Antibody Enzyme-linked Immunosorbent Assay Research Articles

Generation of Hybridomas Producing Monoclonal Antibodies to the NS1 Protein of the Zika Virus

Hybridomas producing monoclonal antibodies (mABs) to the recombinant protein NS1 of the Zika virus synthesized in human embryonic kidney HEK293 (ZV293) cells have been obtained. The specificity of antibodies has been examined by the enzyme-linked immunosorbent assay (ELISA) using the similarly synthesized NS1 proteins of flaviviruses: the Dengue 1–4 serotype virus, Japanese encephalitis virus, yellow fever virus, tick-borne encephalitis virus, and the West Nile fever virus. Two types of hybridomas whose mABs interact with thermolabile (mABs 2F6 and 3F3) and thermostable epitopes (mABs 1B9, 6D7, and 6F2) of the NS1ZV protein have been selected. It has been shown that the 3F3 mAB, which is specific to the thermolabile determinant, can be used as both binding and detecting antibodies in the sandwich ELISA. Based on the 3F3 mAB, an experimental immunochromatographic test for the detection of the NS1ZV antigen has been developed. It has also been found that mABs 1B9, 6D7, and 6F2 against thermostable epitopes of the NS1 protein specifically recognize the NS1ZV protein after its heating for 15 min to 98°C.

High Prevalence of MERS-CoV Infection in Camel Workers in Saudi Arabia.

Middle East respiratory syndrome (MERS), a highly lethal respiratory disease caused by a novel coronavirus (MERS-CoV), is an emerging disease with high potential for epidemic spread. It has been listed by the WHO and the Coalition for Epidemic Preparedness Innovations (CEPI) as an important target for vaccine development. While initially the majority of MERS cases were hospital acquired, continued emergence of MERS is attributed to community acquisition, with camels likely being the direct or indirect source. However, the majority of patients do not describe camel exposure, making the route of transmission unclear. Here, using sensitive immunological assays and a cohort of camel workers (CWs) with well-documented camel exposure, we show that approximately 50% of camel workers (CWs) in the Kingdom of Saudi Arabia (KSA) and 0% of controls were previously infected. We obtained blood samples from 30 camel herders, truck drivers, and handlers with well-documented camel exposure and from healthy donors, and measured MERS-CoV-specific enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), and neutralizing antibody titers, as well as T cell responses. Totals of 16/30 CWs and 0/30 healthy control donors were seropositive by MERS-CoV-specific ELISA and/or neutralizing antibody titer, and an additional four CWs were seronegative but contained virus-specific T cells in their blood. Although virus transmission from CWs has not been formally demonstrated, a possible explanation for repeated MERS outbreaks is that CWs develop mild disease and then transmit the virus to uninfected individuals. Infection of some of these individuals, such as those with comorbidities, results in severe disease and in the episodic appearance of patients with MERS.IMPORTANCE The Middle East respiratory syndrome (MERS) is a coronavirus (CoV)-mediated respiratory disease. Virus transmission occurs within health care settings, but cases also appear sporadically in the community. Camels are believed to be the source for community-acquired cases, but most patients do not have camel exposure. Here, we assessed whether camel workers (CWs) with high rates of exposure to camel nasal and oral secretions had evidence of MERS-CoV infection. The results indicate that a high percentage of CWs were positive for virus-specific immune responses but had no history of significant respiratory disease. Thus, a possible explanation for repeated MERS outbreaks is that CWs develop mild or subclinical disease. These CWs then transmit the virus to uninfected individuals, some of whom are highly susceptible, develop severe disease, and are detected as primary MERS cases in the community.

Treponeme-Associated Hoof Disease of Free-Ranging Elk (Cervus elaphus) in Southwestern Washington State, USA

