Search results for: antigen capture ELISA

Authors: Azizollah Khodakaram- Tafti, Ali Mohammadi, Ghasem Farjanikish

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Bovine viral diarrhea virus (BVDV) is one of the most important viral pathogens of cattle worldwide caused by Pestivirus genus, Flaviviridae family.The aim of the present study was to comparison several diagnostic methods and determine the prevalence of BVDV infection for the first time in dairy herds of Fars province, Iran. For initial screening, a total of 400 blood samples were randomly collected from 12 industrial dairy herds and analyzed using reverse transcription (RT)-PCR on the buffy coat. In the second step, blood samples and also ear notch biopsies were collected from 100 cattle of infected farms and tested by antigen capture ELISA (ACE), RT-PCR and immunohistochemistry (IHC). The results of nested RT-PCR (outer primers 0I100/1400R and inner primers BD1/BD2) was successful in 16 out of 400 buffy coat samples (4%) as acute infection in initial screening. Also, 8 out of 100 samples (2%) were positive as persistent infection (PI) by all of the diagnostic tests similarly including RT-PCR, ACE and IHC on buffy coat, serum and skin samples, respectively. Immunoreactivity for bovine BVDV antigen as brown, coarsely to finely granular was observed within the cytoplasm of epithelial cells of epidermis and hair follicles and also subcutaneous stromal cells. These findings confirm the importance of monitoring BVDV infection in cattle of this region and suggest detection and elimination of PI calves for controlling and eradication of this disease.

Keywords: antigen capture ELISA, bovine viral diarrhea virus, immunohistochemistry, RT-PCR, cattle

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Authors: Ibrahim Aly, Manal Ahmed, Mahmoud M. El-Shall

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Filariasis is a parasitic disease caused by small roundworms. The filarial worms are transmitted and spread by blood-feeding black flies and mosquitoes. Lymphatic filariasis (Elephantiasis) is caused by Wuchereriabancrofti, Brugiamalayi, and Brugiatimori. Elimination of Lymphatic filariasis necessitates an increasing demand for valid, reliable, and rapid diagnostic kits. Nanodiagnostics involve the use of nanotechnology in clinical diagnosis to meet the demands for increased sensitivity, specificity, and early detection in less time. The aim of this study was to evaluate the nano-based enzymelinked immunosorbent assay (ELISA) and novel nano-chip card as a rapid test for detection of filarial antigen in serum samples of human filariasis in comparison with traditional -ELISA. Serum samples were collected from an infected human with filarial gathered across Egypt's governorates. After receiving informed consenta total of 45 blood samples of infected individuals residing in different villages in Gharbea governorate, which isa nonendemic region for bancroftianfilariasis, healthy persons living in nonendemic locations (20 persons), as well as sera from 20 other parasites, affected patients were collected. The microfilaria was checked in thick smears of 20 µl night blood samples collected during 20-22 hrs. All of these individuals underwent the following procedures: history taking, clinical examination, and laboratory investigations, which included examination of blood samples for microfilaria using thick blood film and serological tests for detection of the circulating filarial antigen using polyclonal antibody- ELISA, nano-based ELISA, and nano-chip card. In the present study, a recently reported polyoclonal antibody specific to tegumental filarial antigen was used in developing nano-chip card and nano-ELISA compared to traditional ELISA for the detection of circulating filarial antigen in sera of patients with bancroftianfilariasis. The performance of the ELISA was evaluated using 45 serum samples. The ELISA was positive with sera from microfilaremicbancroftianfilariasis patients (n = 36) with a sensitivity of 80 %. Circulating filarial antigen was detected in 39/45 patients who were positive for circulating filarial antigen using nano-ELISA with a sensitivity of 86.6 %. On the other hand, 42 out of 45 patients were positive for circulating filarial antigen using nano-chip card with a sensitivity of 93.3%.In conclusion, using a novel nano-chip assay could potentially be a promising alternative antigen detection test for bancroftianfilariasis.

Keywords: lymphatic filariasis, nanotechnology, rapid diagnosis, elisa technique

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Authors: Mohamed A. Alhammad, Hadia A. Abou-Donia, Mona H. Hashish, Mohamed N. Massoud

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Background: Rotavirus is the leading etiologic agent of severe diarrheal disease in infants and young children worldwide. The present study was aimed 1) to detect rotavirus infection as a cause of diarrhoea among children under 5 years of age using the two serological methods (ELISA and LA) and the PCR technique (2) to evaluate the three methodologies used for human RV detection in stool samples. Materials and Methods: This study was carried out on 247 children less than 5 years old, diagnosed clinically as acute gastroenteritis and attending Alexandria University Children Hospital at EL-Shatby. Rotavirus antigen was screened by ELISA and LA tests in all stool samples, whereas only 100 samples were subjected to RT-PCR method for detection of rotavirus RNA. Results: Out of the 247 studied cases with diarrhoea, rotavirus antigen was detected in 83 (33.6%) by ELISA and 73 (29.6%) by LA, while the 100 cases tested by RT-PCR showed that 44% of them had rotavirus RNA. Rotavirus diarrhoea was significantly presented with a marked seasonal peak during autumn and winter (61.4%). Conclusion: The present study confirms the huge burden of rotavirus as a major cause of acute diarrhoea in Egyptian infants and young children. It was concluded that; LA is equal in sensitivity to ELISA, ELISA is more specific than LA, and RT-PCR is more specific than ELISA and LA in diagnosis of rotavirus infection.

