Search results for: polyclonal antibody

Authors: Yu-Hsiang Yu

Abstract:

The immunomodulatory effect of propolis has been widely investigated in the past decade. However, the beneficial effects in broilers are still poorly understood. The aim of this study was to evaluate the effect of propolis added in drinking water on immunomodulatory and antibody production in broiler. Total of 48 chicks were randomly allocated into four groups with 12 broilers per group. All birds were intranasal inoculated with Newcastle Disease vaccine at 4 and 14 days old of age. Four groups, including control without any treatment, groups of A, B and F [3 days of anterior (A), 3 days of posterior (P) and 6 days of full (F)] were supplied the propolis at 300 ppm in drinking water when vaccination was performed, respectively. Our results showed that no significant difference was found in growth performance, antibody production and immune organ index among groups. However, propolis treatments in broilers significantly reduced IL-4 expression in spleen at 14 days-old of age and bursa at 28 days-old of age compared with control group. The expression of IFN-gamma in spleen (A, P and F group) and bursal (F group) were elevated compared with control group at 28 days-old of age. In conclusion, our results indicated that propolis-treated birds could bear the capability for immunomodulatory effects by change Th1 subset cytokine expression in vaccination.

Keywords: propolis, broiler, immunomodulatory, vaccination

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Authors: Christian Fercher, Jiaul Islam, Simon R. Corrie

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Fluorescence-based immunodiagnostics are an emerging field in biosensor development and exhibit several advantages over traditional detection methods. While various affinity biosensors have been developed to generate a fluorescence signal upon sensing varying concentrations of analytes, reagentless, reversible, and continuous monitoring of complex biological samples remains challenging. Here, we aimed to genetically engineer biosensors based on single-chain antibody fragments (scFv) that are site-specifically labeled with environmentally sensitive fluorescent unnatural amino acids (UAA). A rational design approach resulted in quantifiable analyte-dependent changes in peak fluorescence emission wavelength and enabled antigen detection in vitro. Incorporation of a polarity indicator within the topological neighborhood of the antigen-binding interface generated a titratable wavelength blueshift with nanomolar detection limits. In order to ensure continuous analyte monitoring, scFv candidates with fast binding and dissociation kinetics were selected from a genetic library employing a high-throughput phage display and affinity screening approach. Initial rankings were further refined towards rapid dissociation kinetics using bio-layer interferometry (BLI) and surface plasmon resonance (SPR). The most promising candidates were expressed, purified to homogeneity, and tested for their potential to detect biomarkers in a continuous microfluidic-based assay. Variations of dissociation kinetics within an order of magnitude were achieved without compromising the specificity of the antibody fragments. This approach is generally applicable to numerous antibody/antigen combinations and currently awaits integration in a wide range of assay platforms for one-step protein quantification.

Keywords: antibody engineering, biosensor, phage display, unnatural amino acids

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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: Xian-Peng Jiang, Catherine C. Baucom, Toby Jiang, Robert L. Elliott

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HLA-G binds to the inhibitory receptors of uterine NK cells and plays an important role in protection of fetal cells from maternal NK lysis. HLA-G also mediates tumor escape, but the immunosuppressive role is often neglected. These studies have focused on the examination of HLA-G expression in human breast carcinoma and HLA-G immunosuppressive role in NK cytolysis. We examined HLA-G expression in breast cell lines by real time PCR, ELISA and immunofluorescent staining. We treated the breast cancer cell lines with anti-human HLA-G antibody or progesterone. Then, NK cytolysis was measured by using MTT assay. We find that breast carcinoma cell lines increase the expression of HLA-G mRNA and protein, compared to normal cells. Blocking HLA-G of the breast cancer cells by the antibody increases NK cytolysis. Progesterone upregulates HLA-G mRNA and protein of human breast cancer cell lines. The increased HLA-G expression suppresses NK cytolysis. In summary, human breast carcinoma overexpress HLA-G immunosuppressive molecules. Blocking HLA-G protein by antibody improves NK cytolysis. In contrast, upregulation of HLA-G expression by progesterone impairs NK cytolytic function. Thus, HLA-G is a new immunosuppressive checkpoint and potential cancer immunotherapeutic target.

Keywords: HLA-G, Breast carcinoma, NK cells, Immunosuppressive checkpoint

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Authors: Hai Xu, Yiwei Wang, Xi Bao, Bihua Deng, Pengcheng Li, Yu Lu

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T7 phage could be used as a perfect vector for peptides expression and haptens presentation. T7-3GnRH recombinant phage was constructed by inserting three copies of Gonadotrophin Releasing Hormone (GnRH) gene into the multiple cloning site of T7 Select 415-1b phage genome. The positive T7-3GnRH phage was selected by using polymerase chain reaction amplification, and the p10B-3GnRH fusion protein was verified by SDS-PAGE and Western-blotting assay. T7-3GnRH vaccine was made and immunized with 10¹⁰ pfu in 0.2 ml per dose in mice. Blood samples were collected at an interval in weeks, and anti-GnRH antibody and testosterone concentrations were detected by ELISA and radioimmunoassay, respectively. The results show that T7-3GnRH phage particles confer a high immunogenicity to the GnRH-derived epitope. Moreover, the T7-3GnRH vaccine induced higher level of anti-GnRH antibody than ImproVac^®. However, the testosterone concentrations in both immunized groups were at a similar level, and the testis developments were significantly inhibited compared to controls. These findings demonstrated that the anti-GnRH antibody could neutralize the endogenous GnRH to down regulate testosterone level and limit testis development, highlighting the potential value of T7-3GnRH in the immunocastration vaccine research.

