenic properties. The aims of this study were to investigate the endothelial transdifferentiation of SCAP, the secretion of pro- and antiangiogenic factors from SCAP, and the paracrine effects of SCAP when exposed to environmental stress to stimulate tissue damage. SCAP were exposed to serum deprivation (SD), glucose deprivation (GD), and oxygen deprivation/hypoxia (OD) conditions, individually or in combination. Endothelial transdifferentiation was evaluated by in vitro capillary-like formation assays, real-time polymerase chain reaction, western blot, and flow cytometric analyses of angiogenesis-related markers; secretome by antibody arrays and enzyme-linked immunosorbent assays (ELISA); and paracrine impact on human umbilical vein endothelial cells (HUVECs) by in vitro transwell migration and capillary-like formation assays. The short-term exposure of SCAP to glucose/oxygen deprivation (GOD) in the presence, but mainly in deprivation, of serum (SGOD) elicited a proangiogenesis effect indicated by expression of angiogenesis-related genes involved in vascular endothelial growth factor (VEGF)/VEGFR and angiopoietins/Tie pathways. This effect was unachievable under SD in normoxia, suggesting that the critical microenvironmental condition inducing rapid endothelial shift of SCAP is the combination of SGOD. Interestingly, SCAP showed high adaptability to these adverse conditions, retaining cell viability and acquiring a capillary-forming phenotype. SCAP secreted higher numbers and amounts of pro- (angiogenin, IGFBP-3, VEGF) and lower amounts of antiangiogenic factors (serpin-E1, TIMP-1, TSP-1) under SGOD compared with SOD or SD alone. Finally, secretome obtained under SGOD was most effective in inducing migration and capillary-like formation by HUVECs. These data provide new evidence on the microenvironmental factors favoring endothelial transdifferentiation of SCAP, uncovering the molecular mechanisms regulating their fate. They also validate the angiogenic properties of their secretome giving insights into preconditioning strategies enhancing their therapeutic potential.

P09.37 Syphilis rapid test validation for treponemal diagnosis in guatemala, 2013–2014

Background International guidelines for syphilis testing recommend non-treponemal tests, and confirmation by more complex treponemal test such as Treponema pallidum haemagglutination (TPHA), enzyme-linked immunosorbent assay (ELISA) or others. Similar to HIV rapid tests, rapid treponemal tests can improve point-of-care testing in laboratories with limited capacities. We present main results of a validation for treponemal diagnosis performed in Guatemala. Methods A syphilis rapid test laboratory validation was performed in two phases during 2013–2014. First stage was performed using frozen serums from National Health Laboratory and second stage was performed using whole blood from pregnant women from peripheral health services from Guatemala City. Four treponemal rapid tests were evaluated in the first stage: Determine TP (DetTP), SD Syphilis (SDSyp), Omega Visitect (OVis), and Rapid Syphilis (RSyp). Only two tests were evaluated in the second stage: DetTP and SDSyp. For confirmation, TPHA was used for diagnosis of treponemal antibodies in serum and treponemal ELISA for plasma. Results A total of 460 serums were evaluated in first stage and 432 whole blood samples in second stage. Compared to TPHA, sensitivity ranged from 87.8% for RSyp to 99.2% in DetTP in serum and 100% for SDSyp and DetTP in whole blood. Specificity was lower, ranging from 74.8% for DetTP to 87.7% for SDSyp in serum and 100% for SDSyp and DetTP in whole blood. Conclusion Rapid treponemal tests are a useful diagnostic tool in syphilis. Sensibility was high enough to be used as a screening test, with good results in whole blood samples. Low prevalence of syphilis in the validation contributed to poor positive predictive values. The use of a reverse algorithm might be useful for implementation of this test in Guatemala. Disclosure of interest statement We declare that we have no conflicts of interest.