Keywords: rotavirus, diarrhea, immunoenzyme techniques, latex fixation tests, RT-PCR

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Authors: Manal Kamel, Sara Maher

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Development of sensitive non-invasive tests for detection of SARS-CoV-2 antigens is imperative to manage the extent of infection throughout the population, yet, it is still challenging. Here, we designed and optimized a sandwich enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2 S1 antigen detection in saliva. Both saliva samples and nasopharyngeal swapswere collected from 170 PCR-confirmed positive and negative cases. Gold nanoparticles (AuNPs) were conjugated with S1protein receptor binding domain (RBD) nanobodies. Recombinant S1 monoclonal antibodies (S1mAb) as primery antibody and gold conjugated nanobodies as secondary antibody were employed in sandwich ELISA. Our developed system were optimized to achieve 87.5 % sensitivity and 100% specificity for saliva samples compared to 89 % and 100% for nasopharyngeal swaps, respectively. This means that saliva could be a suitable replacement for nasopharyngeal swaps No cross reaction was detected with other corona virus antigens. These results revealed that our developed ELISAcould be establishedas a new, reliable, sensitive, and non-invasive test for diagnosis of SARS-CoV-2 infection, using the easily collected saliva samples.

Keywords: COVID 19, diagnosis, ELISA, nanobodies

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Authors: Manal M. Kame, Hanan G. El-Baz, Zeinab A.Demerdash, Engy M. Abd El-Moneem, Mohamed A. Hendawy, Ibrahim R. Bayoumi

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There is a constant need to improve the performance of current diagnostic assays of schistosomiasis as well as develop innovative testing strategies to meet new testing challenges. This study aims at increasing the diagnostic efficiency of monoclonal antibody (MAb)-based antigen detection assays through gold nanoparticles conjugated with specific anti-Schistosoma mansoni monoclonal antibodies. In this study, several hybidoma cell lines secreting MAbs against adult worm tegumental Schistosoma antigen (AWTA) were produced at Immunology Department of Theodor Bilharz Research Institute and preserved in liquid nitrogen. One MAb (6D/6F) was chosen for this study due to its high reactivity to schistosome antigens with highest optical density (OD) values. Gold nanoparticles (AuNPs) were functionalized and conjugated with MAb (6D/6F). The study was conducted on serum samples of 116 subjects: 71 patients with S. mansoni eggs in their stool samples group (gp 1), 25 with other parasites (gp2) and 20 negative healthy controls (gp3). Patients in gp1 were further subdivided according to egg count in their stool samples into Light infection {≤ 50 egg per gram(epg) (n= 17)}, moderate {51-100 epg (n= 33)} and severe infection {>100 epg(n= 21)}. Sandwich ELISA was performed using (AuNPs -MAb) for detection of circulating schistosomal antigen (CSA) levels in serum samples of all groups and the results were compared with that after using MAb/ sandwich ELISA system. Results Gold- MAb/ ELISA system reached a lower detection limit of 10 ng/ml compared to 85 ng/ml on using MAb/ ELISA and the optimal concentrations of AuNPs -MAb were found to be 12 folds less than that of MAb/ ELISA system for detection of CSA. The sensitivity and specificity of sandwich ELISA for detection of CSA levels using AuNPs -MAb were 100% & 97.8 % respectively compared to 87.3% &93.38% respectively on using MAb/ ELISA system. It was found that CSA was detected in 9 out of 71 S.mansoni infected patients on using AuNPs - MAb/ ELISA system and was not detected by MAb/ ELISA system. All those patients (9) was found to have an egg count below 50 epg feces (patients with light infections). ROC curve analyses revealed that sandwich ELISA using gold-MAb was an excellent diagnostic investigator that could differentiate Schistosoma patients from healthy controls, on the other hand it revealed that sandwich ELISA using MAb was not accurate enough as it could not recognize nine out of 71 patients with light infections. Conclusion Our data demonstrated that: Loading gold nanoparticles with MAb (6D/6F) increases the sensitivity and specificity of sandwich ELISA for detection of CSA, thus active (early) and light infections could be easily detected. Moreover this binding will decrease the amount of MAb consumed in the assay and lower the coast. The significant positive correlation that was detected between ova count (intensity of infection) and OD reading in sandwich ELISA using gold- MAb enables its use to detect the severity of infections and follow up patients after treatment for monitoring of cure.

Keywords: Schistosomiasis, nanoparticles, gold, monoclonal antibodies, ELISA

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Authors: Ibrahim Aly, Waleed Elawamy, Hanan Taher, Amira Matar

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Schistosomiasis is infection with blood flukes of the genus Schistosoma, which are acquired trans-cutaneously by swimming or wading in contaminated freshwater. The present study was proposed to produce ultra-sensitive, field-friendly high-throughput rapid immunochromatography diagnostic device for accurate detection of asymptomatic parasite carriers in schistosomiasis pre-elimination settings.For assessing diagnostic potential of rapid device, 50 blood samples from patients with schistosomiasis mansoni, 29 other proven parasitic diseases and 25 blood samples as negative control were from healthy individuals were used. The sensitivity of Quantitative antigen-capture nano-ELISAwas 82 %, and specificity was 87.1 %, where the sensitivity of Nano Dot- ELISA was 86 % and specificity was 90.7 %. The sensitivity of diagnostic device was 78 % and specificity was 85.2 %, with PPV and NPV of 86.2 % and 83.1 %, respectively.The Point of care device resulted in a good performance for the diagnosis of low-intensity infections, it was able to identify 19 out of 25 (76 %) individuals with ⩽7 eggs, 10 out of 14 individuals (71.4 %) with 11–99 eggs and 100 % of individuals with 100–399 eggs.

Keywords: schistosomiasis, immunochromatography, naon-dot-ELISa, diagnostis device

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Authors: Ibrahim Aly, Rabab Zalat, Bahaa EL Deen W. El Aswad, Ismail M. Moharm, Basam M. Masoud, Tarek Diab

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The objective of the present study was to evaluate the novel nanomagnetic beads based–latex agglutination assay (NMB-LAT) as a simple test for diagnosis of S. haematobium as well as standardize the novel nanomagnetic beads based –ELISA (NMB-ELISA). According to urine examination this study included 85 S. haematobium infected patients, 30 other parasites infected patients and 25 negative control samples. The sensitivity of novel NMB-LAT was 82.4% versus 96.5% and 88.2% for NMB-ELISA and currently used sandwich ELISA respectively. The specificity of NMB-LAT was 83.6% versus 96.3% and 87.3% for NMB-ELISA and currently used sandwich ELISA respectively. In conclusion, the novel NMB-ELISA is a valuable applicable diagnostic technique for diagnosis of human schistosomiasis haematobium. The novel NMB-ELISA assay is a suitable applicable diagnostic method in field survey especially when followed by ELISA as a confirmatory test in query false negative results. Trials are required to increase the sensitivity and specificity of NMB-ELISA assay.