A novel foot disease in free-ranging elk ( Cervus elaphus) in southwestern Washington State emerged in 2008 and spread throughout the region. Initial studies showed adult elk had chronic hoof overgrowth, sole ulcers, and sloughed hoof capsules, but no cause was determined. To identify possible causes and characterize the earliest lesions, 9-, 7-, and 3-month-old elk were collected. Nine-month-old elk had sole ulcers (3/9 elk) and sloughed/overgrown hoof capsules (4/9 elk) similar to adults. Histologically, lesions consisted of coronary, heel bulb, and interdigital ulcers with suppurative inflammation, epithelial hyperplasia, deeply invasive spirochetes, and underrunning of the hoof capsule and heel-sole junction. Spirochetes were identified as Treponema via immunohistochemistry and polymerase chain reaction (PCR). Seven-month-old elk had similar underrunning foot ulcers (6/8 elk) with Treponema identified in all lesions but no chronic overgrowth or sloughed hoof capsules. Three-month-old calves had superficial coronary erosions with no inflammation or identifiable spirochetes (3/5 elk) but were culture/PCR positive for Treponema, suggesting possible early lesions. Lesions from 9- and 7-month-old elk included aerobic and anaerobic bacteria, many of which are associated with infectious foot disease in livestock. Antibody enzyme-linked immunosorbent assay of 7- and 3-month-old elk from the enzootic region showed a trend toward increased Treponema antibody titers compared to normal control elk from outside the region, further supporting the significance of Treponema in the pathogenesis of foot disease. Treponeme-associated hoof disease (TAHD) in elk, a debilitating and progressive condition, shares similarities to bovine digital dermatitis and contagious ovine digital dermatitis.

Statistical Learning Methods to Determine Immune Correlates of Herpes Zoster in Vaccine Efficacy Trials.

Using Super Learner, a machine learning statistical method, we assessed varicella zoster virus-specific glycoprotein-based enzyme-linked immunosorbent assay (gpELISA) antibody titer as an individual-level signature of herpes zoster (HZ) risk in the Zostavax Efficacy and Safety Trial. Gender and pre- and postvaccination gpELISA titers had moderate ability to predict whether a 50-59 year old experienced HZ over 1-2 years of follow-up, with equal classification accuracy (cross-validated area under the receiver operator curve = 0.65) for vaccine and placebo recipients. Previous analyses suggested that fold-rise gpELISA titer is a statistical correlate of protection and supported the hypothesis that it is not a mechanistic correlate of protection. Our results also support this hypothesis.

Expanded Diagnostic Approach to Hepatitis E Virus Detection in Patients with Acute-on-Chronic Liver Failure: A Pilot Study

Introduction: Acute decompensation of pre-existing chronic liver disease (CLD), known as acute-on-chronic liver failure (ACLF), is associated with high mortality. Hepatitis E virus (HEV) as a potential cause was studied. Objectives: The objectives of this study are to evaluate the role of HEV in ACLF patients using an IgM anti-HEV antibody enzyme-linked immunosorbent assay (ELISA), HEV antigen ELISA, and a quantitative HEV polymerase chain reaction (PCR). Materials and Methods: In this prospective cross-sectional study, blood samples were collected from 50 ACLF (cases) as defined by the standard guidelines (APASL, 2014) and 50 patients with stable CLD (controls) from January 2015 to August 2016, after obtaining informed consent. Two IgM ELISAs (MP Diagnostics HEV IgM ELISA 3.0, Singapore and Wantai HEV IgM ELISA, Beijing, China) were compared using plasma from cases and controls. In addition, an HEV antigen detection by ELISA (Wantai, Beijing, China) and a real-time PCR for quantification of HEV RNA in plasma and stool were employed. Results: Ethanol was the leading cause of acute insult in ACLF (54%) cases. HEV infection accounted for 20% of cases. Ten ACLF patients (20%) had 1–3 markers of HEV versus two (4%) among controls (P = 0.0138). Among ACLF cases, one had HEV viraemia (403 IU/ml), faecal shedding (2790 IU/ml) and detectable HEV antigenaemia. Agreement between the two anti-HEV IgM ELISAs was 0.638 (kappa value). Conclusion: This study shows that alcohol is a major contributing factor for both underlying CLD and ACLF while HEV is the most common infectious cause for ACLF, suggesting a need for a vaccination in such patients, whenever made available.

An Improved Enzyme-Linked Immunoassay for the Detection of Leptospira-Specific Antibodies.

Leptospirosis is a neglected zoonotic disease with worldwide endemicity and continues to be a significant public health burden on resource-limited populations. Previously, we produced three highly purified recombinant antigens (rLipL32, rLipL41, and rLigA-Rep) and evaluated their performance of detecting Leptospira-specific antibodies in enzyme-linked immunosorbent assay (ELISA) as compared with the microscopic agglutination test (MAT). The overall sensitivity of this assay approached 90%. Recently, another recombinant antigen (rLigB-Rep) was prepared. We tested each individual antigen and a 1:1:1:1 mixture of these four antigens for the detection of Leptospira-specific antibodies in ELISA. The performance of these recombinant antigens was evaluated with a much larger febrile patient panel (337 MAT-confirmed positive sera and 92 MAT-negative sera from febrile patients). Combining the detection results of immunoglobulin M and immunoglobulin G from these four individual antigens, the overall sensitivity was close to 90% but the specificity was only 66%, based on the MAT reference method. The overall sensitivity and specificity of the four-antigen mixture were 82% and 86%, respectively. The mixture of four antigens also exhibited a broader reactivity with MAT-positive samples of 18 serovars from six major pathogenic Leptospira species. Given the limitations of MAT, the data were further analyzed by Bayesian latent class model, showing that ELISA using a 1:1:1:1 mixture still maintained high sensitivity (79%) and specificity (88%) as compared with the sensitivity (90%) and specificity (83%) of MAT. Therefore, ELISA using a mixture of these four antigens could be a very useful test for seroprevalence studies.