Keywords: Gonadotrophin Releasing Hormone (GnRH), Immunocastration, T7 phage, Phage vaccine

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Authors: Takashi Kaminagayoshi, Shigeyuki Haruyama

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To improve the manufacturing efficiency of biologics, such as antibody drugs, a quality engineering framework was designed. Within this framework, critical steps and parameters in the manufacturing process were studied. Identification of these critical steps and critical parameters allows a deeper understanding of manufacturing capabilities, and suggests to process development department process control standards based on actual manufacturing capabilities as part of a PDCA (plan-do-check-act) cycle. This cycle can be applied to each manufacturing process so that it can be standardized, reducing the time needed to establish each new process.

Keywords: antibody drugs, biologics, manufacturing efficiency, PDCA cycle, quality engineering

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Authors: Atefeh Esmailnejad, Gholam Reza Nikbakht Brujeni

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The major histocompatibility complex (MHC) is the best-characterized genetic region associated with susceptibility and/or resistance to a wide range of infectious diseases, autoimmune diseases and immune responses to vaccines. It has been demonstrated that there is an association between the MHC and resistance to Marek disease, Newcastle disease, Rous sarcoma tumor, Avian leucosis, Fowl cholera, Salmonellosis and Pasteurellosis in chicken. The present study evaluated the MHC polymorphism and its association with antibody response to Newcastle (ND) vaccine in Iranian native chickens. The MHC polymorphism was investigated using LEI0258 microsatellite locus by PCR-based fragment analysis. LEI0258 microsatellite marker is a genetic indicator for MHC, which is located on microchromosome 16 and strongly associated with serologically defined MHC haplotypes. Antibody titer against ND vaccine was measured by Haemaglutination Inhibition (HI) assay. Statistical analysis was performed using SPSS software (version 21). Total of 13 LEI0258 microsatellite alleles were identified in 72 samples which indicated a high genetic diversity in the population. The association study revealed a significant influence of MHC alleles on immune responses to Newcastle vaccine. 311 and 313 bp alleles were significantly associated with elevated immune responses to Newcastle vaccine (p<0.05). These results would be applicable in designing and improving the populations under selective breeding.

Keywords: chicken, LEI0258, MHC, Newcastle vaccine

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Authors: Christina Roman, Deepak Koirala, Joseph A. Piccirilli

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Phage displaying synthetic Fab libraries have been used to obtain Fabs that bind to specific RNA targets with high affinity and specificity. These Fabs have been demonstrated to facilitate RNA crystallization. However, the antibody framework used in the construction of these phage display libraries contains numerous bulky, flexible, and charged residues, which facilitate solubility and hinder aggregation. These residues can interfere with crystallization due to the entropic cost associated with burying them within crystal contacts. To systematically reduce the surface entropy of the Fabs and improve their crystallization properties, a protein engineering strategy termed surface entropy reduction (SER) is being applied to the Fab framework. In this approach, high entropy residues are mutated to smaller ones such as alanine or serine. Focusing initially on Fab BL3-6, which binds an RNA AAACA pentaloop with 20nM affinity, the SER P server (http://services.mbi.ucla.edu/SER/) was used and analysis was performed on existing RNA-Fab BL3-6 co-crystal structures. From this analysis twelve surface entropy reduced mutants were designed. These SER mutants were expressed and are now being measured for their crystallization and diffraction performance with various RNA targets. So far, one mutant has generated 3.02 angstrom diffraction with the yjdF riboswitch RNA. Ultimately, the most productive mutations will be combined into a new Fab framework to be used in a optimized phage displayed Fab library.

Keywords: antibody fragment, crystallography, RNA, surface entropy reduction

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Authors: Nasser A. Al-Shabib

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Food allergens are most common non-native form when exposed to the immune system. Most food proteins undergo various treatments (e.g. thermal or proteolytic processing) during food manufacturing. Such treatments have the potential to impact the chemical structure of food allergens so as to convert them to more denatured or unfolded forms. The conformational changes in the proteins may affect the allergenicity of treated-allergens. However, most allergenic proteins possess high resistance against thermal modification or digestive enzymes. In the present study, ovomucoid (a major allergenic protein of egg white) was heated in phosphate-buffered saline (pH 7.4) at different temperatures, aqueous solutions and on different surfaces for various times. The results indicated that different antibody-based methods had different sensitivities in detecting the heated ovomucoid. When using one particular immunoassay‚ the immunoreactivity of ovomucoid increased rapidly after heating in water whereas immunoreactivity declined after heating in alkaline buffer (pH 10). Ovomucoid appeared more immunoreactive when dissolved in PBS (pH 7.4) and heated on a stainless steel surface. To the best of our knowledge‚ this is the first time that antibody-based methods have been applied for the detection of ovomucoid adsorbed onto different surfaces under various conditions. The results obtained suggest that use of antibodies to detect ovomucoid after food processing may be problematic. False assurance will be given with the use of inappropriate‚ non-validated immunoassays such as those available commercially as ‘Swab’ tests. A greater understanding of antibody-protein interaction after processing of a protein is required.