Keywords: diagnosis, iatex agglutination, nanomagnetic beads, sandwich ELISA

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Authors: Zatovska T. V., Nesterova N. V., Baranova G. V., Zagorodnya S. D.

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In recent years, biosensor technologies based on the phenomenon of surface plasmon resonance (SPR) are becoming increasingly used in biology and medicine. Their application facilitates exploration in real time progress of binding of biomolecules and identification of agents that specifically interact with biologically active substances immobilized on the biosensor surface (biochips). Special attention is paid to the use of Biosensor analysis in determining the antibody-antigen interaction in the diagnostics of diseases caused by viruses and bacteria. According to WHO, the diseases that are caused by the herpes simplex virus (HSV), take second place (15.8%) after influenza as a cause of death from viral infections. Current diagnostics of HSV infection include PCR and ELISA assays. The latter allows determination the degree of immune response to viral infection and respective stages of its progress. In this regard, the searches for new and available diagnostic methods are very important. This work was aimed to develop Biosensor chip for detection of specific antibodies to HSV-1 in the human blood serum. The proteins of HSV1 (strain US) were used as antigens. The viral particles were accumulated in cell culture MDBK and purified by differential centrifugation in cesium chloride density gradient. Analysis of the HSV1 proteins was performed by polyacrylamide gel electrophoresis and ELISA. The protein concentration was measured using De Novix DS-11 spectrophotometer. The device for detection of antigen-antibody interactions was an optoelectronic two-channel spectrometer ‘Plasmon-6’, using the SPR phenomenon in the Krechman optical configuration. It was developed at the Lashkarev Institute of Semiconductor Physics of NASU. The used carrier was a glass plate covered with 45 nm gold film. Screening of human blood serums was performed using the test system ‘HSV-1 IgG ELISA’ (GenWay, USA). Development of Biosensor chip included optimization of conditions of viral antigen sorption and analysis steps. For immobilization of viral proteins 0.2% solution of Dextran 17, 200 (Sigma, USA) was used. Sorption of antigen took place at 4-8°C within 18-24 hours. After washing of chip, three times with citrate buffer (pH 5,0) 1% solution of BSA was applied to block the sites not occupied by viral antigen. It was found direct dependence between the amount of immobilized HSV1 antigen and SPR response. Using obtained biochips, panels of 25 positive and 10 negative for the content of antibodies to HSV-1 human sera were analyzed. The average value of SPR response was 185 a.s. for negative sera and from 312 to. 1264 a.s. for positive sera. It was shown that SPR data were agreed with ELISA results in 96% of samples proving the great potential of SPR in such researches. It was investigated the possibility of biochip regeneration and it was shown that application of 10 mM NaOH solution leads to rupture of intermolecular bonds. This allows reuse the chip several times. Thus, in this study biosensor chip for detection of specific antibodies to HSV1 was successfully developed expanding a range of diagnostic methods for this pathogen.

Keywords: biochip, herpes virus, SPR

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Authors: Ping-Lun Jiang, Hung-Jun Lin, Shen-Fu Lin, Mei-Yin Chien, Ting-Wei Li, Chun-Han Lin, Der-Zen Liu

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Mucosal vaccine induces both mucosal (secretory IgA) and systemic immune responses and it is considered an ideal vaccination strategy for prevention of infectious diseases. One important point to be considered in mucosal vaccination is effective antigen delivery system which can manage effective delivery of antigen to antigen-presenting cells (APCs) of mucosal. In the present study, cationic gelatin nanoparticles were prepared as ideal carriers for more efficient antigen delivery. The average diameter of cationic gelatin nanoparticle was approximate 190 nm, and the zeta potential was about +45 mV, then ovalbumin (OVA) was physically absorbed onto cationic gelatin nanoparticle. The OVA absorption rate was near 95% the zeta potential was about +20 mV. We show that cationic gelatin nanoparticle effectively facilitated antigen uptake by mice bone marrow-derived dendritic cells (mBMDCs) and RAW264.7 cells and induced higher levels of pro-inflammatory cytokines. C57BL/6 mice twice immunized intranasally with OVA-absorbed cationic gelatin nanoparticle induced high levels of OVA-specific IgG in the serum and IgA in their in the nasal and lung wash fluid. These results indicate that nasal administration of cationic gelatin nanoparticles induced both mucosal and systemic immune responses and cationic gelatin nanoparticles might be a potential antigen delivery carrier for further clinical applications.

Keywords: antigen delivery, antigen-presenting cells, gelatin nanoparticle, mucosal vaccine

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Authors: Bongmun Kang, Kagnari Yamakawa, Yoshihisa Hagihara, Yuji Ito, Michimasa Kishimoto, Yoichi Kumada

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The PMMA-tag was genetically fused with the C-terminal region of VHH molecules. This antibody, VHH, is known as a single-chain domain, which is devoid of light chains. The PMMA-tag, which could affect the isoelectric point (pI) changeable with a charge of amino acid in VHHs were closely related to the solubility of VHH molecules during refolding. The genetic fusion of PMMA-tag to C-terminal region of VHHs significantly affects the recovery of their soluble protein during refolding by 50 mM TAPS at pH 8.5. It could be refolded with a recovery of more than 95% by dialysis at pH 8.5. A marked difference in the antigen-binding activities in the adsorption state was significantly high in VHH-PM compared to the wild type of VHH. There are approximately 8-fold differences in the antigen-binding activities in the adsorption state between VHH-PM and VHH.