HITTING the Diagnosis: Testing for Heparin-Induced Thrombocytopenia in Cancer Patients.

To evaluate the use of a pretest probability score (4Ts score) in cancer patients to guide ordering of laboratory screening tests for heparin-induced thrombocytopenia (HIT). A retrospective chart review was conducted for patients (n = 140) in whom laboratory testing for HIT was requested. 4Ts scores were calculated and correlated with heparin-endogenous platelet factor 4 antibody enzyme-linked immunosorbent assay (ELISA) test results. All patients with a high pretest probability of HIT (4Ts score = 6-7) had positive ELISA results, compared to 26.1% of patients with intermediate (4Ts score = 4-5) and 4.3% of patients with low (4Ts score ≤3) pretest probability. No patients with 4Ts scores of 2 or less had positive ELISA results. HIT can be ruled out in cancer patients (negative predictive value and sensitivity = 100%) with low pretest probability, defined by 4Ts scores of 2 or less, significantly reducing the need for laboratory testing in this patient population.

Antibodies anti-trypanosomatides in domestic cats in Paraná: who is at highest risk of infection?

The aim of this study were to detect antibodies anti-Leishmania spp. and anti-Trypanosoma cruzi in two different populations of domestic cats (Felis catus domesticus) from North Paraná referred for surgical castration and to determine which characteristics of the animals studied may be associated with seropositivity. Serum samples from 679 cats were analyzed using enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody test (IFAT) in series. Associations between age, sex, race, year of care and animal group were verified using the simple logistic regression. Percentage of 8.5% (58/679) of cats were positive for Leishmania spp. and 7.6% (51/673) for T. cruzi by the tests ELISA and IFAT. Animals collected by non-governmental animal protection organizations presented more seropositivity for Leishmania spp. (p<0.0001). Results shown that Leishmania spp. and T. cruzi are present in domestic cats in the northern part of the state of Paraná, as well as, owners of non-governmental animal protection organizations may be more exposed to leishmaniasis when compared to other animal owners evaluated in the present study.

Sarcodon imbricatus polysaccharides improve mouse hematopoietic function after cyclophosphamide-induced damage via G-CSF mediated JAK2/STAT3 pathway

Sarcodon imbricatus, a rare medicinal and edible fungus, has various pharmacological bioactivities. We investigated the effects of S. imbricatus polysaccharides (SIPS) on hematopoietic function and identified the underlying mechanisms using in vitro experiments with CHRF, K562, and bone marrow mononuclear cells (BMMNCs) and in vivo experiments with a mouse model of cyclophosphamide-induced hematopoietic dysfunction. We found that SIPS induced proliferation and differentiation of CHRF and K562 cells and upregulated the expression of hematopoietic-related proteins, including p90 ribosomal S6 kinases (RSK1p90), c-Myc, and ETS transcription factor, in the two cell lines. After 28 days of treatment, SIPS enhanced the bodyweight and thymus indices of the mice, alleviated enlargement of the spleen and liver, and contributed to the recovery of peripheral blood to normal levels. More importantly, the percentages of B lymphocytes and hematopoietic stem cells or hematopoietic progenitor cells were significantly elevated in bone marrow. Based on an antibody chip analysis and enzyme-linked immunosorbent assay, SIPS were found to successfully regulate 12 cytokines to healthy levels in serum and spleen. The cytokines included the following: interleukins 1Ra, 2, 3, 4, 5, and 6, tumor necrosis factor α, interferon−γ, granulocyte colony-stimulating factor (G-CSF) and macrophage colony-stimulating factor (M-CSF), C-C motif chemokine1, and monocyte chemoattractant protein−1. Moreover, SIPS upregulated the phosphorylation levels of janus kinase 2 (JAK2) and the signal transducer and activator of transcription 3 (STAT3) in the spleen, and similar results were validated in CHRF cells, K562 cells, and BMMNCs. The data indicate that SIPS activated the JAK2/STAT3 pathway, possibly by interactions among multiple cytokines, particularly G-CSF. We found that SIPS was remarkably beneficial to the bone marrow hematopoietic system, and we anticipate that it could improve myelosuppression induced by long-term radiotherapy or chemotherapy.

