Retrospective immunohistochemical investigation on dolphin morbillivirus infection by comparing the performance of heterologous monoclonal and polyclonal antibodies – Short communication

2021 ◽  
Author(s):  
Federica Giorda ◽  
Giovanni Di Guardo ◽  
Katia Varello ◽  
Alessandra Pautasso ◽  
Eva Sierra ◽  
...  
Keyword(s):  
Short Communication ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Binding Activity ◽  
N Protein ◽  
Target Organs ◽  
Dolphin Morbillivirus ◽  
Free Ranging ◽  
The Brain ◽  
Histology And Immunohistochemistry

AbstractDolphin morbillivirus (DMV) is a pathogen of great concern in free-ranging cetaceans. Confirmation and staging of morbillivirus infections rely on histology and immunohistochemistry (IHC), following molecular detection. As at the present time no specific antibodies (Abs) against DMV are available, two heterologous Abs have been used worldwide for the examinations of morbillivirus infections of cetaceans. One is a monoclonal Ab (MoAb) prepared against the N protein of canine distemper virus (CDV), whereas the other is a polyclonal Ab raised in rabbits against rinderpest virus (RPV). Both Abs are known to show cross-reactivity with DMV. In this study we compared the labelling quality and the neuroanatomical distribution of staining with these two Abs by means of IHC analysis. To this end, serial sections of the target organs from ten free-ranging stranded cetaceans, previously diagnosed as being infected with DMV by PCR and/or serology, were subjected to IHC. The brain, lungs and lymph nodes of one animal were found to be positive with both Abs. From two other animals, the brain and the spleen, respectively, tested positive only with the polyclonal Ab. In the positive brain tissues, multifocal immunostaining was observed, with similar staining location and extent, with the two antibodies tested. Our results suggest that the polyclonal anti-RPV Ab might have a stronger binding activity to DMV than the anti-CDV MoAb. Nevertheless, the elaboration and use of specific anti-DMV Abs might be essential to guarantee conclusive results in diagnostic and pathogenetic investigations.

scholarly journals In-House Immunofluorescence Assay for Detection of SARS-CoV-2 Antigens in Cells from Nasopharyngeal Swabs as a Diagnostic Method for COVID-19

Diagnostics ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 2346
Author(s):  
Athene Hoi-Ying Lam ◽  
Jian-Piao Cai ◽  
Ka-Yi Leung ◽  
Ricky-Ruiqi Zhang ◽  
Danlei Liu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Nucleocapsid Protein ◽  
Diagnostic Method ◽  
Polyclonal Antibodies ◽  
Respiratory Viruses ◽  
Cross Reactivity ◽  
Immunofluorescence Assay ◽  
Respiratory Epithelial Cells ◽  
N Protein ◽  
Respiratory Epithelial

Immunofluorescence is a traditional diagnostic method for respiratory viruses, allowing rapid, simple and accurate diagnosis, with specific benefits of direct visualization of antigens-of-interest and quality assessment. This study aims to evaluate the potential of indirect immunofluorescence as an in-house diagnostic method for SARS-CoV-2 antigens from nasopharyngeal swabs (NPS). Three primary antibodies raised from mice were used for immunofluorescence staining, including monoclonal antibody against SARS-CoV nucleocapsid protein, and polyclonal antibodies against SARS-CoV-2 nucleocapsid protein and receptor-binding domain of SARS-CoV-2 spike protein. Smears of cells from NPS of 29 COVID-19 patients and 20 non-infected individuals, and cells from viral culture were stained by the three antibodies. Immunofluorescence microscopy was used to identify respiratory epithelial cells with positive signals. Polyclonal antibody against SARS-CoV-2 N protein had the highest sensitivity and specificity among the three antibodies tested, detecting 17 out of 29 RT-PCR-confirmed COVID-19 cases and demonstrating no cross-reactivity with other tested viruses except SARS-CoV. Detection of virus-infected cells targeting SARS-CoV-2 N protein allow identification of infected individuals, although accuracy is limited by sample quality and number of respiratory epithelial cells. The potential of immunofluorescence as a simple diagnostic method was demonstrated, which could be applied by incorporating antibodies targeting SARS-CoV-2 into multiplex immunofluorescence panels used clinically, such as for respiratory viruses, thus allowing additional routine testing for diagnosis and surveillance of SARS-CoV-2 even after the epidemic has ended with low prevalence of COVID-19.