Keywords: VHH, PMMA-tag, isoelectric point, pH, Solubility, refolding, immobilization, ELISA

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Authors: Meisam Omidi, M. Mirijalili, Mohammadmehdi Choolaei, Z. Sharifi, F. Haghiralsadat, F. Yazdian

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In this work, we have used arrays of micromechanical piezoresistive cantilever with different geometries to detect carcinoembryonic antigen (CEA), which is known as an important biomarker associated with various cancers such as the colorectal, lung, breast, pancreatic, and bladder cancer. The sensing principle is based on the surface stress changes induced by antigen–antibody interaction on the microcantilevers surfaces. Different concentrations of CEA in a human serum albumin (HSA) solution were detected as a function of the deflection of the beams. According to the experiments, it was revealed that microcantilevers have surface stress sensitivities in the order of 8 (mJ/m). This matter allows them to detect CEA concentrations as low as 3 ng/mL or 18 pM. This indicates the fact that the self-sensing microcantilever approach is beneficial for pathological tests.

Keywords: micromechanical biosensors, carcinoembryonic antigen (CEA), surface stress

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Authors: Chang, Ya-Ju, Hsieh, Pei-Hsin, Wu, Jui-Chuang, Chen-Yang, Yui Whei

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A mesoporous silica material was prepared to apply to the lateral-flow immunochromatography for detecting a model biosample. The probe antibody is immobilized on the silica surface as the test line to capture its affinity antigen, which laterally flows through the chromatography strips. The antigen is labeled with nano-gold particles, such that the detection can be visually read out from the test line without instrument aids. The result reveals that the mesoporous material provides a vast area for immobilizing the detection probes. Biosening surfaces corresponding with a positive proportion of detection signals is obtained with the biosample loading.

Keywords: mesoporous silica, immunochromatography, lateral-flow strips, biosensors, nano-gold particles

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Authors: Yuriy A. Knirel

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Enteric bacteria Escherichia coli is the predominant facultative anaerobe of the colonic flora, and some specific serotypes are associated with enteritis, hemorrhagic colitis, and hemolytic uremic syndrome. Shigella spp. are human pathogens that cause diarrhea and bacillary dysentery (shigellosis). They are in effect E. coli with a specific mode of pathogenicity. Strains of Salmonella enterica are responsible for a food-borne infection (salmonellosis), and specific serotypes cause typhoid fever and paratyphoid fever. All these bacteria are closely related in respect to structure and genetics of the lipopolysaccharide, including the O-polysaccharide part (O‑antigen). Being exposed to the bacterial cell surface, the O antigen is subject to intense selection by the host immune system and bacteriophages giving rise to diverse O‑antigen forms and providing the basis for typing of bacteria. The O-antigen forms of many bacteria are unique, but some are structurally and genetically related to others. The sequenced O-antigen gene clusters between conserved galF and gnd genes were analyzed taking into account the O-antigen structures established by us and others for all S. enterica and Shigella and most E. coli O-serogroups. Multiple genetic mechanisms of diversification of the O-antigen forms, such as lateral gene transfer and mutations, were elucidated and are summarized in the present paper. They include acquisition or inactivation of genes for sugar synthesis or transfer or recombination of O-antigen gene clusters or their parts. The data obtained contribute to our understanding of the origins of the O‑antigen diversity, shed light on molecular evolutionary relationships between the O-antigens of enteric bacteria, and open a way for studies of the role of gene polymorphism in pathogenicity.

Keywords: enteric bacteria, O-antigen gene cluster, polysaccharide biosynthesis, polysaccharide structure

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Authors: Nermin Isik, Ozlem Derinbay Ekici, Oguzhan Avci

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The aim of this study was to determination of role infection in neonatal calves in Central Anatolian, Turkey. A total 300 fecal samples were collected from diarrheic neonatal calves, aged between 0–90 days from Konya, Karaman, and Aksaray from January to April 2014. Fecal specimens from calves with clinically diarrheic symptoms were examined for the presence of Bovine Coronavirus, Bovine Rotavirus, Cryptosporidium sp., and E. coli by commercially available capture direct enzyme linked immunosorbent assay (ELISA) kit and Modified Ziehl Neelsen method (MZN). Calves were grouped according to their age as follows: 1-14, 15-29, and 30-90 days. Cryptosporidium sp. infection was detected in 52.8%, 58.8%, and 39.2% by ELISA and 33.9%, 47%, 26.7% by MZN in the respective age groups. The seroprevalance of Rotavirus (12.5 %, 40 %, 12.5 %), Coronavirus (2.5%, 0%, 3.5%) and E. coli (5%, 4.7%, 8.9%) infections were determined according to the age groups respectively. Cryptosporidium sp. was the most detected enteropathogen (52 %) of calves and coronavirus was the least detected (2 %). The detection rate of the mixed enfection was 12.3%. In conclusion, it must be evaluated by mix infections in calves with diarrhea. These results will provide an important contribution against the factors that cause diarrhea

Keywords: cryptosporidium sp., bovine coronavirus, bovine rotavirus, E.coli, calve, ELISA

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Authors: Maria Margaretha, Dana Indra Sensuse, Lukman

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Knowledge capture is a step in knowledge life cycle to get knowledge in the organization. Tacit and explicit knowledge are needed to organize in a path, so the organization will be easy to choose which knowledge will be use. There are many challenges to capture knowledge in the organization, such as researcher must know which knowledge has been validated by an expert, how to get tacit knowledge from experts and make it explicit knowledge, and so on. Besides that, the technology will be a reliable tool to help the researcher to capture knowledge. Some paper wrote how ontology in knowledge management can be used for proposed framework to capture and reuse knowledge. Organization has to manage their knowledge, process capture and share will decide their position in the business area. This paper will describe further from literature review about the tool of ontology that will help the organization to capture its knowledge.