Field Efficacy of an Attenuated Infectious Bronchitis Variant 2 Virus Vaccine in Commercial Broiler Chickens.

Egyptian poultry suffer from frequent respiratory disease outbreaks associated with Infectious Bronchitis Virus (IBV) variant 2 strains (Egy/VarII). Different vaccination programs using imported vaccines have failed to protect the flocks from field challenge. Recent studies confirmed a successful protection using homologous strains as live attenuated vaccines. In this study, a newly developed live attenuated IB-VAR2 vaccine representing the GI-23 Middle East IBV lineage was evaluated in day-old commercial broilers in an IBV-endemic area. A commercial broiler flock was vaccinated with the IB-VAR2 vaccine at day-old age followed by IB-H120 at day 16. The vaccinated flock was monitored on a weekly basis till the slaughter age. The health status and growth performance were monitored, and selected viral pathogen real-time RT-PCR (rRT-PCR) detection was conducted on a weekly basis. Finally, the flock was compared to a nearby farm with only the classical IB-H120 vaccination program. Results showed that the IB-VAR2 vaccine was tolerable in day-old broiler chicks. The IBV virus rRT-PCR detection was limited to the trachea as compared to its nephropathogenic parent virus. Respiratory disease problems and high mortalities were reported in the IB-H120-only vaccinated flock. An exposure to a wild-type Egy/VarII strain was confirmed in both flocks as indicated by partial IBV S1 gene sequence. Even though the IB-VAR2-vaccinated flock performance was better than the flock that received only IB-H120, the IBV ELISA (enzyme-linked immunosorbent assay) and log2 Haemagglutination inhibition (HI) antibody mean titers remained high (3128 ± 2713 and ≥9 log2, respectively) until the 28th day of age. The current study demonstrates the safety and effectiveness of IB-VAR2 as a live attenuated vaccine in day-old commercial broilers. Also, the combination of IB-VAR2 and classical IBV vaccines confers a broader protective immune response against IBV in endemic areas.

Clinical study of the Liangxue-Zhuyu decoction combined with montelukast in the treatment of henoch schonlein purpura

Objective To study the efficacy of Liangxue-Zhuyu decoction combined with montelukast in the treatment of henoch schonlein purpura (HSP) and its influence on immunologic function. Methods A total of 95 HSP children treated in department of pediatrics from February 2014 to December 2016 were enrolled, and then were randomly divided into observation group (n=48) and control group (n=47), meanwhile another 48 healthy children admitted to a medical examination were selected as the healthy group. The patients in the control group were given the treatment of oral montelukast sodium chewable tablets before sleeping (the dose:2-6 years old 4 mg/d, 6-14 years old 5 mg/d), and the patients in the observation group accepted the therapy of Liangxue-Zhuyu decoction based on the treatment of the control group. The course of the treatment in the two groups were 30d. The time of clinical symptom regression and adverse reactions were recorded, and the clinical efficacy was evaluated. Monoclonal antibody immunofluorescence method and Enzyme-linked immunosorbent assay (ELISA) were used to detect the levels of serum T lymphocyte subsets and INF-γ and IL-10 before and after treatment, and their differences among three groups were compared. Results The effective rates of the observation group was 95.8% (46/48), which was significantly higher than 76.6% (36/47) of control group (χ2=7.441, P<0.05). The subsided time of the symptoms, including purpura (5.0 ± 1.3 d vs. 6.6 ± 2.6 d, t=3.816), abdominal pain (3.1 ± 2.1 d vs. 4.1 ± 2.6 d, t=2.139), joint pain (4.3 ± 1.5 d vs. 5.4 ± 1.9 d, t=3.416), fecal occult blood (4.5 ± 1.5 d vs. 6.1 ± 1.9 d, t=4.404) of the observation group were significantly shorter than those of the control group (P<0.05). After the treatment, the CD4 (45.14% ± 9.32% vs. 41.02% ± 9.97%), CD8 (25.10% ± 4.69% vs. 21.52% ± 5.71%) of the observation group was significantly higher than those of the control group, and the CD4/CD8 (1.62 ± 0.32 vs. 1.95 ± 0.35) and IL-10 (33.07 ± 1.86 pg/ml vs. 53.92 ± 2.98 pg/ml) of the observation group was significantly lower than this of the control group (P<0.05). Conclusions The efficacy of the Liangxue-Zhuyu decoction combined with montelukast for HSP was definite, and the effect was rapid with little side effects. The mechanism might be related to regulating the secretion of T lymphocyte and IL-10, thereby to correct Th1/Th2 cell balance and disturbance of immune function. Key words: Purpura, schoenlein-henoch; Liangxue-Zhuyu decoction; Montelukast sodium chewable tablets; T-Lymphocytes; Interferon-gamma; Interleukin-10