Synovial Fluid and Serum Concentrations of Interleukin-1 Receptor Antagonist and Interleukin-1ß in Naturally Occurring Equine Osteoarthritis and Septic Arthritis

The objective of this study was to investigate the concentrations of interleukin-1 receptor antagonist (IL-1Ra) and interleukin-1ß (IL-1ß) in synovial fluid (SF) and serum (SE) of horses with different grades of osteoarthritis (OA) and septic arthritis. Based on SF analysis, radiographic, and arthroscopic scores, 40 horses were classified into three groups as follows: mild OA, advanced OA, and septic arthritis. Horses without orthopedic problems served as a control group. Equine-specific antibody enzyme-linked immunosorbent assays were used to determine the concentration of IL-1Ra and the catabolic cytokine IL-1ß in SF and SE. Results were further compared with levels of the previously described biomarkers C-telopeptide fragments of type II collagen (CTX-II) and myeloperoxidase. In the present study, the SF of healthy joints, those with nonseptic OA, and those with septic arthritis contained significantly different levels of IL-1Ra. Serum concentrations of IL-1Ra were only significantly elevated in horses with septic arthritis when compared with the control group. Different levels of IL-1ß were detected in SE of control horses compared with those with various degrees of joint disease. Synovial fluid concentrations of IL-1ß were only moderately elevated in the groups of horses with joint disease. In addition to lameness examination and standard diagnostic procedures, the determination of IL-1Ra concentration in SF, in combination with further biomarkers, might be useful to assess the extent of intrasynovial inflammation.

Multidistrict Outbreak of Marburg Virus Disease-Uganda, 2012.

In October 2012, a cluster of illnesses and deaths was reported in Uganda and was confirmed to be an outbreak of Marburg virus disease (MVD). Patients meeting the case criteria were interviewed using a standard investigation form, and blood specimens were tested for evidence of acute or recent Marburg virus infection by reverse transcription-polymerase chain reaction (RT-PCR) and antibody enzyme-linked immunosorbent assay. The total count of confirmed and probable MVD cases was 26, of which 15 (58%) were fatal. Four of 15 laboratory-confirmed cases (27%) were fatal. Case patients were located in 4 different districts in Uganda, although all chains of transmission originated in Ibanda District, and the earliest case detected had an onset in July 2012. No zoonotic exposures were identified. Symptoms significantly associated with being a MVD case included hiccups, anorexia, fatigue, vomiting, sore throat, and difficulty swallowing. Contact with a case patient and attending a funeral were also significantly associated with being a case. Average RT-PCR cycle threshold values for fatal cases during the acute phase of illness were significantly lower than those for nonfatal cases. Following the institution of contact tracing, active case surveillance, care of patients with isolation precautions, community mobilization, and rapid diagnostic testing, the outbreak was successfully contained 14 days after its initial detection.

Vaccination with a Live Attenuated Cytomegalovirus Devoid of a Protein Kinase R Inhibitory Gene Results in Reduced Maternal Viremia and Improved Pregnancy Outcome in a Guinea Pig Congenital Infection Model.