Keywords: knowledge capture, ontology, technology, organization

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Authors: Azad A. Meerkhan, Alaa Hani Razak, Bayan M. S. Younis

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The efficiency of enzyme-linked immunosorbent assay (ELISA) in sheep infected with Fasciola hepatica was studied. 232 jaundiced sheep among 5208 sheep slaughter in the Duhok abattoir (regardless of the age and gender) between the period of May. 2012 to Oct. 2012 were examined by direct examination (Searching of adult flukes in the bile duct) and by Enzyme-linked immunosorbent assay (ELISA) to detect the prevalence of fascioliasis in the studied population which showed a high observed infection ratio in Sep. 2012 (12.2%) with the high (ELISA) result of infection in May. 2012 (25.36%). Significant differences were found between the two ways in all of the months with the highest difference in May. 2012 and the net deference between the both ways was 6.91%.

Keywords: fascioliasis, Fasciola hepatica, layers, liver fluk, ELISA, direct examination

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Authors: Eric Chabrol, Sidonia B.G. Eckle, Renate de Boer, James McCluskey, Jamie Rossjohn, Mirjam H.M. Heemskerk, Stephanie Gras

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αβ and γδ T-cells are disparate T-cell lineages that, via their use of either αβ or γδ T-cell antigen receptors (TCRs) respectively, can respond to distinct antigens. Here we characterise a new population of human T-cells, term δβ T-cells, that express TCRs comprising a TCR-δ variable gene fused to a Joining-α/Constant-α domain, paired with an array of TCR-β chains. We characterised the cellular, functional, biophysical and structural characteristic feature of this new T-cells population that reveal some new insight into TCR diversity. We provide molecular bases of how δβ T-cells can recognise viral peptide presented by Human Leukocyte Antigen (HLA) molecule. Our findings highlight how components from αβ and γδTCR gene loci can recombine to confer antigen specificity thus expanding our understanding of T-cell biology and TCR diversity.

Keywords: new delta-beta TCR, HLA, viral peptide, structural immunology

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Authors: Wenxiao Liu, Kun Zhang, Yongqing Li

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Bovine herpesvirus 1, a member of the genus Variellovirus of the subfamily Alphaherpesvirinae, has caused severe economic cost to the bovine industry. In this study, BoHV-1 glycerol protein gD was expressed in insect cells, and the purified gD was immunized in the Balb/C mice to generate monoclonal antibodies. Based on hybridoma cell fusion techniques, 20 monoclonal antibodies against Bovine herpesvirus 1 have been obtained. Further, mAb 3F8 with neutralizing activity and gD were applied to develop a blocking enzyme-linked immunosorbent assay (Elisa) for detecting neutralizing antibodies against BoHV-1, which shows a significant correlation between the blocking Elisa and VNT. The sensitivity and specificity of the test were estimated to be 94.59% and 93.42%, respectively. Furthermore, antibody pairing tests revealed that mAb 1B6 conjugated to fluorescence microspheres was used as the capture antibody, and mAb 3F9 was used as the detectable antibody to establish the immunochromatographic assay (ICS). The ICS was conducted to detect BoHV-1 in bovine samples with high sensitivity, specificity, and good stability. Clinical sample testing revealed that the results of ICS and real-time PCR have a coincidence rate of 95.42%. Our research confirmed that the ICS is a rapid and reliable method for the diagnosis of BoHV-1. In conclusion, our results lay a solid foundation for the prevention and control of BoHV-1 infection.

Keywords: bovine disease, BoHV-1, ELISA, ICS assay

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Authors: Piamsiri Sawaisorn, Tienrat Tangchaikeeree, Waraporn Chan-On, Chaniya Leepiyasakulchai, Rachanee Udomsangpetch, Suradej Hongeng, Kulachart Jangpatarapongsa

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Human Vγ9Vδ2 T lymphocytes are regarded as promising effector cells for cancer immunotherapy since they have the ability to eliminate several tumor cells through non-peptide antigen recognition and non-major histocompatibility complex (MHC) restriction. An issue of recent interest is the capability to activate γδ T cells by use of a group of drugs, such as pamidronate, that cause accumulation of phosphoantigen which is recognized by γδ T cell receptors. Moreover, their antigen presenting cell-like phenotype and function have been confirmed in many clinical trials. In this study, Vγ9Vδ2 T cells derived from normal peripheral blood mononuclear cells were activated with pamidronate and the expanded Vγ9Vδ2 T cells can recognize and kill chronic myeloid leukemia (CML) cells treated with pamidronate through their cytotoxic activity. To support the strong role played by Vγ9Vδ2 T cells against cancer, we provide the evidence that Vγ9Vδ2 T cells activated with CML cell lysate antigen can efficiently express antigen presenting cell (APC) phenotype and function. In conclusion, pamidronate can be used in intentional activation of human Vγ9Vδ2 T cells and can increase the susceptibility of CML cells to cytotoxicity of Vγ9Vδ2 T cells. The activated Vγ9Vδ2 T cells by cancer cells lysate can show their APC characteristics, and so greatly increase the interest in exploring their therapeutic potential in hematologic malignancy.

Keywords: γδ T lymphocytes, antigen-presenting cells, chronic myeloid leukemia, cancer, immunotherapy

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Authors: Seyed Mehrzad Pahlavani, Mozaffar Haji Jafari Anaraki, Sayma Mohammadi

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A total of 34700 day-old broilers were exposed to green, blue and yellow light using a light-emitting diode system for 6 weeks to investigate the effects of light wave length on antibody responses to Newcastle disease by HI test and the correlation with ELISA. 3 poultry house broiler farms with the same conditions was selected and the lightening system of each was set according to the requirement. Blood samples were taken from 20 chicks on days 1, 24 and 46 and the Newcastle virus specific antibody was titered in serum using HI an ELISA test. On day 24, the probability value of more than 0/05 was observed in HI and ELISA tests of all groups while at the end of breeding period, the average HI serum antibody titer was more in the green light than the yellow one while the blue light was not significantly different from both. At the last titration, the green light has got the highest titer of Newcastle antibodies. There were no significant differences of Newcastle antibody titers between all groups and ages in broiler pullets in ELISA. According to the sampling and analysis of HI and ELISA serum tests, there were no significant relationships between all broiler pullets breeding in green, blue and yellow light on days 24 and 46 and the P-value was more than 0/05. It is suggested that the monochromatic light is effective on broilers immunity against Newcastle disease.