The first 142 amino acids of glutamate decarboxylase do not contribute to epitopes recognized by autoantibodies associated with Type 1 diabetes.

Glutamate decarboxylase (GAD) antibodies are the most widely used predictive marker for Type 1 diabetes, but many individuals currently found to be GAD antibody-positive are unlikely to develop diabetes. We have shown previously that radioimmunoassays using N-terminally truncated 35 S-GAD65 (96-585) offer better disease specificity with similar sensitivity to full-length 35 S-GAD65 (1-585). To determine whether assay performance could be improved further, we evaluated a more radically truncated 35 S-GAD65 (143-585) radiolabel. Samples from people with recent-onset Type 1 diabetes (n = 157) and their first-degree relatives (n = 745) from the Bart's-Oxford family study of childhood diabetes were measured for GAD antibodies using 35 S-labelled GAD65 (143-585). These were screened previously using a local radioimmunoassay with 35 S-GAD65 (1-585). A subset was also tested by enzyme-linked immunosorbent assay (ELISA), which performs well in international workshops, but requires 10 times more serum. Results were compared with GAD antibody measurements using 35 S-GAD65 (1-585) and 35 S-GAD65 (96-585). Sensitivity of GAD antibody measurement was maintained using 35 S-GAD65 (143-585) compared with 35 S-GAD65 (1-585) and 35 S-GAD65 (96-585). Specificity for Type 1 diabetes was improved compared with 35 S-GAD65 (1-585), but was similar to 35 S-GAD65 (96-585). Relatives found to be GAD antibody-positive using these truncated labels were at increased risk of diabetes progression within 15 years, compared with those positive for GAD(1-585) antibody only, and at similar risk to those found GAD antibody-positive by ELISA. The first 142 amino acids of GAD65 do not contribute to epitopes recognized by Type 1 diabetes-associated GAD antibodies. Low-volume radioimmunoassays using N-terminally truncated 35 S-GAD65 are more specific than those using full-length GAD65 and offer practical alternatives to the GAD antibody ELISA for identifying children at increased risk of Type 1 diabetes.

Zinc transporter 8 autoantibody (ZnT8A) by ELISA for diagnosing type 1 diabetes among Chinese people

This study aimed to evaluate the utility of enzyme-linked immunosorbent assay (ELISA) to measure ZnT8A for diagnosing type 1 diabetes among Chinese people. We recruited a group of 95 patients with type 1 diabetes, 130 patients with type 2 diabetes, and 110 subjects without diabetes. We measured ZnT8A level by ELISA and glutamic acid decarboxylase antibody (GADA) level by radioimmunoassay. We collected data on their history-based variables, body mass index (BMI), fasting blood glucose, glycosylated hemoglobin, and lipid levels. 24.2% were positive for ZnT8A in type 1 diabetics, compared to 0.0% in type 2 diabetics and 0.9% in the participants without diabetes (both p < 0.001). And the type 1 diabetics had higher ZnT8A level compared with the latter two groups (both p < 0.001). The frequency of ZnT8A in the “classical” type 1 diabetics was higher than that in patients with latent autoimmune diabetes in adults (45.0 vs. 18.7%, p < 0.05). The frequency/level of ZnT8A was higher in the youngest group (all p < 0.05). The ROC curve area was 0.892. The combination of ZnT8A and GADA increased the diagnostic sensitivity. The ZnT8A level was correlated with the GADA level. ZnT8A-positive type 1 diabetics had younger age at diagnosis (p = 0.022), lower BMI scores (p = 0.016), and more frequent ketosis (p = 0.034) and needed more insulin (p = 0.041) than ZnT8A-negative type 1 diabetics. This study demonstrated the value of ZnT8A in addition to GADA for the diagnosis of type 1 diabetes.