Keywords: ovomucoid, thermal treatment, solutions, surfaces

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Authors: Gulyas Erzsebet

Abstract:

The multimerisation of proteins is a common feature of many cellular processes; however, it could also impair protein functions and/or be associated with the occurrence of diseases. Thus, development of a research tool monitoring the appearance/presence of multimeric protein forms has great importance for a variety of research fields. Such a tool is potentially applicable in the ante-mortem diagnosis of certain conformational diseases, such as transmissible spongiform encephalopathies (TSE) and Alzheimer’s disease. These conditions are accompanied by the appearance of aggregated protein multimers, present in low concentrations in various tissues. This detection is particularly relevant for TSE where the handling of tissues derived from affected individuals and of meat products of infected animals have become an enormous health concern. Here we demonstrate the potential of such a multimer detection approach in TSE by developing a facile approach. The Biospiral-Detect system resembles a traditional sandwich ELISA, except that the capturing antibody that is attached to a solid surface and the detecting antibody is directed against the same or overlapping epitopes. As a consequence, the capturing antibody shields the epitope on the captured monomer from reacting with the detecting antibody, therefore monomers are not detected. Thus, MDS is capable of detecting only protein multimers with high specificity. We developed an alternative system as well, where RNA aptamers were employed instead of monoclonal antibodies. In order to minimize degradation, the 3' and 5' ends of the aptamer contained deoxyribonucleotides and phosphorothioate linkages. When compared the monoclonal antibodies-based system with the aptamers-based one, the former proved to be superior. Thus all subsequent experiments were conducted by employing the Biospiral -Detect modified sandwich ELISA kit. Our approach showed an order of magnitude higher sensitivity toward mulimers than monomers suggesting that this approach may become a valuable diagnostic tool for conformational diseases that are accompanied by multimerization.

Keywords: diagnosis, ELISA, Prion, TSE

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Authors: Paul J Yazaki, Michael Bouvet, John Shively

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Surgery for solid gastrointestinal (GI) cancers such as pancreatic, colorectal, and gastric adenocarcinoma remains the mainstay of curative therapy. Complete resection of the primary tumor with negative margins (R0 resection), its draining lymph nodes, and distant metastases offers the optimal surgical benefit. Real-time fluorescence guided surgery (FGS) promises to improve GI cancer outcomes and is rapidly advancing with tumor-specific antibody conjugated fluorophores that can be imaged using near infrared (NIR) technology. Carcinoembryonic Antigen (CEA) is a non-internalizing tumor antigen validated as a surface tumor marker expressed in >95% of colorectal, 80% of gastric, and 60% of pancreatic adenocarcinomas. Our humanized anti-CEA hT84.66-M5A (M5A) monoclonal antibody (mAb)was conjugated with the NHS-IRDye800CW fluorophore and shown it can rapidly and effectively NIRoptical imageorthotopically implanted human colon and pancreatic cancer in mouse models. A limitation observed is that these NIR-800 dye conjugated mAbs have a rapid clearance from the blood, leading to a narrow timeframe for FGS and requiring high doses for effective optical imaging. We developed a novel antibody-fluorophore conjugate by incorporating a PEGylated sidearm linker to shield or mask the IR800 dye’s hydrophobicity which effectively extended the agent’s blood circulation half-life leading to increased tumor sensitivity and lowered normal hepatic uptake. We hypothesized that our unique anti-CEA linked to the fluorophore, IR800 by PEGylated sidewinder, M5A-SW-IR800 will become the next generation optical imaging agent, safe, effective, and widely applicable for intraoperative image guided surgery in CEA expressing GI cancers.

Keywords: optical imaging, anti-CEA, cancer, fluorescence-guided surgery

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Authors: Naveen Kumar B. T., Anuj Tyagi, Niraj Kumar Singh, Visanu Boonyawiwat, Shanthanagouda A. H., Orawan Boodde, Shankar K. M., Prakash Patil, Shubhkaramjeet Kaur

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Since 2009, Acute Hepatopancreatic Necrosis Disease (AHPND) outbreaks have increased rapidly, and these have led to the major economic losses to the global shrimp industry. In comparison to other treatments, passive immunity and monoclonal antibody (MAb) based farmer level kit have proved their importance in controlling and treating the diseases in the shrimp industry. In the present study, MAbs were produced against the recombinant PirB protein Vibrio parahaemolyticus strain causing AHPND. Briefly, Balb/C mice were immunized with rPirB at 15 days interval, and antibody titer was determined by ELISA. Spleen cells from mice showing high antibody titer were fused with SP2O myeloma cells for hybridoma production. Among 130 hybridomas, four showed high antibody titer and positive reactivity in an immunoblot assay. In Western blot assay, three out of four MAbs (4C4, 2C2 and 4G3) showed reactivity to rPirB protein. However, in the natural host, only Mab clone 4G3 show strong reactivity (with a strain of V. parahemolyticus causing EMS/AHPND). These clones also showed reactivity with less than 20 kDa proteins in AHPND free V. parahaemolyticus (Thailand stain). Further, on from MAb 4G3 clone, four panels of single cell MAbs clones (G3F5, G3B8, G3H2, and G3D6) were produced of which three showed strong positive reactivity to rPirB protein in the Western blot. These MAbs have potential for controlling and prevention of the AHPND through passive immunity and development of filed level rapid diagnostic kits.

Keywords: shrimp, economic loss, AHPND, MAb

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Authors: Ishani Das, Avinash Padhi, Sitabja Mukherjee, Santosh Kar, Avinash Sonawane

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Activation of cell mediated and humoral immune responses to Mycobacterium tuberculosis (Mtb) are critical for protection. Herein, we show that mice immunized with Mtb lipid bound chitosan nanoparticles(NPs) induce secretion of prominent Th1 and Th2 cytokines in lymph node and spleen cells, and also induced significantly higher levels of IgG, IgG1, IgG2 and IgM in comparison to control mice measured by ELISA. Furthermore, significantly enhanced γδ-T cell activation was observed in lymph node cells isolated from mice immunized with Mtb lipid coated chitosan-NPs as compared to mice immunized with chitosan-NPs alone or Mtb lipid liposomes through flow cytometric analysis. Also, it was observed that in comparison to CD8+ cells, significantly higher CD4+ cells were present in both the lymph node and spleen cells isolated from mice immunized with Mtb lipid coated chitosan NP. In conclusion, this study represents a promising new strategy for efficient delivery of Mtb lipids using chitosan NPs to trigger enhanced cell mediated and antibody response against Mtb lipids.