Keywords: monochromatic light, Newcastle disease, HI test, ELISA test

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Authors: Feixiong Chen, Naoufel Haddour, Marie Frenea-Robin, Yves MéRieux, Yann Chevolot, Virginie Monnier

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Neonatal thrombopenia occurs when the mother generates antibodies against her baby’s platelet antigens. It is particularly critical for newborns because it can cause coagulation troubles leading to intracranial hemorrhage. In this case, diagnosis must be done quickly to make platelets transfusion immediately after birth. Before transfusion, platelet antigens must be tested carefully to avoid rejection. The majority of thrombopenia (95 %) are caused by antibodies directed against Human Platelet Antigen 1a (HPA-1a) or 5b (HPA-5b). The common method for antigen platelets detection is polymerase chain reaction allowing for identification of gene sequence. However, it is expensive, time-consuming and requires significant blood volume which is not suitable for newborns. We propose to develop a point-of-care device based on double functionalized magnetic colloids with 1) antibodies specific to antigen platelets and 2) highly sensitive electroactive molecules in order to be detected by an electrochemical microsensor. These magnetic colloids will be used first to isolate platelets from other blood components, then to capture specifically platelets bearing HPA-1a and HPA-5b antigens and finally to attract them close to sensor working electrode for improved electrochemical signal. The expected advantages are an assay time lower than 20 min starting from blood volume smaller than 100 µL. Our functionalization procedure based on amine dendrimers and NHS-ester modification of initial carboxyl colloids will be presented. Functionalization efficiency was evaluated by colorimetric titration of surface chemical groups, zeta potential measurements, infrared spectroscopy, fluorescence scanning and cyclic voltammetry. Our results showed that electroactive molecules and antibodies can be immobilized successfully onto magnetic colloids. Application of a magnetic field onto working electrode increased the detected electrochemical signal. Magnetic colloids were able to capture specific purified antigens extracted from platelets.

Keywords: Magnetic Nanoparticles , Electroactive Molecules, Antibody, Platelet

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Authors: Andreas Latz, Daniela Heinz, Fatima Hashemi, Melek Baygül

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Introduction: Feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), and feline coronavirus (FCoV) are serious infectious diseases affecting cats worldwide. Transmission of these viruses occurs primarily through close contact with infected cats (via saliva, nasal secretions, faeces, etc.). FeLV, FIV, and FCoV infections can occur in combination and are expressed in similar clinical symptoms. Diagnosis can therefore be challenging: Symptoms are variable and often non-specific. Sick cats show very similar clinical symptoms: apathy, anorexia, fever, immunodeficiency syndrome, anemia, etc. Sample volume for small companion animals for diagnostic purposes can be challenging to collect. In addition, multiplex diagnosis of diseases can contribute to an easier, cheaper, and faster workflow in the lab as well as to the better differential diagnosis of diseases. For this reason, we wanted to develop a new diagnostic tool that utilizes less sample volume, reagents, and consumables than multiplesingleplex ELISA assays Methods: The Multiplier from Dynextechonogies (USA) has been used as platform to develop a Multiplex diagnostic tool for the detection of antibodies against FIV and FCoV/FIP and antigens for FeLV. Multiplex diagnostics. The Dynex®Multiplier®is a fully automated chemiluminescence immunoassay analyzer that significantly simplifies laboratory workflow. The Multiplier®ease-of-use reduces pre-analytical steps by combining the power of efficiently multiplexing multiple assays with the simplicity of automated microplate processing. Plastic beads have been coated with antigens for FIV and FCoV/FIP, as well as antibodies for FeLV. Feline blood samples are incubated with the beads. Read out of results is performed via chemiluminescence Results: Bead coating was optimized for each individual antigen or capture antibody and then combined in the multiplex diagnostic tool. HRP: Antibody conjugates for FIV and FCoV antibodies, as well as detection antibodies for FeLV antigen, have been adjusted and mixed. 3 individual prototyple batches of the assay have been produced. We analyzed for each disease 50 well defined positive and negative samples. Results show an excellent diagnostic performance of the simultaneous detection of antibodies or antigens against these feline diseases in a fully automated system. A 100% concordance with singleplex methods like ELISA or IFA can be observed. Intra- and Inter-Assays showed a high precision of the test with CV values below 10% for each individual bead. Accelerated stability testing indicate a shelf life of at least 1 year. Conclusion: The new tool can be used for multiplex diagnostics of the most important feline infectious diseases. Only a very small sample volume is required. Fully automation results in a very convenient and fast method for diagnosing animal diseases.With its large specimen capacity to process over 576 samples per 8-hours shift and provide up to 3,456 results, very high laboratory productivity and reagent savings can be achieved.

Keywords: Multiplex, FIV, FeLV, FCoV, FIP

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Authors: Albina V. Moskvicheva, Gevorg G. Kazarian, Anna R. Valeeva, Marina A. Efimova, Malik N. Mukminov, Eduard A. Shuralev, Rustam Kh. Ravilov, Kamil S. Khaertynov

Abstract:

Improving the early diagnosis of tuberculosis and solving a number of problems associated with the differential diagnosis of Mycobacterium bovis infection, nonspecific tuberculin reactions caused by sensitization of the body by non-tuberculosis mycobacteria, is urgent. The filtrates and extracts of M. bovis cell surface components are promising antigens with diagnostic potential. The purpose of this study was to isolate and characterize antigenic proteins and determine the dominant M. bovis antigens recognized by the humoral immune system. The mycobacterial cells were homogenized on FastPrep-24. Gel-filtration chromatography was used to fractionate the lysates of cell surface component extracts and proteins isolated from M. bovis culture supernatant. The separated fractions were analyzed using two-dimensional gel electrophoresis followed by determination of antigen serological activity using immunoblot with specific hyperimmune rabbit blood serum. As a result of electrophoretic separation of components by molecular weight, 23 antigen fractions were obtained. Analysis of densitograms showed that the fractions contained two zones of antigens with pronounced serological activity, corresponding to molecular weights of 28 and 21 kDa. The high serological activity of the 28 kDa antigen was established by immunoblot using hyperimmune blood sera. Separated and characterized by M. bovis specific antigen with a molecular weight of 28 kDa was added to the collection of specific marker antigens for M. bovis.