Quantifying malaria endemicity in Ethiopia through combined application of classical methods and enzyme-linked immunosorbent assay: an initial step for countries with low transmission initiating elimination programme

BackgroundIn the context of reduced transmission of malaria, it is essential to re-evaluate and determine the level of transmission as it guides re-orientation of control measures which is appropriate to local disease epidemiology. However, little is known about level of malaria transmission in Ethiopia. The present study aimed to investigate the level of malaria transmission through combined application of classical methods and enzyme-linked immunosorbent assay (EIA) in low transmission settings of Ethiopia.MethodsThis study was conducted in June 2016 on 763 apparently healthy children 2–9 years of age. Children were recruited from ten sites representing different malaria transmission settings in Ethiopia. Splenomegaly rate, infection rate and EIA antibody test were used to determine endemicity. The data were analysed using SPSS 21.0 and Stata 12.0.ResultsThe overall prevalence of malaria parasitaemia was 2.49% (95% CI 1.38–3.59) and 2.36% (95% CI 1.28–3.44) as detected using rapid diagnostic test and microscopy, respectively. Plasmodium falciparum accounted for 62.63% of the infections. The prevalence of parasitaemia significantly varied by altitude and localities; the highest (5.8%) in areas below 1500 m above sea level. Overall, splenomegaly rate was 1.70% (95% CI 0.78–0.2.66%), making the overall malaria transmission hypoendemic. Infection rate was higher among males (2.7%), but rate of splenomegaly was higher in females. Incongruent with spleen rate and parasitaemia, EIA showed a higher level of cumulative exposure to malaria with spatially localized and highly heterogeneous transmission. Overall, 126 (18.75%, 95% CI 15.79–21.71) of the children were positive for total malaria antibodies with significant variations with altitude, age and sex; the higher in areas of < 1500 m asl (25.8%), children ≥ 5 years (22.1%) and among males (20.9%).ConclusionsSplenomegaly and parasitaemia are not good measures to show variations in the levels of malaria transmission in reduced and/or low endemic settings. The malaria antibody (i.e. serological) test seems to be a good measure of malaria endemicity showing greater degree of heterogeneity and localized risk of transmission. Thus, malaria elimination efforts need to be supported with serological indicators to identify patterns of foci of transmission to set priorities for interventions.

S196. ASSOCIATION BETWEEN INTRACELLULAR INFECTIOUS AGENTS AND SCHIZOPHRENIA IN KOREA

BackgroundA number of studies have reported association between Toxoplasma gondii (T. gondii) and Chlamydia infection and the risk of schizophrenia. The aim of the present study was to compare the prevalence of T. gondii and Chlamydia infection between the schizophrenia and normal control subjects and to compare the clinical features between seropositive and seronegative Korean schizophrenia patients.MethodsThe rate of serum reactivity to T. gondii, Chlamydia trachomatis (C. trachomatis), Chlamydia pneumonia in 96 schizophrenia and 50 control subjects was investigated using enzyme-linked immunosorbent assay and indirect fluorescent antibody technique. The clinical symptoms of the schizophrenia patients were scored with Positive and Negative Syndrome Scale and a comparative analysis was carried out.ResultsA significant positive association between immunoglobulin G (IgG) antibodies to T. gondii and C. trachomatis in schizophrenia was found, and the odds ratio of schizophrenia associated with IgG antibody was found to be 3.22 and 2.86, respectively. The Toxoplasma-seropositive schizophrenia patient had higher score on the negative subscale N1 and N7 and general psychopathology subscale G13, while C. trachomatis-seropositive schizophrenia patient had higher score on the general psychopathology subscale G10.DiscussionThe results from the present study suggest significant association between T. gondii, C. trachomatis infection and schizophrenia. In future, further studies are needed to elucidate the correlation between the two types of infection and schizophrenia.

Quantification of Histidine-Rich Protein 3 of Plasmodium falciparum.

Malaria is a life-threatening infectious disease and continues to be a major public health crisis in many parts of the tropical world. Plasmodium falciparum is responsible for the majority of mortality and morbidity associated with malaria. During the intraerythrocytic cycle, P. falciparum releases three proteins with high histidine content as follows: histidine-rich protein 1 (HRP1), histidine-rich protein 2 (HRP2), and histidine-rich protein 3 (HRP3). Currently, most of the diagnostic tests of P. falciparum infection target HRP2, and a number of monoclonal antibodies (mAbs) against HRP2 have been developed for use in HRP2 detection and quantification. When parasites have HRP2 deletions, the detection of HRP3 could augment the sensitivity of the detection system. The combination of both HRP2 and HRP3 mAbs in the detection system will enhance the test sensitivity. In the HRP quantitative enzyme-linked immunosorbent assay (ELISA), both HRP2 and HRP3 contribute to the result, but the relative contribution of HRP2 and HRP3 was unable to investigate, because of the nonavailability of HRP3 specific antibody ELISA. Hence an ELISA test system based on HRP3 is also essential for detection and quantification. There is not much documented in the literature on HRP3 antigen and HRP3 specific mAbs and polyclonal antibodies (pAbs). In the present study, recombinant HRP3 was expressed in Escherichia coli and purified with Ni-NTA agarose column. The purified rHRP3 was used for the generation and characterization of monoclonal and pAbs. The purification of monoclonal and pAbs was done using a mixed-mode chromatography sorbent, phenylpropylamine HyperCel™. With the purified antibodies, a sandwich ELISA was developed. The sandwich ELISA method was explored to detect and quantify HRP3 of P. falciparum in the spent medium. The generated mAbs could be potentially used for the detection and quantification of P. falciparum HRP3.