Development of a vaccine to prevent congenital cytomegalovirus infection is a major public health priority. Live vaccines attenuated through mutations targeting viral mechanisms responsible for evasion of host defense may be both safe and efficacious. Safety and vaccine efficacy were evaluated using a guinea pig cytomegalovirus (GPCMV) model. Recombinant GPCMV with a targeted deletion of gp145 (designated Δ145), a viral protein kinase R (PKR) inhibitor, was generated. Attenuation was evaluated following inoculation of 10(7) PFU of Δ145 or parental virus into guinea pigs immunosuppressed with cyclophosphamide. Efficacy was evaluated by immunizing GPCMV-naive guinea pigs twice with either 10(5) or 10(6) PFU of Δ145, establishing pregnancy, and challenging the guinea pigs with salivary gland-adapted GPCMV. The immune response, maternal viral load, pup mortality, and congenital infection rates in the vaccine and control groups were compared. Δ145 was substantially attenuated for replication in immunocompromised guinea pigs. Vaccination with Δ145 induced enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody levels comparable to those achieved in natural infection. In the higher- and lower-dose vaccine groups, pup mortality was reduced to 1/24 (4%) and 4/29 (14%) pups, respectively, whereas it was 26/31 (81%) in unvaccinated control pups (P < 0.0001 for both groups versus the control group). Congenital infection occurred in 20/31 (65%) control pups but only 8/24 (33%) pups in the group vaccinated with 10(6) PFU (P < 0.05). Significant reductions in the magnitude of maternal DNAemia and pup viral load were noted in the vaccine groups compared to those in the controls. Deletion of a GPCMV genome-encoded PKR inhibitor results in a highly attenuated virus that is immunogenic and protective as a vaccine against transplacental infection. Previous attempts to develop successful immunization against cytomegalovirus have largely centered on subunit vaccination against virion proteins but have yielded disappointing results. The advent of bacterial artificial chromosome technologies has enabled engineering of recombinant cytomegaloviruses (CMVs) from which virus genome-encoded immune modulation genes have been deleted, toward the goal of developing a safe and potentially more efficacious live attenuated vaccine. Here we report the findings of studies of such a vaccine against congenital CMV infection based on a virus with a targeted deletion in gp145, a virus genome-encoded inhibitor of protein kinase R, using the guinea pig model of vertical CMV transmission. The deletion virus was attenuated for dissemination in immunocompromised guinea pigs but elicited ELISA and neutralizing responses. The vaccine conferred protection against maternal DNAemia and congenital transmission and resulted in reduced viral loads in newborn guinea pigs. These results provide support for future studies of attenuated CMV vaccines.

Chemokine CXCL-1: activity in the vitreous during proliferative vitreoretinopathy.

The aim of this study was to investigate CXCL-1 chemokine levels in the vitreous during rhegmatogenous retinal detachment (RRD) with and without proliferative vitreoretinopathy (PVR) and identify possible correlations with clinical parameters (extent and duration or RRD and PVR grade). Vitreous samples from patients with primary RRD with or without PVR were collected and assayed using a double antibody enzyme-linked immunosorbent assay (ELISA). Eleven vitreous samples from organ donors were employed as a control group. CXCL-1 levels were measured in 35 vitreous samples from 35 RRD patients. Mean CXCL-1 levels (64·82 ± 6·47 pg/ml) were significantly higher (P = 0·048) compared to controls. There was a significant positive correlation between CXCL-1 levels and the extent of the detachment (r = 0·794, P = 0·006). Peak CXCL-1 levels coincided with 3+ quadrant RRD, an interim of 29-60 days' duration and PVR grade B. Increased CXCL-1 levels may be indicative of mild inflammation in the detached retina and the adjacent vitreous. The results of the present study may provide novel insight into the complex interactions taking place during the early and late stages of RRD complicated by PVR.

Challenges for bovine viral diarrhoea virus antibody detection in bulk milk by antibody enzyme-linked immunosorbent assays due to changes in milk production levels.