Keywords: antigen, gel-filtration chromatography, immunoblot, Mycobacterium bovis

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Authors: Zuzana Chaloupková, Zdeňka Marková, Václav Ranc, Radek Zbořil

Abstract:

Prostate cancer is now one of the most serious oncological diseases in men with an incidence higher than that of all other solid tumors combined. Diagnosis of prostate cancer usually involves detection of related genes or detection of marker proteins, such as PSA. One of the new potential markers is PSMA (prostate specific membrane antigen). PSMA is a unique membrane bound glycoprotein, which is considerably overexpressed on prostate cancer as well as neovasculature of most of the solid tumors. Commonly applied methods for a detection of proteins include techniques based on immunochemical approaches, including ELISA and RIA. Magnetically assisted surface enhanced Raman spectroscopy (MA-SERS) can be considered as an interesting alternative to generally accepted approaches. This work describes a utilization of MA-SERS in a detection of PSMA in human blood. This analytical platform is based on magnetic nanocomposites Fe3O4@Ag, functionalized by a low-molecular selector labeled as JB303. The system allows isolating the marker from the complex sample using application of magnetic force. Detection of PSMA is than performed by SERS effect given by a presence of silver nanoparticles. This system allowed us to analyze PSMA in clinical samples with limits of detection lower than 1 ng/mL.

Keywords: diagnosis, cancer, PSMA, MA-SERS, Ag nanoparticles

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Authors: Rizky Kusuma Cahyani

Abstract:

Hepatitis B is a liver inflammatory disease caused by hepatitis B virus (HBV). This infection can be prevented by vaccination which contains HBV surface protein (sHBsAg). However, vaccine supply is limited. Several attempts have been conducted to produce local sHBsAg. However, the purity degree and protein yield are still inadequate. Therefore optimization of HBsAg purification steps is required to obtain high yield with better purification fold. In this study, optimization of purification was done in 2 steps, precipitation using variation of NaCl concentration (0,3 M; 0,5 M; 0,7 M) and PEG (3%, 5%, 7%); ion exchange chromatography (IEC) using NaCl 300-500 mM elution buffer concentration.To determine HBsAg protein, bicinchoninic acid assay (BCA) and enzyme-linked immunosorbent assay (ELISA) was used in this study. Visualization of HBsAg protein was done by SDS-PAGE analysis. Based on quantitative analysis, optimal condition at precipitation step was given 0,3 M NaCl and PEG 3%, while in ion exchange chromatography step, the optimum condition when protein eluted with NaCl 500 mM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicates that the presence of protein HBsAg with a molecular weight of 25 kDa (monomer) and 50 kDa (dimer). The optimum condition for purification of sHBsAg produced in Pichia pastoris gave a yield of 47% and purification fold 17x so that it would increase the production of hepatitis B vaccine to be more optimal.

Keywords: hepatitis B virus, HBsAg, hepatitis B surface antigen, Pichia pastoris, purification

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Authors: Mini P. Singh, Manasi Majumdar, Kapil Goyal, Pvm Lakshmi, Deepak Bhatia, Radha Kanta Ratho

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India is endemic for Hepatitis E virus and frequent water borne outbreaks are reported. The conventional diagnosis rests on the detection of serum anti-HEV IgM antibodies which may take 7-10 days to develop. Early diagnosis in such a situation is desirable for the initiation of prompt control measures. The present study compared three diagnostic methods in 60 samples collected during two suspected HEV outbreaks in the vicinity of Chandigarh, India. The anti-HEV IgM, HEV antigen and HEV-RNA could be detected in serum samples of 52 (86.66%), 16 (26.66%) and 18 (30%) patients respectively. The suitability of saliva samples for antibody detection was also evaluated in 21 paired serum- saliva samples. A total of 15 serum samples showed the presence of anti HEV IgM antibodies, out of which 10 (10/15; 66.6%) were also positive for these antibodies in saliva samples (χ2 = 7.636, p < 0.0057), thus showing a concordance of 76.91%. The positivity of reverse transcriptase PCR and HEV antigen detection was 100% within one week of illness which declined to 5-10% thereafter. The outbreak was attributed to HEV Genotype 1, Subtype 1a and the clinical and environmental strains clustered together. HEV antigen and RNA were found to be an early diagnostic marker with 96.66% concordance. The results indicate that the saliva samples can be used as an alternative to serum samples in an outbreak situation.

Keywords: HEV-antigen, outbreak, phylogenetic analysis, saliva

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Authors: Fatemeh Keshavarz

Abstract:

Background: Schizophrenia is a disease of the nervous system, and immune system disorders can affect its pathogenesis. Activation of microglia, proinflammatory cytokines, disruption of the blood-brain barrier (BBB) due to inflammation, activation of autoreactive B cells, and consequently the production of autoantibodies against system antigens are among the immune processes involved in neurological diseases. interleukin 32 (IL-32) a proinflammatory cytokine that important player in the activation of the innate and adaptive immune responses. This study aimed to measure the serum level of IL-32 as well as the frequency of autoantibody positivity against several nervous system antigens in patients with schizophrenia. Material and Methods: This study was conducted on 40 patients with schizophrenia and 40 healthy individuals in the control group. Serum IL-32 levels were measured by ELISA. The frequency of autoantibodies against Hu, Ri, Yo, Tr, CV2, Amphiphysin, SOX1, Zic4, ITPR1, CARP, GAD, Recoverin, Titin, and Ganglioside antigens were measured by indirect immunofluorescence method. Results: Serum IL-32 levels in patients with schizophrenia were significantly higher compared to the control group. Autoantibodies were positive in 8 patients for GAD antigen and 5 patients for Ri antigen, which showed a significant relationship compared to the control group. Autoantibodies were also positive in 2 patients for CV2, in 1 patient for Hu, and in 1 patient for CARP. Negative results were reported for other antigens. Conclusion: Our findings suggest that elevated the serum IL-32 level and autoantibody positivity against several nervous system antigens may be involved in the pathogenesis of schizophrenia.

