The choice of an antibody for a protein-based research study is one of the most crucial steps in any project. Seemingly straightforward, the process is actually quite nuanced and filled with ...potential pitfalls. In this chapter, we will discuss five major topics that require consideration in the antibody selection process. These include overall study objectives and resources, details of both species and clonality, suitability in applications, and available detection methods. Each section will provide background information on the topic as well as specifics of antibody use in the laboratory. This chapter may be used as a guide to help vet antibody candidates for your project so you can stain with confidence.
The flowers of Nyctanthes arbor-tristis (L.) heals mouth ulcers. Its tinctures promote gastric secretions, and improve lung expectoration when taken orally. It has traditionally been used to treats ...scabies and other skin problems. The leaves of NAT(L.) plant are used in Ayurvedic medicine to treat sciatica, chronic fever, rheumatism, internal worm infections, and as a laxative, diaphoretic, and diuretic. The bark used in treatment of snakebite and bronchitis. In addition to traditional uses, pharmacologically this plant has potent antimalarial, antiarthritic, anticancer and antidiabetic activity. However, the mechanistic antiproliferative potentials of NAT(L.) flower as anticancer therapeutics has not yet been explored.
The current study is based on a broad range of scientific literature that highlights the nutritional and therapeutic benefits of NAT (L.). Present investigation was carried out to determine the therapeutic efficacy of NAT (L.) against breast adenocarcinoma cells and T-cell lymphoma.
The ethyl-acetate extract of NAT(L.) was tested against breast cancer cells to assess the anticancer potential. To evaluate apoptosis, intracellular ROS levels and mitochondrial dynamics, fluorescence microscopy and flow cytometry were employed. Additionally, cell cycle analysis and western blotting were also performed. Furthermore, in vivo antitumor efficacy of flower extracts was investigated in T-cell lymphoma-bearing BALB/c mice model.
Our present study revealed that NAT (L.) exert anticancer activity against breast cancer cells effectively at IC50 320 μg/ml while having less impact on normal cells with IC50 more than 480 μg/ml. Fluorescence imaging showed that NAT (L.) treatment elicits a concentration-dependent rise in the occurrence of apoptotic cell deaths with altered mitochondrial dynamics and was subsequently confirmed by flow cytometry. Further, flow cytometric analysis delineates ethyl acetate flower extract exposure promotes arrest of cells in S phase of the cell cycle. The differential expression of apoptotic proteins such as Bax, Bcl-2, cleaved PARP-1, cleaved caspase 3, Cytochrome-c, p53 and VEGF A were influenced by NAT (L.) treatment. The in vivo antitumor activity study delineates that NAT(L.) therapy significantly increased the life span of T-cell lymphoma bearing mice while reducing tumor load and belly size growth pattern without causing significant other distinct side effects as evident by histopathological studies.
Our current findings unveil that NAT(L.) ethyl acetate flower extract potentially induces mitochondrial pathway of apoptosis, promote cell cycle arrest, reduces tumor load of mice, enhances survivability and could be a promising agent against the triple negative breast cancer and lymphoma.
Graphical representation of NAT(L.) EFE preparation and plausible mechanism of apoptosis after treatment. Display omitted
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•Edible insects are increasingly promoted as an efficient and sustainable food source.•Even if allergy to insects by ingestion is recognized, only a few studies are actually available.•Allergenicity ...of arginine kinase (AK) from mealworms and crickets is comparatively investigated.•Whole insects and purified proteins sources show different allergenic reactions based on entomological staff sera.•Low specific allergenicity to AK is discussed in relation to insect safety for human food.
Insects are seen as a solution to the increasing demand for protein sources for food. However, entomophagy has unfortunately been linked to allergic reactions in Europe with people with professional contacts. As mealworms (Tenebrio molitor) and crickets (Acheta domesticus) have recently become commercially available (both whole or in food formulation) in several European countries, this research assessed the cross allergenicity of arginine kinase (AK). Based on the collection of sera from a entomology laboratory staff, oven cooked insects but also purified AK fractions were tested. Immunoblotting against the protein extracts revealed different Immunoglobulin E reactivity of sera according to the insect target species: two bands (40 and 14 kDa) for crickets and a pattern including light responses at 17, 25 and 37 kDa for mealworms. Focusing on AK, low specific allergenicity was here illustrated and discussed in relation to the development of a safe edible insect consumption by humans.
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Les sarcoglycanopathies sont des maladies musculaires génétiques à transmission autosomique récessive. Elles sont dues à une déficience d’une protéine du complexe sarcoglycane entraînant une ...instabilité de la membrane cellulaire.