Keywords: antibody response, chitosan nanoparticles, cytokines, mycobacterium tuberculosis lipids

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Authors: Gholamreza Hashemitabr, Reza Valadan, Alireza Rafiei, Mohammad Reza Bassami

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Breast cancer is the most common malignancy among women worldwide. Cancer cells use a complex multilayer network of epidermal growth factor receptors (EGFRs) signaling pathways to support their survival and growth. The overlapping networks of EGFRs signaling pathways account for the failure of most ErbB-targeted therapies. The aim of this study was to enrich a pool of recombinant antibody fragments against common epitopes of ErbB1 and ErbB2 in order to simultaneous blockade of ErbBs signaling pathways. ErbB1 and ErbB2 were expressed stably in VERO cells. Selection of recombinant antibodies was performed on live cells expressing either of ErbB1 and ErbB2 receptors using subtractive phage display approach. The results of PCR and DNA fingerprinting in the last round of panning showed that most clones contained insert (80% and 85% for ErbB1 and ErbB2 respectively) with an identical restriction pattern. The selected clones showed positive reaction to both ErbB1 and ErbB2 receptors in phage-ELISA test. Furthermore, the resulting soluble antibody fragments recognized common epitopes of both immunoprecipitated ErbB1 and ErbB2 in western blot. Additionally, the antibodies directed against the dimerization domain of ErbB1 demonstrated a significant absorbance in EGF-stimulated VERO/ErbB1 cells than non-stimulated cells (1.91 and 1.09 respectively). Moreover, the results of dimerization inhibition test showed that these antibodies blocked ErbB1 and ErbB2 dimerization on the surface of ErbB1 and ErbB2 expressing VERO cells. Regarding the importance of pan-ErbB approach to cancer therapy, the antibodies developed here might provide novel therapeutics for simultaneous blockade of ErbBs signaling pathways.

Keywords: breast cancer, single-chain antibody, ErbB1, ErbB2, epitope

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Authors: Barbra Bhebhe, Melvyn Quan

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A serological survey was done to detect antibodies against Shuni virus (SHUV) from cattle in Western Kenya. In Kenya the disease status of SHUV in cattle has never been established. It is a zoonotic virus and even though studies have been carried out as early as the 1960s, little research has been published and SHUV is still not a well-recognised Orthobunyavirus. One hundred serum samples were collected from healthy cattle in Kenya and tested for antibodies against SHUV by a serum neutralization assay. All antibody titre values were greater than 1:160, with most of the samples greater than 1:320. Of the samples tested, 87 % had titres greater than 1:320, 12% had a titre of 1:320 and 2% had a titre of 1:160. Samples were classified as positive if the antibody titre was ≥ 1:10 and negative if < 1:10. This study suggests that cattle are exposed commonly to SHUV, which may be endemic in Kenya.

Keywords: Shuni virus, Orthobunyavuruses, serum neutralization test, cell-culture

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Authors: Lucian Mocan, Teodora Mocan, Matea Cristian, Cornel Iancu

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We present method of nanoparticle enhanced laser thermal ablation of Klebsiella pneumoniae infections, using gold nanoparticles combined with a specific growth factor and demonstrate its selective therapeutic efficacy. Ab (antibody solution) bound to GNPs (gold nanoparticles) was administered in vitro and determined the specific delivery of the nano-bioconjugate into the microorganism. The extent of necrosis was considerable following laser therapy, and at the same time, normal cells were not seriously affected. The selective photothermal ablation of the infected tissue was obtained after the selective accumulation of Ab bound to GNPs into bacteria following perfusion. These results may represent a major step in antibiotherapy treatment using nanolocalized thermal ablation by laser heating.

Keywords: gold nanoparticles, Klebsiella pneumoniae, nanoparticle functionalization, laser irradiation, antibody

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Authors: Dan W. Rhodes, Juliane Hey, Magali Karagueuzian, Florianne Martinez, Yael Sandowski, Vanessa Roulet, Mahmoud Badawi, Mohammed-Amine Chakir, Valérie Simon, Jérémie Gautier, Françoise Le Boulaire, Catherine Coignard, Claire Vincent, Sandrine Greaume, Isabelle Voisin

Abstract:

Background: Beckman Coulter, Inc. (BEC) has recently developed a fully automated second-generation anti-HCV test on a new immunoassay platform. The objective of this multicenter study conducted in Europe was to evaluate the performance of the ACCESS anti-HCV assay on the recently CE-marked DxI 9000 ACCESS Immunoassay Analyzer as an aid in the diagnosis of HCV (Hepatitis C Virus) infection and as a screening test for blood and plasma donors. Methods: The clinical specificity of the ACCESS anti-HCV assay was determined using HCV antibody-negative samples from blood donors and hospitalized patients. Sample antibody status was determined by a CE-marked anti-HCV assay (Abbott ARCHITECTTM anti-HCV assay or Abbott PRISM HCV assay) with an additional confirmation method (Immunoblot testing with INNO-LIATM HCV Score - Fujirebio), if necessary, according to pre-determined testing algorithms. The clinical sensitivity was determined using known HCV antibody-positive samples, identified positive by Immunoblot testing with INNO-LIATM HCV Score - Fujirebio. HCV RNA PCR or genotyping was available on all Immunoblot positive samples for further characterization. The false initial reactive rate was determined on fresh samples from blood donors and hospitalized patients. Thirty (30) commercially available seroconversion panels were tested to assess the sensitivity for early detection of HCV infection. The study was conducted from November 2019 to March 2022. Three (3) external sites and one (1) internal site participated. Results: Clinical specificity (95% CI) was 99.7% (99.6 – 99.8%) on 5852 blood donors and 99.0% (98.4 – 99.4%) on 1527 hospitalized patient samples. There were 15 discrepant samples (positive on ACCESS anti-HCV assay and negative on both ARCHITECT and Immunoblot) observed with hospitalized patient samples, and of note, additional HCV RNA PCR results showed five (5) samples had positive HCV RNA PCR results despite the absence of HCV antibody detection by ARCHITECT and Immunoblot, suggesting a better sensitivity of the ACCESS anti-HCV assay with these five samples compared to the ARCHITECT and Immunoblot anti-HCV assays. Clinical sensitivity (95% CI) on 510 well-characterized, known HCV antibody-positive samples was 100.0% (99.3 – 100.0%), including 353 samples with known HCV genotypes (1 to 6). The overall false initial reactive rate (95% CI) on 6630 patient samples was 0.02% (0.00 – 0.09%). Results obtained on 30 seroconversion panels demonstrated that the ACCESS anti-HCV assay had equivalent sensitivity performances, with an average bleed difference since the first reactive bleed below one (1), compared to the ARCHITECTTM anti-HCV assay. Conclusion: The newly developed ACCESS anti-HCV assay from BEC for use on the DxI 9000 ACCESS Immunoassay Analyzer demonstrated high clinical sensitivity and specificity, equivalent to currently marketed anti-HCV assays, as well as a low false initial reactive rate.

Keywords: DxI 9000 ACCESS Immunoassay Analyzer, HCV, HCV antibody, Hepatitis C virus, immunoassay

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Authors: Myrto Vlazaki

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Tetanus is a non-transmissible, preventable bacterial disease with high mortality. In the U.K., the demographic group systematically accounting for a large proportion of the infections notified to the authorities over the years have been the elderly (> 60 years old). The 2009 seroepidemiological study for tetanus in England reports a gender-age interaction for the +70, with males having significantly higher anti-tetanus antibody levels than females. A systematic review of the literature was carried out to characterise: I. the seroepidemiology of tetanus in economically developed countries with similar immunisation schemes to the U.K., introduced in the 1960’s. II. the factors leading to differential vaccine uptake between males and females in 1910-1945 (corresponding to ages of 60-95 in 2005). III. the immune response elicited by anti-tetanus immunisation in males and females IV. the value of catch-up immunisation in the elderly Similar age- and gender- differences in anti-tetanus antibody levels are noted in other countries. Gender differences in immune responses elicited by vaccination are not consistent with the finding that elder females are less well protected against tetanus compared to their male counterparts. Attention is drawn to the selective anti-tetanus immunisation scheme introduced in the U.K. in 1938, specific to the World War II conscripts. The age-specific immunity gap observed amongst the +70 could be explained as the by-product of that early scheme targetting mostly males. Introducing anti-tetanus vaccination in the +70 in the U.K. could help bridge the immunity gap between males and females and reduce the overall tetanus susceptibility of this age group.

Keywords: elderly, immunisation, gender-specific differences, seroepidemiology, tetanus, World War II

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Authors: Hamed Ahari, Behrouz Akbari Adreghani, Vadood Razavilar, Amirali Anvar, Sima Moradi, Hourieh Shalchi

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Considering the ever increasing population and industrialization of the developmental trend of humankind's life, we are no longer able to detect the toxins produced in food products using the traditional techniques. This is due to the fact that the isolation time for food products is not cost-effective and even in most of the cases, the precision in the practical techniques like the bacterial cultivation and other techniques suffer from operator errors or the errors of the mixtures used. Hence with the advent of nanotechnology, the design of selective and smart sensors is one of the greatest industrial revelations of the quality control of food products that in few minutes time, and with a very high precision can identify the volume and toxicity of the bacteria. Methods and Materials: In this technique, based on the bacterial antibody connection to nanoparticle, a sensor was used. In this part of the research, as the basis for absorption for the recognition of bacterial toxin, medium sized silica nanoparticles of 10 nanometer in form of solid powder were utilized with Notrino brand. Then the suspension produced from agent-linked nanosilica which was connected to bacterial antibody was positioned near the samples of distilled water, which were contaminated with Staphylococcus aureus bacterial toxin with the density of 10-3, so that in case any toxin exists in the sample, a connection between toxin antigen and antibody would be formed. Finally, the light absorption related to the connection of antigen to the particle attached antibody was measured using spectrophotometry. The gene of 23S rRNA that is conserved in all Staphylococcus spp., also used as control. The accuracy of the test was monitored by using serial dilution (l0-6) of overnight cell culture of Staphylococcus spp., bacteria (OD600: 0.02 = 107 cell). It showed that the sensitivity of PCR is 10 bacteria per ml of cells within few hours. Result: The results indicate that the sensor detects up to 10-4 density. Additionally, the sensitivity of the sensors was examined after 60 days, the sensor by the 56 days had confirmatory results and started to decrease after those time periods. Conclusions: Comparing practical nano biosensory to conventional methods like that culture and biotechnology methods(such as polymerase chain reaction) is accuracy, sensitiveness and being unique. In the other way, they reduce the time from the hours to the 30 minutes.