Keywords: schizophrenia, microglia, autoantibodies, IL-32

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Authors: Ojo Oo, Akinde S. B., Kiilani A. O., Jayeola Jo, Jogbodo T. M., Ajani Ka, Olaniyan So, Adeagbo Oy, Bolarinwa Ra, Durosomo Ha, Sule W. F.

Abstract:

Lockdown imposed to control SARS-CoV-2 transmission hampered malaria control services in Nigeria. Considering COVID-19 vaccination, we assessed Plasmodium falciparum (Pf) antigen and SARS-CoV-2 immunoglobulin-G (IgG) positivity among adults in Osogbo, Osun State, Nigeria. Consenting attendees of four Healthcare Centres were consecutively enrolled for blood sampling; relevant socio-demographic/behavioral/clinical/environmental data were collected with a questionnaire. Samples were tested, using commercial rapid test kits, for Pf antigen and SARS-CoV-2 IgG and results were analyzed using logistic regression. Participants' mean age was 40.99 years (n=200), and they were predominantly females (84.5%), traders/businessmen/women (86.0%), with self-reported receipt of COVID-19 vaccine from 123 (61.5%). Pf antigen positivity was 17.5% (95% CI: 12.23–22.77%) with age (p=0.004), marital status (p=0.004), report of stagnant water around the workplace (p=0.041) and bush around homes (p=0.008) being associated. SARS-CoV-2 IgG positivity was 56.5% (95% CI: 49.63–63.37%) with age (p=0.012) and receipt of COVID-19 vaccination (p=0.001) being associated. Although the vaccinated had a 22.8 times higher likelihood of IgG positivity, no factor was predictive of COVID-19 vaccine receipt. We report 17.5% Pf antigen positivity with four predictors, and 56.5% SARS-CoV-2 IgG positivity with two predictors.

Keywords: COVID-19, vaccine, IgG, Plasmodium falciparum, SARS-CoV-2

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Authors: Maysam Mard-Soltani, Mohamad Javad Rasaee, Saeed Khalili, Abdol Karim Sheikhi, Mehdi Hedayati

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Production of specific antibody responses against hTSH is a cumbersome process due to the high identity between the hTSH and the other members of the glycoprotein hormone family (FSH, LH and HCG) and the high identity between the human hTSH and host animals for antibody production. Therefore, two polyclonal antibodies were purified against two recombinant proteins. Four possible ELISA tests were designed based on these antibodies. These ELISA tests were checked against hTSH and other glycoprotein hormones, and their sensitivity and specificity were assessed. Bioinformatics tools were used to analyze the immunological properties. After the immunogen region selection from hTSH protein, c terminal of B hTSH was selected and applied. Two recombinant genes, with these cut pieces (first: two repeats of C terminal of B hTSH, second: tetanous toxin+B hTSH C terminal), were designed and sub-cloned into the pET32a expression vector. Standard methods were used for protein expression, purification, and verification. Thereafter, immunizations of the white New Zealand rabbits were performed and the serums of them were used for antibody titration, purification and characterization. Then, four ELISA tests based on two antibodies were employed to assess the hTSH and other glycoprotein hormones. The results of these assessments were compared with standard amounts. The obtained results indicated that the desired antigens were successfully designed, sub-cloned, expressed, confirmed and used for in vivo immunization. The raised antibodies were capable of specific and sensitive hTSH detection, while the cross reactivity with the other members of the glycoprotein hormone family was minimum. Among the four designed tests, the test in which the antibody against first protein was used as capture antibody, and the antibody against second protein was used as detector antibody did not show any hook effect up to 50 miu/l. Both proteins have the ability to induce highly sensitive and specific antibody responses against the hTSH. One of the antibody combinations of these antibodies has the highest sensitivity and specificity in hTSH detection.

Keywords: hTSH, bioinformatics, protein expression, cross reactivity

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Authors: Simin Khodabakhshi, Hossein Rassi

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Hyperlipidemia refers to any of several acquired or genetic disorders that result in a high level of lipids circulating in the blood. Helicobacter pylori infection is a contributing factor in the progression of hyperlipidemia with serum lipid changes. The aim of this study was to detect of Helicobacter pylori by PCR and serological methods in patients with hyperlipidemia. In this case-control study, 174 patients with hyperlipidemia and 174 healthy controls were studied. Also, demographics, physical and biochemical parameters were performed in all samples. The DNA extracted from blood specimens was amplified by H pylori cagA specific primers. The results show that H. pylori cagA positivity was detected in 79% of the hyperlipidemia and in 56% of the control group by ELISA test and 49% of the hyperlipidemia and in 24% of the control group by PCR test. Prevalence of H. pylori infection was significantly higher in hyperlipidemia as compared to controls. In addition, patients with hyperlipidemia had significantly higher values for triglyceride, total cholesterol, LDL-C, waist to hip ratio, body mass index, diastolic and systolic blood pressure and lower levels of HDL-C than control participants (all p < 0.0001). Our result detected the ELISA was a rapid and cost-effective detection and considering the high prevalence of cytotoxigenic H. pylori strains, cag A is suggested as a promising target for PCR and ELISA tests for detection of infection with toxigenic strains. In general, it can be concluded that molecular analysis of H. pylori cagA and clinical parameters are important in early detection of hyperlipidemia and atherosclerosis with H. pylori infection by PCR and ELISA tests.

Keywords: Helicobacter pylori, hyperlipidemia, PCR, ELISA

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