BackgroundBovine viral diarrhoea (BVD) is considered eradicated from Denmark. Currently, very few (if any) Danish cattle herds could be infected with BVD virus (BVDV). The Danish antibody blocking enzyme-linked immunosorbent assay (ELISA) has been successfully used during the Danish BVD eradication program, initiated in 1994. During the last decade, the cattle herd size has increased while the prevalence of BVDV has decreased. In this study, we investigated how these changes could affect the performance of the Danish blocking ELISA and of the SVANOVIR®BVDV-Ab indirect ELISA. The latter has successfully been used to eradicate BVD in Sweden.Data (2003–2010) on changes in median herd size and milk production levels, occurrence of viremic animals and bulk milk surveillance were analysed. Additionally, the Danish blocking ELISA and the SVANOVIR ELISA were compared analyzing milk and serum samples. The prevalence of antibody positive milking cows that could be detected by each test was estimated, by diluting positive individual milk samples and making artificial milk pools.ResultsDuring the study period, the median herd size increased from 74 (2003) to 127 cows (2010), while the prevalence of BVDV infected herds decreased from 0.51 to 0.02 %. The daily milk yield contribution of a single seropositive cow to the entire daily bulk milk was reduced from 1.61 % in 2003 to 0.95 % in 2010 due to the increased herd size. It was observed that antibody levels in bulk milk decreased at national level. Moreover, we found that when testing bulk milk, the SVANOVIR®BVDV-Ab can detect a lower prevalence of seropositive lactating cows, compared to the Danish blocking ELISA (0.78 % vs. 50 %). Values in the SVANOVIR®BVDV-Ab better relate to low concentrations of antibody positive milk (R2 = 94-98 %), than values in the blocking ELISA (R2 = 23–75 %). For sera, the two ELISAs performed equally well.ConclusionsThe SVANOVIR ELISA is recommended for analysis of bulk milk samples in the current Danish situation, since infected dairy herds e.g. due to import of infected cattle can be detected shortly after BVDV introduction, when only few lactating cows have seroconverted. In sera, the two ELISAs can be used interchangeably.

Excessive antigen reactivity may underlie the clinical aggressiveness of chronic lymphocytic leukemia stereotyped subset #8

AbstractSubset #8 is a distinctive subset of patients with chronic lymphocytic leukemia (CLL) defined by the expression of stereotyped IGHV4-39/IGKV1(D)-39 B-cell receptors. Subset #8 patients experience aggressive disease and exhibit the highest risk for Richter transformation among all CLL. In order to obtain biological insight into this behavior, we profiled the antigen reactivity and signaling capacity of subset #8 vs other clinically aggressive stereotyped subsets, namely subsets #1 and #2. Twenty-seven monoclonal antibodies (mAbs) from subsets #1, #2, and #8 CLL clones were prepared as recombinant human immunoglobulin G1 and used as primary antibodies in enzyme-linked immunosorbent assays against representatives of the major classes of established antigenic targets for CLL. Subset #8 CLL mAbs exhibited broad polyreactivity as they bound to all antigens tested, in clear contrast with the mAbs from the other subsets. Antigen challenge of primary CLL cells indicated that the promiscuous antigen-binding activity of subset #8 mAbs could lead to significant cell activation, again in contrast to the less responsive CLL cells from subsets #1 and #2. These features constitute a distinctive profile for CLL subset #8, supporting the existence of distinct mechanisms of aggressiveness in different immunogenetic subsets of CLL.

Development of a Novel Urine Alzheimer-Associated Neuronal Thread Protein ELISA Kit and Its Potential Use in the Diagnosis of Alzheimer's Disease.

Alzheimer's disease (AD) is an age-related chronic degenerative disease that damages the nervous system. A noninvasive and simple method for early detection of AD is extremely important for the diagnosis and prognosis of AD. Thus, we aimed to develop an enzyme-linked immunosorbent assay (ELISA) kit to detect urine Alzheimer-associated neuronal thread protein (AD7C-NTP), and to evaluate its clinical value for the diagnosis of AD. Immunogenic AD7C-NTP peptide fragments were synthesized by the solid-phase method and used for immunizing mice or rabbits to generate anti-AD7C-NTP antibodies. The urine AD7C-NTP ELISA kit was then established; the generated mouse anti-AD7C-NTP antibody was used as a capture antibody, the biotin-labeled rabbit anti-AD7C-NTP antibody was used as a detection antibody, and avidin labeled by horseradish peroxidase was used as a substrate. The first morning urine specimens of 121 AD patients and 118 age-matched controls were collected, and the urine AD7C-NTP levels were detected by the above ELISA kit. Mouse and rabbit anti-AD7C-NTP antibody ELISA titer was found to be 1:8,000 and 1:32,000, respectively. A single band with a relative molecular mass of 41 kDa was found in human brain specimens by Western blot assay, which was identified as AD7C-NTP antibody. The urine AD7C-NTP concentration of the AD patients was higher than that of the age-matched controls, the sensitivity was 89.3% and the specificity was 84.7%. Our study demonstrated that our newly developed urine AD7C-NTP ELISA kit has suggested potential for diagnosing AD in a Chinese population, suggesting it may be a useful diagnostic kit for detecting early AD.