Keywords: exotoxin, nanobiosensor, recognition, Staphylococcus aureus

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Authors: Joseph L. Mathew, Shourjendra N. Banerjee, R. K. Ratho, Sourabh Dutta, Vanita Suri

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Background: In India and many other developing countries, a single dose of measles vaccine is administered to infants at 9 months of age. This is based on the assumption that maternal transplacentally transferred antibodies will protect infants until that age. However, our previous data showed that most infants lose maternal anti-measles antibodies before 6 months of age, making them susceptible to measles before vaccination at 9 months. Objective: This prospective study was designed to compare susceptibility in pre-term vs term infants, at different time points. Material and Methods: Following Institutional Ethics Committee approval and a formal informed consent process, venous blood was drawn from a cohort of 45 consecutive term infants and 45 consecutive pre-term infants (both groups delivered by the vaginal route); at birth, 3 months, 6 months and 9 months (prior to measles vaccination). Serum was separated and anti-measles IgG antibody levels were measured by quantitative ELISA kits (with sensitivity and specificity > 95%). Susceptibility to measles was defined as antibody titre < 200mIU/ml. The mean antibody levels were compared between the two groups at the four time points. Results: The mean gestation of term babies was 38.5±1.2 weeks; and pre-term babies 34.7±2.8 weeks. The respective mean birth weights were 2655±215g and 1985±175g. Reliable maternal vaccination record was available in only 7 of the 90 mothers. Mean anti-measles IgG antibody (±SD) in terms babies was 3165±533 IU/ml at birth, 1074±272 IU/ml at 3 months, 314±153 IU/ml at 6 months, and 68±21 IU/ml at 9 months. The corresponding levels in pre-term babies were 2875±612 IU/ml, 948±377 IU/ml, 265±98 IU/ml, and 72±33 IU/ml at 9 months (p > 0.05 for all inter-group comparisons). The proportion of susceptible term infants at birth, 3months, 6months and 9months was 0%, 16%, 67% and 96%. The corresponding proportions in the pre-term infants were 0%, 29%, 82%, and 100% (p > 0.05 for all inter-group comparisons). Conclusion: Majority of infants are susceptible to measles before 9 months of age suggesting the need to anticipate measles vaccination, but there was no statistically significant difference between the proportion of susceptible term and pre-term infants, at any of the four-time points. A larger study is required to confirm these findings and compare sero-protection if vaccination is anticipated to be administered between 6 and 9 months.

Keywords: measles, preterm, susceptibility, term infant

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Authors: Sahar Essa, Hussain A. Safar, Raj Raghupathy

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Human cytomegalovirus (CMV) continues to be a source of severe complications in immunologically immature and immunocompromised hosts. Effective CMV vaccines that help diminish CMV disease in transplant patients and avoid congenital infection are of great importance. Though the exact roles of defense mechanisms are unidentified, viral-specific antibodies and cytokine responses are known to be involved in controlling CMV infections. CMV envelope glycoprotein B (UL55/gB), matrix proteins (UL83/pp65, UL99/pp28, UL32/pp150), and assembly protein UL80a/pp38 are known to be targets of antiviral immune responses. We immunized mice intraperitoneally with these five CMV-related proteins (commercial) for their ability to induce specific antibody responses (in-house immunoassay) and cytokine production (commercial assay) in a mouse model. We observed a significant CMV-antigen-specific antibody response to pp38 and pp65 (E/C ˃2.0, p˂0.001). Mice immunized with pp38 had significantly higher concentrations of GM-CSF, IFN-α, IL-2 IL-4, IL-5, and IL-17A (p˂0.05). Mice immunized with pp65 showed significantly higher concentrations of GM-CSF, IFN-γ, IL-2 IL-4, IL-10, IL-12, IL-17A, and TNF-α. Th1 to Th2 cytokines ratios revealed a Th1 cytokine bias in mice immunized with pp38, pp65, pp150, and gB. We suggest that stimulation with multiple CMV-related proteins, which include pp38, pp65, and gB antigens, will allow both humoral and cellular immune responses to be efficiently activated, thus serving as appropriate CMV antigens for future vaccines.

Keywords: cytomegalovirus, UL99/pp28, UL80a/pp38, UL83/pp65, UL32/pp150, UL55/gB, CMV-antigen-specific antibody, CMV antigen-specific cytokine responses

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Authors: Chuan Yin

Abstract:

Cancer stem cells (CSCs) represent a subpopulation of cancer cells that possess the characteristics associated with normal stem cells. CD133 is a phenotype of melanoma CSCs responsible for melanoma metastasis and drug resistance. Although adriamycin (ADR) is commonly used drug in melanoma therapy, but it is ineffective in the treatment of melanoma CSCs. In this study, we constructed CD133 antibody conjugated ADR immunoliposomes (ADR-Lip-CD133) to target CD133+ melanoma CSCs. The results showed that the immunoliposomes possessed a small particle size (~150 nm), high drug encapsulation efficiency (~90%). After 72 hr treatment on the WM266-4 melanoma tumorspheres, the IC50 values of the drug formulated in ADR-Lip-CD133, ADR-Lip (ADR liposomes) and ADR are found to be 24.42, 57.13 and 59.98 ng/ml respectively, suggesting that ADR-Lip-CD133 was more effective than ADR-Lip and ADR. Significantly, ADR-Lip-CD133 could almost completely abolish the tumorigenic ability of WM266-4 tumorspheres in vivo, and showed the best therapeutic effect in WM266-4 melanoma xenograft mice. It is noteworthy that ADR-Lip-CD133 could selectively kill CD133+ melanoma CSCs of WM266-4 cells both in vitro and in vivo. ADR-Lip-CD133 represent a potential approach in targeting and killing CD133+ melanoma CSCs.