SPECIFIC PROPERTIES OF TUBULIN IN THE PREFRONTAL CORTEX IN CONTROL, SCHIZOPHRENIA AND VASCULAR DEMENTIA

2020 ◽  
pp. 65-71
Author(s):  
Burbaeva G.Sh. ◽  
Androsova L.V. ◽  
Vorobyeva E.A. ◽  
Savushkina O.K.
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Prefrontal Cortex ◽  
Vascular Dementia ◽  
Binding Activity ◽  
Nephelometric Method ◽  
Rate Of Polymerization ◽  
Mental Diseases ◽  
And Control ◽  
The Brain

The aim of the study was to evaluate the rate of polymerization of tubulin into microtubules and determine the level of colchicine binding (colchicine-binding activity of tubulin) in the prefrontal cortex in schizophrenia, vascular dementia (VD) and control. Colchicine-binding activity of tubulin was determined by Sherlinе in tubulin-enriched extracts of proteins from the samples. Measurement of light scattering during the polymerization of the tubulin was carried out using the nephelometric method at a wavelength of 450-550 nm. There was a significant decrease in colchicine-binding activity and the rate of tubulin polymerization in the prefrontal cortex in both diseases, and in VD to a greater extent than in schizophrenia. The obtained results suggest that not only in Alzheimer's disease, but also in other mental diseases such as schizophrenia and VD, there is a decrease in the level of tubulin in the prefrontal cortex of the brain, although to a lesser extent than in Alzheimer's disease, and consequently the amount of microtubules.

184 Two types of anti-TIGIT antibodies with distinct binding epitope and functional activities

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A198-A198
Author(s):  
Tingting Zhong ◽  
Xinghua Pang ◽  
Zhaoliang Huang ◽  
Na Chen ◽  
Xiaoping Jin ◽  
...  
Keyword(s):  
T Cells ◽  
Mixed Culture ◽  
Cell Activity ◽  
Cross Reactivity ◽  
Binding Activity ◽  
Jurkat Cells ◽  
List Type ◽  
Dependent Manner ◽  
Vitro Assay

BackgroundTIGIT is an inhibitory receptor mainly expressed on natural killer (NK) cells, CD8+ T cells, CD4+ T cells and Treg cells. TIGIT competes with CD226 for binding with CD155. In cancers, CD155 has been reported to up-regulate on tumor cells, and TIGIT was found to increase on TILs.1 Activation of TIGIT/CD155 pathway would mediate immunosuppression in tumor; while blockade of TIGIT promotes anti-tumor immune response.MethodsAK126 and AK113 are two humanized anti-human TIGIT monoclonal antibodies developed by Akesobio. Binding activity of AK126 and AK113 to human TIGIT, and competitive binding activity with CD155 and CD112, were performed by using ELISA, Fortebio, and FACS assays. Cross-reactivity with cynomolgus monkey TIGIT and epitope binning were also tested by ELISA assay. In-vitro assay to investigate the activity to promote IL-2 secretion was performed in mixed-culture of Jurkat-TIGIT cells and THP-1 cells.ResultsAK126 and AK113 could specifically bind to human TIGIT with comparative affinity and effectively blocked the binding of human CD155 and CD112 to human TIGIT. X-ray crystal structure of TIGIT and PVR revealed the C’-C’’ loop and FG loop regions of TIGIT are the main PVR interaction regions.2 The only amino acid residue differences in these regions between human and monkey TIGIT are 70C and 73D. AK126 binds to both human and monkey TIGIT, AK113 binds only to monkey TIGIT. This suggests that these residues are required for AK113 binding to human TIGIT, but not required for AK126. Interestingly, results from cell-based assays indicated that AK126 and AK113 showed significantly different activity to induce IL-2 secretion in mixed-culture of Jurkat-TIGIT cells and THP-1 cells (figure 1A and B), in which AK126 had a comparable capacity of activity to 22G2, a leading TIGIT mAb developed by another company, to induce IL-2 secretion, while, AK113 showed a significantly higher capacity than 22G2 and AK126.Abstract 184 Figure 1Anti-TIGIT Antibodies Rescues IL-2 Production in Vitro T-Cell Activity Assay in a dose dependent manner. Jurkat-TIGIT cells (Jurkat cells engineered to over-express human TIGIT) were co-cultured with THP-1 cells, and stimulated with plate-bound anti-CD3 mAb in the presence of TIGIT ligand CD155 (A) or CD112 (B) with anti-TIGIT antibodies. After incubated for 48h at 37° C and 5.0% CO2, IL-2 levels were assessed in culture supernatants by ELISA. Data shown as mean with SEM for n = 2.ConclusionsWe discovered two distinct types of TIGIT antibodies with differences in both epitope binding and functional activity. The mechanism of action and clinical significance of these antibodies require further investigation.ReferencesSolomon BL, Garrido-Laguna I. TIGIT: a novel immunotherapy target moving from bench to bedside. Cancer Immunol Immunother 2018;67:1659–1667.Stengel KF, Harden-Bowles K, Yu X, et al. Structure of TIGIT immunoreceptor bound to poliovirus receptor reveals a cell-cell adhesion and signaling mechanism that requires cis-trans receptor clustering. Proc Natl Acad Sci USA 2012;109:5399–5404.