Étudier les caractéristiques phénotypiques et immunologiques des différents types des sarcoglycnopathies par étude immunohistochimique (IHC) et par Western Blot Multiplex (WB).
Notre étude est menée sur 29 patients appartenant à 18 familles tunisiennes présentant les différents types des sarcoglycanopathies. Nous avons étudié les caractéristiques cliniques de nos patients. L’atteinte cardiaque et respiratoire était recherchée par la réalisation d’une échographie cardiaque et d’une épreuve fonctionnelle respiratoire. La déficience protéique en protéines du complexe sarcoglycane chez nos patients a été démontrée par IHC et par WB.
Par l’IHC et WB nous avons révélé l’absence des sarcoglycanes : α chez 9 patients, β chez 3 patients et la γ chez 11 patients. Aucune déficience n’a été détectée pour la sarcoglycane δ. Cependant, nous avons détecté l’absence de plus d’une protéine chez 6 patients. L’atteinte clinique était variable chez nos patients : 67 % des patients avaient une hypertrophie des mollets, une cardiomyopathie était notée chez 21 % patient et un seul patient avait une macroglossie.
Conformément aux données de la littérature, la sarcoglycanopathie γ était la forme la plus fréquente dans notre série. Une variabilité phénotypique était notée chez nos patients. L’atteinte cardiaque était aussi fréquente dans notre groupe. L’immunomarquage seul ne peut pas être spécifique d’un déficit protéique. Dans ce cas, une étude génétique s’avère indispensable pour la confirmation de diagnostic.
Malgré la similarité de phénotype entre les sarcoglycanopathies certaines particularités permettent de les distinguer. Une étude immunologique était indispensable pour orienter le diagnostic. L’étude génétique reste nécessaire pour le confirmer.
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The objective of this study was to analyze the immune responses of bucks to small ruminant lentivirus (SRLV) with a focus on the reproductive system of males with recent and chronic infection. A ...total of 12 bucks were selected, six seronegative and six seropositive with chronic natural infection for more than 18 months (chronic infection group). After selecting the animals, the six seronegative males were intravenously inoculated with caprine arthritis-encephalitis virus (CAEV)-Co viral strain at a titer of 10-5,6 TCID50/mL. After viral inoculation, this group was called the recent infection group and was monitored weekly with the chronically infected group for 180 days with blood serum and seminal plasma Western Blot (WB) analysis. Of the animals with chronic SRLV infection, 18.94% (50/264) showed anti-SRLV antibodies in at least one of the samples, and 81.06% (214/264) were negative. Anti-SRLV antibodies were detected in 27.27% (36/132) of the blood serum samples from this group, while 10.60% (14/132) were reactive in the seminal plasma WB test. The animals inoculated with CAEV-Co became seropositive after the third week of viral inoculation. In this group, 31.06% (41/132) of seminal plasma samples had anti-SRLV antibodies, and of these, 70.73% (29/41) coincided with blood serum results. Of the remaining 29.27% (12/41), the seminal plasma sample of only three animals (RIA2, RIA3, and RIA5) had anti-SRLV antibodies. One of the animals with a recent infection presented anti-SRLV antibodies only in seminal plasma samples, possibly due to virus compartmentalization. Intermittent viral shedding was observed in both biological samples, regardless of the infection stage. The immune response in bucks with recent SRLV infection is more significant than in chronically infected animals. Regardless of the stage of infection, there is a fluctuation in antibody levels, therefore, this creates a risk of false-negative samples when performing the diagnosis.
•Ultrasound (PUS) could increase the WHC and tenderness of beef during curing.•LF-NMR analysis showed that PUS could induce the higher P21 values of beef.•Higher ratio of myosin oligomer was related ...to the increased WHC of beef by PUS.•PUS could increase the MFI values and the extent of myofibril proteolysis of beef.
The objective of this study was to explore the mechanisms of power ultrasound (PUS, 150 and 300W) and treatment time (30 and 120min) on the water-holding capacity (WHC) and tenderness of beef during curing. Beef muscle at 48h post mortem was subjected to PUS treatment at a frequency of 20kHz. Analysis of compression loss and shear force showed that PUS-assisted curing significantly increased the WHC and the tenderness of beef compared to static brining (p<0.05). According to the analysis of LF-NMR, PUS treatment could increase the P21 values which indicated an improvement in water-binding ability of beef muscle. SDS-PAGE and LC-ESI-MS/MS analysis suggested that PUS induced moderate oxidation of myosin causing polymerization, which may contribute to increased water retention. On the other hand, an increased tenderness of beef is suggested by the increased MFI values and proteolysis of desmin and troponin-T. Transmission electron microscopy (TEM) further supported the effects of PUS on WHC and tenderness changes due to the swelling and disruption of myofibrils. Thus, these results provide knowledge about the mechanism for improving WHC and tenderness of beef by PUS curing, which could be employed as an emerging technology for various meat curing processes.