Keywords: cancer stem cells, melanoma, immunoliposomes, CD133

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Authors: Jai Myung Yang, Na Young Kim, Sung Ho Shin

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The adverse reactions of current live smallpox vaccines and potential use of smallpox as a bioterror weapon have heightened the development of new effective vaccine for this infectious disease. In the present study, DNA vaccine vector was produced which was optimized for expression of the vaccinia virus L1 antigen in the mouse model. A plasmid IgM-tL1R, which contains codon-optimized L1R gene, was constructed and fused with an IgM signal sequence under the regulation of a SV40 enhancer. The expression and secretion of recombinant L1 protein was confirmed in vitro 293 T cell. Mice were administered the DNA vaccine by electroporation and challenged with vaccinia virus. We observed that immunization with IgM-tL1R induced potent neutralizing antibody responses and provided complete protection against lethal vaccinia virus challenge. Isotyping studies reveal that immunoglobulin G2 (IgG2) antibody predominated after the immunization, indicative of a T helper type 1 response. Our results suggest that an optimized DNA vaccine, IgM-tL1R, can be effective in stimulating anti-vaccinia virus immune response and provide protection against lethal orthopoxvirus challenge.

Keywords: DNA vaccine, electroporation, L1R, vaccinia virus

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Authors: Yung-Chih Kuo, I-Hsuan Lee

Abstract:

Solid lipid nanoparticles (SLNs) conjugated with aprotinin (Apr) and melanotransferrin antibody (Anti-MTf) were used to carry doxorubicin (Dox) across the blood–brain barrier (BBB) for glioblastoma multiforme (GBM) chemotherapy. Dox-entrapped SLNs with grafted Apr and Anti-MTf (Apr-Anti-MTf-Dox-SLNs) were applied to a cultured monolayer comprising human brain-microvascular endothelial cells (HBMECs) with regulation of human astrocyte (HAs) and to a proliferated colony of U87MG cells. Based on the average particle diameter, zeta potential, entrapping efficiency of Dox, and grafting efficiency of Apr and Anti-MTf, we found that 40% (w/w) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in lipids were appropriate for fabricating Apr-Anti-MTf-Dox-SLNs. In addition, Apr-Anti-MTf-Dox-SLNs could prevent Dox from fast dissolution and did not induce a serious cytotoxicity to HBMECs and HAs when compared with free Dox. Moreover, the treatments with Apr-Anti-MTf-Dox-SLNs enhanced the ability of Dox to infuse the BBB and to inhibit the growth of GBM. The current Apr-Anti-MTf-Dox-SLNs can be a promising pharmacotherapeutic preparation to penetrate the BBB for malignant brain tumor treatment.

Keywords: solid lipid nanoparticle, glioblastoma multiforme, blood–brain barrier, doxorubicin

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Authors: Ying Sun, Biao Cheng, Qing Lu, Xuefei Tao, Xiaoyu Lai, Cheng Guo, Dan Wang

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Fibrinogen-like protein 2 (FGL2) has recently been identified to play an important role in inflammatory diseases such as atherosclerosis through a thrombin-dependent manner. Here, a murine monoclonal antibody was raised against the critical residue Ser(89) of FGL2, and the effects of the anti-FGL2 mAb (SPF89) were analyzed in human umbilical vein endothelial cells (HUVECs) and THP-1 cells. Firstly, it was proved that SPF89, which belongs to the IgG1 subtype with a KD value of 44.5 pM, could specifically show the expression levels of protein FGL2 in different cell lines of known target gene status. The lipopolysaccharide (LPS)-mediated endothelial cell proliferation was significantly inhibited with a decline of phosphorylation nuclear factor-κB (NF-κB) in a dose-dependent manner after SPF89 treatment. Furthermore, SPF89 reduced LPS-induced expression of adhesion molecules and inflammatory cytokines such as vascular cell adhesion molecule-1, tumor necrosis factor-α, Matrix metalloproteinase MMP-2, Integrin αvβ3, and interleukin-6 in HUVECs. In macrophage-like THP-1 cells, SPF89 effectively inhibited LPS and low-density lipoprotein-induced foam cell formation. However, these anti-inflammatory and anti-atherosclerotic effects of anti-FGL2 mAb in HUVECs and THP-1 cells were significantly reduced after treatment with an NF-κB inhibitor PDTC. All the above suggest, by efficiently inhibiting LPS-induced pro-inflammatory effects in vascular endothelial cells by attenuating NF-κB dependent pathway, the new anti-FGL2 mAb SPF89 could to be a potential therapeutic candidate for protecting the vascular endothelium against inflammatory diseases such as atherosclerosis. This work was supported by the Program of Sichuan Science and Technology Department (2017FZ0069) and Collaborative Innovation Program of Sichuan for Elderly Care and Health(YLZBZ1511).

Keywords: monoclonal antibody, fibrinogen like protein 2, inflammation, endothelial cells

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Authors: Mohammadjavad Mehrabanpour, Maryam Ranjbar Bushehri, Dorsa Mehrabanpour

Abstract:

Respiratory diseases are the most important problems in the country’s poultry industry, particularly when it comes to broiler flocks. Ornithobacterium rhinotracheale (ORT) is a species that causes poor performance in growth rate, egg production, and mortality. This pathogen causes a respiratory infection including pulmonary alveolar inflammation, and pneumonia of birds throughout the world. Newcastle disease (ND) is a highly contagious disease in poultry, and also, it causes considerable losses to the poultry industry. The aim of this study was to evaluate the simultaneous occurrence of ORT and ND and NDV isolation by inoculation in embryonated eggs and confirmed by RT-PCR in broiler chicken flocks in Fars province. In this study, 318 blood and 85 tissue samples (brain, trachea, liver, and cecal tonsils) were collected from 15 broiler chicken farms. Survey serum antibody titers against ORT by using a commercial enzyme-linked immunosorbent assay (ELISA) kit performed. Evaluation of antibody titer against ND virus is performed by hemagglutination inhibition test. Virus isolation with chick embryo eggs 9-11 and RT-PCR method were carried out. A total of 318 serum samples, 135 samples (42.5%) were positive for antibodies to ORT and titer of HI antibodies against NDV in 122 serum samples (38/4%) were 7-10 (log2) and 61 serum samples (19/2%) had occurrence antibody titer against Newcastle virus and ORT. Results of the present study indicated that 20 tissue samples were positive in embryonated egg and in rapid hemagglutination (HA) test. HI test with specific ND positive serum confirmed that 6 of 20 samples. PCR confirmed that all six samples were positive and PCR products of samples indicated 535-base pair fragments in electrophrosis. Due to the great economic importance of these two diseases in the poultry industry, it is necessary to design and implement a comprehensive plan for prevention and control of these diseases.