Biotechnology for obtaining hybrid positive control samples for immunoassay for detecting antibodies against Chlamydia trachomatis

The enzyme-linked immunosorbent assay (ELISA) is the most informative and versatile method of serological diagnostics. The possibility of detection by ELISA of specific antibodies of different classes allow one to differentiate primary infectious processes and their remission, exacerbation and chronic disease (holding of differential diagnosis). This approach is implemented in the methodology for evaluation of patients for the presence of humoral immune response against TORCH-infections pathogens (toxoplasmosis, rubella, cytomegalovirus, herpes simplex viruses infections, and some others). Therefore, testing for the presence of specific IgG and IgM antibodies against TORCH-infection pathogens in blood serum is an important element of mother and child protection. The essential problem in the production of IgM-capture ELISA diagnostic kits is obtaining positive control. The classic version of positive control is human blood serum (plasma) containing specific antibodies. But specific IgM-positive sera are insignificant raw material. This fact can significantly limit the production of diagnostic kits, especially in case of large-scale manufacture. We have suggested a methodological approach to the use of synthetic positive controls in IgM-capture ELISA kits based on conjugate of normal human IgM and monoclonal antibodies against horseradish peroxidase. It was found that it is possible to realize such a task by means of NHS ester-maleimide-mediated conjugation (by sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), reductive amination-mediated conjugation (by sodium periodate) and glutaraldehyde-mediated conjugation. It was found that conjugates of normal human IgM and monoclonal antibodies against horseradish peroxidase obtained using NHS ester-mediated maleimide conjugation and periodate method are homogeneous in molecular weight, whereas conjugate synthesized by glutaraldehyde method comprises at least three types of biopolymers with close molecular weight. It was found that synthetic positive control obtained by different methods are characterized by higher titre compared to IgM-positive high-titre serum. However, positive control obtained by NHS ester-mediated maleimide conjugation has the best titration profile characteristics. We have suggested a methodological approach to the use of synthetic positive controls in indirect ELISA kits based on conjugate of normal human IgM (IgA) and monoclonal antibodies against major outer membrane protein of Ch. trachomatis. It was found that it is possible to realize such task by means of NHS ester-maleimide-mediated conjugation (by sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and reductive amination-mediated conjugation (by sodium periodate). It was found that synthetic positive control obtained by different methods are characterized by higher titre compared to IgM- and IgA-positive high-titre serum. However, positive control obtained by NHS ester-mediated maleimide conjugation has the best titration profile characteristics, both at the release time and after a week’s storage at 37 °C.

Human dental pulp stromal cell conditioned medium alters endothelial cell behavior