Integration of genetically modified virus-like-particles with an optical resonator for selective bio-detection

A novel virus-like particle (TMV-VLP) receptor layer has been integrated with an optical microdisk resonator transducer for biosensing applications. This bioreceptor layer is functionalized with selective peptides that encode unique recognition affinities. Integration of bioreceptors with sensor platforms is very challenging due their very different compatibility regimes. The TMV-VLP nanoreceptor exhibits integration robustness, including the ability for self-assembly along with traditional top-down microfabrication processes. An optical microdisk resonator has been functionalized for antibody binding with this receptor, demonstrating resonant wavelength shifts of (Δλo) of 0.79 nm and 5.95 nm after primary antibody binding and enzyme-linked immunosorbent assay (ELISA), respectively, illustrating label-free sensing of this bonding event. This demonstration of label-free sensing with genetically engineered TMV-VLP shows the flexibility and utility of this receptor coating when considering integration with other existing transducer platforms.

Development of mature BDNF-specific sandwich ELISA.

Mature brain-derived neurotrophic factor (mBDNF) plays a vital role in the nervous system, whereas proBDNF elicits neurodegeneration and neuronal apoptosis. Although current enzyme-linked immunosorbent assay (ELISA) has been widely used to measure BDNF levels, it cannot differentiate mBDNF from proBDNF. As the function of proBDNF differs from mBDNF, it is necessary to establish an ELISA assay specific for the detection of mBDNF. Therefore, we aimed to establish a new mBDNF-specific sandwich ELISA. In this study, we have screened and found a combination of antibodies for a sandwich ELISA. A monoclonal antibody and sheep anti-BDNF were chosen as capture and detection antibody for sandwich ELISA respectively. The new ELISA showed no cross-reactivity to human recombinant NT-3, NT-4, nerve growth factor and negligible cross-reactivity (0.99-4.99%) for proBDNF compared to commercial ELISA kits (33.18-91.09%). The application of the new mBDNF ELISA was shown through the measurement of mBDNF levels in different brain regions of rats and in the brain of β-site amyloid precursor protein cleaving enzyme 1 (BACE1)(-/-) and WT mice and compared to western blot. Overall, this new ELISA will be useful for the measurement of mBDNF levels with high specificity. As the function of proBDNF differs from mBDNF (mature BDNF), it is necessary to establish an ELISA specific for the detection of mBDNF. Here, we present a novel sandwich ELISA which detects mBDNF with high specificity. This new ELISA will be useful for the measurement of mBDNF levels with high specificity in various human and animal tissues. proBDNF, precursor of BDNF; BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; NGF, nerve growth factor.

Direct infection of primary salivary gland epithelial cells by human T lymphotropic virus type I in patients with Sjögren's syndrome.