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Background
: IgG detection to determine immune status to
Toxoplasma gondii
infection and seroconversion mainly relies on ELISA techniques and, if necessary, on a confirmatory test, Western blot. This ...study evaluated the performance of the
recom
Line
Toxoplasma
IgG immunoblot (IB-
recom
Line) (Mikrogen) as a confirmatory test on a large number of sera. A total of 171 sera were selected (113 patients) and had previously been analyzed by two ELISA tests, ARCHITECT (Abbott) and VIDAS (bioMérieux) ± LDBIO-Toxo II IgG Western blot (WB-LDBIO) (LDBio). The sera were classified into three groups: group 1 included 50 sera without difficulty in interpreting the IgG results (patients with documented past infection or uninfected); group 2 included 47 sera with difficulty in interpreting the ELISA results; and group 3 included 74 sequential sera from 25 pregnant women with seroconversion.
Results
: In group 1, overall IgG agreements were 94% and 90% with ARCHITECT and VIDAS, respectively. In group 2, low agreement was observed between IB-
recom
Line and WB-LDBIO, with eight false-positive and 13 false-negative results. In group 3, 4/13 seroconversions were detected earlier with IB-
recom
Line compared to other tests.
Conclusions
: IB-
recom
Line allowed for earlier diagnosis of toxoplasmic seroconversion compared to both ELISA tests and WB-LDBIO but led to insufficient performance to confirm the immune status when ELISA results were discordant or equivocal.
Contexte
: La détection des IgG pour déterminer le statut immunitaire vis-à-vis de l’infection à
Toxoplasma gondii
et de la séroconversion repose principalement sur les techniques ELISA et, si nécessaire, sur un test de confirmation, le Western blot. Cette étude a évalué les performances de l’immunoblot
recom
Line
Toxoplasma
IgG (IB-
recom
Line) (Mikrogen) en tant que test de confirmation sur un grand nombre de sérums. Un total de 171 sérums ont été sélectionnés (113 patients) et ont été préalablement analysés par deux tests ELISA, ARCHITECT (Abbott) et VIDAS (bioMérieux) ± LDBIO-Toxo II IgG Western blot (WB-LDBIO) (LDBio). Les sérums ont été classés en trois groupes : le groupe 1 comprenait 50 sérums sans difficulté d’interprétation des résultats IgG (patients avec antécédents d’infection documentée ou non infectés); le groupe 2 comprenait 47 sérums avec des difficultés d’interprétation des résultats ELISA; le groupe 3 comprenait 74 sérums séquentiels de 25 femmes enceintes séroconverties.
Résultats
: Dans le groupe 1, les concordances globales des IgG étaient respectivement de 94 % et 90 % avec ARCHITECT et VIDAS. Dans le groupe 2, une faible concordance a été observée entre IB-
recom
Line et WB-LDBIO, avec huit faux positifs et 13 faux négatifs. Dans le groupe 3, 4/13 séroconversions ont été détectées plus tôt avec IB-
recom
Line par rapport aux autres tests.
Conclusions
: IB-
recom
Line a permis un diagnostic plus précoce de la séroconversion toxoplasmique par rapport aux tests ELISA et WB-LDBIO, mais a conduit à des performances insuffisantes pour confirmer le statut immunitaire lorsque les résultats ELISA étaient discordants ou équivoques.
Since its first description, Western blot has been widely used in molecular labs. It constitutes a multistep method that allows the detection and/or quantification of proteins from simple to complex ...protein mixtures. Western blot quantification method constitutes a critical step in order to obtain accurate and reproducible results. Due to the technical knowledge required for densitometry analysis together with the resources availability, standard office scanners are often used for the imaging acquisition of developed Western blot films. Furthermore, the use of semi-quantitative software as ImageJ (Java-based image-processing and analysis software) is clearly increasing in different scientific fields. In this work, we describe the use of office scanner coupled with the ImageJ software together with a new image background subtraction method for accurate Western blot quantification. The proposed method represents an affordable, accurate and reproducible approximation that could be used in the presence of limited resources availability.
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