BackgroundAngiogenesis is of utmost importance for tissue regeneration and repair. Human dental pulp stromal cells (hDPSCs) possess angiogenic potential, as they secrete paracrine factors that may alter the host microenvironment. However, more insight into how hDPSCs guide endothelial cells (ECs) in a paracrine fashion is yet to be obtained. Therefore, the current study aimed to investigate the effect(s) of conditioned medium derived from hDPSCs (hDPSC-CM) on EC behavior in vitro.MethodshDPSCs were harvested from third molars scheduled for surgical removal under informed consent. The angiogenic profile of hDPSC-CM was identified using human angiogenesis antibody array and enzyme-linked immunosorbent assay (ELISA). Using real-time reverse transcription-polymerase chain reaction (RT-PCR) and ELISA, the mRNA and protein expression level of specific angiogenic biomarkers was determined in human umbilical vein endothelial cells (HUVECs) exposed to hDPSC-CM. The effect of hDPSC-CM on HUVEC attachment, proliferation and migration was evaluated by crystal violet staining, MTT, transwell migration along with real-time cell monitoring assays (xCELLigence; ACEA Biosciences, Inc.). A Matrigel assay was included to examine the influence of hDPSC-CM on HUVEC network formation. Endothelial growth medium (EGM-2) and EGM-2 supplemented with hDPSC-CM served as experimental groups, whereas endothelial basal medium (EBM-2) was set as negative control.ResultsA wide range of proangiogenic and antiangiogenic factors, including vascular endothelial growth factor, tissue inhibitor of metalloproteinase protein 1, plasminogen activator inhibitor (serpin E1), urokinase plasminogen activator and stromal cell-derived factor 1, was abundantly detected in hDPSC-CM by protein profiling array and ELISA. hDPSC-CM significantly accelerated the adhesion phases, from sedimentation to attachment and spreading, the proliferation rate and migration of HUVECs as shown in both endpoint assays and real-time cell analysis recordings. Furthermore, Matrigel assay demonstrated that hDPSC-CM stimulated tubulogenesis, affecting angiogenic parameters such as the number of nodes, meshes and total tube length.ConclusionsThe sustained proangiogenic and promaturation effects of hDPSC-CM shown in this in vitro study strongly suggest that the trophic factors released by hDPSCs are able to trigger pronounced angiogenic responses, even beyond EGM-2 considered as an optimal culture condition for ECs.

Interleukin-38 expression and clinical significance in serum of patients with chronic obstructive pulmonary disease

Objective: To investigate the serum level of interleukin-38 (IL-38) and its clinical significance in patients with chronic obstructive pulmonary disease (COPD). Methods: Totally 72 patients with acute exacerbation of COPD (AECOPD group) and 65 patients with stable COPD (S-COPD group) were recruited from Tianjin Chest Hospital from June 2016 to August 2017. In the same period 40 elderly healthy subjects were selected as control group (C group). The general data and laboratory examination results of these subjects were recorded. Serum IL-38 was measured by double antibody enzyme-linked immunosorbent assays (ELISA). The inter-group differences of above parameters were analyzed. Pearson correlation or Spearman rank correlation analysis was used to analyze the correlation between IL-38 and each variable, and multivariate stepwise regression analysis was used to determine the influencing factors of serum IL-38 in COPD patients. Results: The serum level of IL-38 was higher in AECOPD group than in S-COPD group[(57.88±13.72) vs (51.75±14.06) ng/L, P<0.05], and was higher in either of the two COPD groups than in C group[(46.37±13.18) ng/L](both P<0.05). Correlation analysis of single factor showed that serum IL-38 levels were positively correlated with body mass index (r=0.190, P<0.05), and negatively correlated with C reactive protein (CRP), fibrinogen (FIB), forced expiratory volume in one second as a percentage of estimated value (FEV1%pred) and the number of acute exacerbations in the past 1 year (r=-0.344, -0.176, -0.195, -0.229, all P<0.05). The CRP level and the number of acute exacerbations in the past 1 year were independent factors affecting the serum level of IL-38 (β=-0.204, -0.183, both P<0.05) in patients with COPD. Conclusion: IL-38 is compensatory increased in serum of patients with COPD and may be used as one of the serological markers for evaluation of COPD.

Epitope-Specific Humoral Responses to Human Cytomegalovirus Glycoprotein-B Vaccine With MF59: Anti-AD2 Levels Correlate With Protection From Viremia.

The human cytomegalovirus (HCMV) virion envelope protein glycoprotein B (gB) is essential for viral entry and represents a major target for humoral responses following infection. Previously, a phase 2 placebo-controlled clinical trial conducted in solid organ transplant candidates demonstrated that vaccination with gB plus MF59 adjuvant significantly increased gB enzyme-linked immunosorbent assay (ELISA) antibody levels whose titer correlated directly with protection against posttransplant viremia. The aim of the current study was to investigate in more detail this protective humoral response in vaccinated seropositive transplant recipients. We focused on 4 key antigenic domains (AD) of gB (AD1, AD2, AD4, and AD5), measuring antibody levels in patient sera and correlating these with posttransplant HCMV viremia. Vaccination of seropositive patients significantly boosted preexisting antibody levels against the immunodominant region AD1 as well as against AD2, AD4, and AD5. A decreased incidence of viremia correlated with higher antibody levels against AD2 but not with antibody levels against the other 3 ADs. Overall, these data support the hypothesis that antibodies against AD2 are a major component of the immune protection of seropositives seen following vaccination with gB/MF59 vaccine and identify a correlate of protective immunity in allograft patients.