scholarly journals Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom

Toxins ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 254
Author(s):  
Shelby S. Szteiter ◽  
Ilse N. Diego ◽  
Jonathan Ortegon ◽  
Eliana Salinas ◽  
Abcde Cirilo ◽  
...  
Keyword(s):  
Snake Venom ◽  
Polyclonal Antibody ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Snake Envenomation ◽  
Disintegrin Domain ◽  
The Common ◽  
Neutralization Ability ◽  
New Strategies

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

The Role of Tanycytes in the Hypothalamus-Pituitary-Thyroid Axis and the Possibilities for Their Genetic Manipulation

2019 ◽  
Vol 128 (06/07) ◽  
pp. 388-394
Author(s):  
Helge Müller-Fielitz ◽  
Markus Schwaninger
Keyword(s):  
Energy Homeostasis ◽  
Genetic Manipulation ◽  
Heart Function ◽  
Feedback Regulation ◽  
Target Organs ◽  
Pituitary Thyroid Axis ◽  
Thyroid Axis ◽  
Hpt Axis ◽  
The Brain

AbstractThyroid hormone (TH) regulation is important for development, energy homeostasis, heart function, and bone formation. To control the effects of TH in target organs, the hypothalamus-pituitary-thyroid (HPT) axis and the tissue-specific availability of TH are highly regulated by negative feedback. To exert a central feedback, TH must enter the brain via specific transport mechanisms and cross the blood-brain barrier. Here, tanycytes, which are located in the ventral walls of the 3rd ventricle in the mediobasal hypothalamus (MBH), function as gatekeepers. Tanycytes are able to transport, sense, and modify the release of hormones of the HPT axis and are involved in feedback regulation. In this review, we focus on the relevance of tanycytes in thyrotropin-releasing hormone (TRH) release and review available genetic tools to investigate the physiological functions of these cells.

scholarly journals Characterization of a Gi-protein from Trypanosoma cruzi epimastigote membranes

1992 ◽  
Vol 287 (2) ◽  
pp. 443-446 ◽  
Author(s):  
O A Coso ◽  
A Díaz Añel ◽  
H Martinetto ◽  
J P Muschietti ◽  
M Kazanietz ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Pertussis Toxin ◽  
Polyclonal Antibodies ◽  
Gel Filtration ◽  
Binding Activity ◽  
Gi Protein ◽  
Liver Membranes ◽  
Blocking Capacity ◽  
Polypeptide Band