To investigate whether human T lymphotropic virus type I (HTLV-I) directly infects salivary gland epithelial cells (SGECs) and induces the niche of the salivary glands in patients with Sjögren's syndrome (SS). SGECs were cultured with the HTLV-I-producing CD4+ T cell line HCT-5 or with Jurkat cells. Antibody arrays, immunofluorescence analysis, and enzyme-linked immunosorbent assay (ELISA) were used to determine the profiles of inflammation-related molecules, and the profiles of apoptosis-related molecules were determined by antibody array and immunofluorescence analysis. The presence of HTLV-I-related molecules was assessed by immunofluorescence analysis and in situ polymerase chain reaction. Apoptosis of SGECs was evaluated by TUNEL staining. Among the SGECs, 7.8 ± 1.3% (mean ± SD) were positive for HTLV-I-related proteins after 96-hour coculture with HCT-5 cells. Nuclear NF-κB p65 was also detected in 10% of the SGECs. The presence of HTLV-I proviral DNA in SGECs after coculture with HCT-5 cells was detected by in situ polymerase chain reaction. After coculture of SGECs with HCT-5, the expression of cytokines and chemokines, including soluble intercellular adhesion molecule 1, RANTES, and interferon γ-induced protein 10 kd (IP-10/CXCL10) was increased in a time-dependent manner. The expression of proapoptotic molecules (e.g., cytochrome c and Fas) and antiapoptotic molecules (e.g., Bcl-2, Heme oxygenase 2, and Hsp27) was increased in the SGECs cocultured with HCT-5, showing that apoptosis of SGECs was not detected after coculture with HCT-5 or Jurkat cells. HTLV-I is thought to infect SGECs and alter their cellular functions. These changes may induce the niche of SS and contribute to the development of SS in anti-HTLV-I antibody-positive individuals.

The Enzyme Linked Immunosorbent and Chemiluminescence Assay for the Detection of Human Chorionic Gonadotrophin Using Soybean Peroxidase as Label Enzyme

Soybean peroxidase (SBP) is characterized by relatively high heat resistance, wide substrate scope, good stability and broad pH applicability. These advantages led SBP to be used as labeledantibody for immunoassays. In this work, monoclonal antibody was linked with SBP to beta-HCG (Mab II-SBP) for enzyme-linked immunosorbent assay (ELISA) or chemiluminescence assay to explore its potential as an alternative to the widely used horseradish peroxidase (HRP) based ELISA. The Mab II-SBP was prepared by the sodium periodate oxidation method. The effects of enzyme coupling rate and molar ratio of Mab II to enzyme on assay performance were investigated. The optimum mass concentration ratio of Mab II and SBP was determined to be 1:3. The best coat concentration of the monoclonal antibody to alfa-HCG antibody (Mab I) was 4 μg/mL. Under optimum conditions, the HCG concentration was determined by the proposed ELISA assay with linear response range from 0.18 to 18 ng/mL and detection limit of 0.07 ng/mL at a signal-to-noise ratio of 3. Both enhancers, 3-(10’-phenothiazinyl) propane- 1-sulfonate (SPTZ) and 4-morpholinopyridine (MORPH), amplified chemiluminescence intensity nearly 200-fold in comparison to the SBP-luminol-H2O2 system. The present work demonstrates the feasibility and rationale of SBP-labeling antibodies for ELISA and chemiluminescence assays.

Genomic scan for identifying candidate genes for paratuberculosis resistance in sheep

Breeding objectives relating to health, functional traits and welfare need to receive priority in the research programs and selection schemes, but very few reports are available on natural resistant genotypes in livestock, where some important diseases cause severe economic losses and pose serious zoonotic threats. In this study, diagnosis of paratuberculosis was performed on 759 adult sheep, from a single flock, with the serum antibody enzyme-linked immunosorbent assay; 100 sheep were selected among the extreme divergent animals for the S/P ratio obtained from the test, and were genotyped on the Illumina Ovine SNP50K BeadChip. A genome-wide scan was then performed on the individual marker genotypes, in the attempt to identify genomic regions associated with disease resistance in sheep. For each marker, the allelic substitution effect was calculated by regressing the S/P value on the number of copies of the reference allele. The position on the OARv3.1 Genome Assembly was searched for 32 markers, which showed a statistically significant allelic substitution effect (Raw P &lt; 0.0006 and FDR P &lt; 0.09). All markers were located within, or close to, annotated genes. Five of these genes, SEMA3, CD109, PCP4, PRDM2 and ITFG2 are referred in literature to play a role in either disease resistance or cell-mediated immune response.