Keywords: ELISA, Ornithobacterium rhinotracheale, newcastle disease, seroprevalence

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Authors: Arun Kharate, Sarata Kumar Sahu, Susen Kumar Panda, Niranjan Sahoo, H. K. Panda

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In the present study, an attempt has been made for isolation and identification of feline panleukopaenia virus (FPLV) from wild felids of Nandankanan zoo, Odisha, India, along with prevalence study of FPLV. Fecal samples collected from wild felids (26 tigers, 22 lions, 5 leopards, 3 hyenas, 1 jaguar, 2 foxes and 1 wild cat) were subjected to hemagglutinnation test and fluorescent antibody test. In hemagglutinnation test 13 (50%) samples from tiger, 14 (63.63%) samples from lions, 1 (20%) sample from leopards, 1 (50%) from fox, 3 (100%) samples from hyenas and 1 (100%) sample from wild cat were positive. On fluorescent antibody test (FAT), 15 (57.69%) samples from tiger, 18 (81.81%) from lions, 2 (40%) from leopards, 1 (50%) from fox, 3 (100%) from hyenas and 1 (100%) from wild cat were positive. FPLV was isolated using MDBK cell line and preliminary characterization was done on the basis of characteristic cytopathic effect. The virus samples were quantified through titration in MDBK cells. Serological confirmation of FPLV isolates was carried out by HI test, micro-SNT and indirect-ELISA. Physico-chemical characters like pH and temperature resistance along molecular identification using specific FPLV primers was carried out. Seroprevalence study of 36 serum samples employing HI test, micro SNT and indirect-ELISA revealed prevalence of 38.8, 44.4 and 72.2% respectively. During study period an adult tigress and a tiger cub died suspected of feline panleukopenia. The necropsy findings in both animals showed hemorrhagic gastroenteritis. The cytological examination revealed presence of intranuclear inclusion bodies in the intestinal epithelial cells. Spleen, mesenteric lymph node and intestine were positive for feline panleukopenia by FAT. The investigation revealed that feline panleukopenia was prevalent in wild felines of Nandankanan zoo.

Keywords: Feline panleukopenia, fluorescent antibody test, hemagglutination test, indirect-ELISA, Nandankanan zoo

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Authors: Ranjeeta Kumari, Makesh M, Gayatri Tripathi, K V Rajendran, Megha Bedekar

Abstract:

A comparative study on DNA immunization with recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) construct of Edwardsiella tarda (pGPD group) and a bicistronic construct expressing GAPDH plus IFN-γ of Labeo rohita as adjuvant (pGPD+IFN group) was undertaken in Labeo rohita along with the control animals. Successful co-expression of two genes that is GAPDH and IFN-γ was confirmed in SSN-1 cells line by RT-qPCR and western blot. The protective immune response of host to DNA vaccine construct was determined by RPS and specific antibody production. Fishes immunized with plasmids via intramuscular injection (I/M) exhibited a considerable relative percentage survivability of 66.66% in pGPD+IFN immunized group and 53.34% in pGPD immunized group after challenge with E. tarda. Antibody response was also significantly high in pGPD+IFN group at all time points under study. This was analysed by competitive ELISA, using anti GAPDH monoclonal antibodies. The experiment revealed that the GAPDH gene of E. tarda is one of the ideal candidates for generating protective immune response in L. rohita. Further addition of Interferon gamma to DNA vaccine construct can enhance the immune response in host.

Keywords: DNA vaccine, Edwardsiella tarda, Labeo rohita, zoonosis, immune response

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Authors: Adia Fatima, Naila Chand, Rifat Ullah Khan

Abstract:

The goal of this study was to determine how fennel seed supplementation affected broiler growth, carcass quality, antioxidant status, and antibody titer in heat-stressed broilers. A total of 720 one-day-old broiler chickens were weighed and assigned to 28-floor pens (25 broiler chickens per pen). The broiler chickens were housed in a thermoneutral (TN) environment and were exposed to heat stress (HS). For 23 hours, the broiler chickens were kept under fluorescent lighting. For 35d, HS broiler chickens were fed a control diet and three levels of fennel seeds powder at rates of 15g/kg (Fen-15), 20 g/kg (Fen-20), and 25 g/kg (Fen-25). Overall feed intake, weight gain, and dressing % were considerably greater (P < 0.05) in Fen-25 and TN, but FCR was significantly reduced (P<0.01) in the same groups. When TN, Fen-20, and Fen-25 were compared to the control, malondialdehyde (MDA), paraoxonase (PON1), and antibody titer against New Castle disease (ND) were considerably (P < 0.05) greater. Further, the linear and quadratic response was for feed intake, weight gain, FCR, MDA, PON1, and ND titer. It was concluded that Fen-20 and Fen-25 increased broiler growth, carcass quality, antioxidant status, and immunological response under HS conditions.

Keywords: heat stress, growth, antioxidant, immunity

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Authors: Manal Kamel, Sara Maher

Abstract:

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

Procedia PDF Downloads 135