A guanosine 5′-[gamma-[35S]thio]triphosphate-binding activity was detergent-extracted from Trypanosoma cruzi membranes. This binding activity was co-eluted from gel-filtration columns with a factor which, in a heterologous reconstitution system, blocks glucagon stimulation of adenylate cyclase activity in liver membranes. ADP-ribosylation of these membranes by pertussis toxin eliminated this blocking capacity. Incubation of T. cruzi membranes with activated pertussis toxin and [adenylate-32P]NAD+ led to the incorporation of radioactivity into a labelled product with an apparent M(r) of approx. 43,000. Crude membranes were electrophoresed on SDS/polyacrylamide gels and analysed, by Western blotting, with GA/1 anti-alpha common, AS/7 anti-alpha t, anti-alpha i1 and anti-alpha i2 polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 anti-beta antibody, of about 30 kDa, was also detected in the membrane fraction.

scholarly journals Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants

1989 ◽  
Vol 259 (3) ◽  
pp. 847-853 ◽  
Author(s):  
I Benveniste ◽  
A Lesot ◽  
M P Hasenfratz ◽  
F Durst
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Higher Plants ◽  
Polyclonal Antibodies ◽  
Reductase Activity ◽  
Jerusalem Artichoke ◽  
Cross Reactivity ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Cytochrome P 450

Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals.

Brain size/body weight in the dingo (Canis dingo): comparisons with domestic and wild canids

2017 ◽  
Vol 65 (5) ◽  
pp. 292 ◽  
Author(s):  
Bradley P. Smith ◽  
Teghan A. Lucas ◽  
Rachel M. Norris ◽  
Maciej Henneberg
Keyword(s):  
Body Size ◽  
Brain Size ◽  
Cuon Alpinus ◽  
Size Relationship ◽  
Wild Canids ◽  
Free Ranging ◽  
The Impact ◽  
Endocranial Volume ◽  
The Brain

Endocranial volume was measured in a large sample (n = 128) of free-ranging dingoes (Canis dingo) where body size was known. The brain/body size relationship in the dingoes was compared with populations of wild (Family Canidae) and domestic canids (Canis familiaris). Despite a great deal of variation among wild and domestic canids, the brain/body size of dingoes forms a tight cluster within the variation of domestic dogs. Like dogs, free-ranging dingoes have paedomorphic crania; however, dingoes have a larger brain and are more encephalised than most domestic breeds of dog. The dingo’s brain/body size relationship was similar to those of other mesopredators (medium-sized predators that typically prey on smaller animals), including the dhole (Cuon alpinus) and the coyote (Canis latrans). These findings have implications for the antiquity and classification of the dingo, as well as the impact of feralisation on brain size. At the same time, it highlights the difficulty in using brain/body size to distinguish wild and domestic canids.

scholarly journals Rapid immunoassays for detection of anabolic nortestosterone in dietary supplements

2013 ◽  
Vol 31 (No. 5) ◽  
pp. 514-519 ◽  
Author(s):  
B. Holubová ◽  
S. Göselová ◽  
L. Ševčíková ◽  
M. Vlach ◽  
M. Blažková ◽  
...  
Keyword(s):  
Dietary Supplements ◽  
Rapid Detection ◽  
Visual Detection ◽  
Limit Of Detection ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Rapid Analysis ◽  
Experimental Conditions ◽  
Immunochromatographic Strip ◽  
Strip Test

An enzyme immunoassay (ELISA) and an immunochromatographic strip were designed for a rapid detection of nortestosterone in dietary supplements. Two polyclonal antibodies and two types of nortestosterone-protein coating conjugates were tested to develop the most appropriate method. Under optimal experimental conditions, the most sensitive ELISA achieved the IC<sub>50 </sub>and the limit of detection values of 6.41 and 0.09 ng/ml, respectively. The assay specificity was tested measuring cross-reactivity of several steroids. The interference with the assay was negligible (&lt; 0.1%), except for cross-reactivity with another frequently abused steroid testosterone (23%). The optimised gold particle-based immunochromatographic strip provided in semi-quantitative test a visual detection limit of 1&nbsp;ng/ml. None of these methods showed the interference using a filtrate of the suspension of non-contaminated sample. After the validation for particular matrices, the ELISA and the strip test could be useful tools for a rapid analysis of nortestosterone in crude extracts of dietary supplements.

Sign in / Sign up

Export Citation Format

Share Document