Hybridoma Screening by Antigen Capture: Capture or Sandwich ELISA in 96-Well Plates

In an antigen capture assay for hybridoma screening, the detection method identifies the presence of the antigen. Often this is achieved by labeling the antigen directly. In this assay, the polyvinyl chloride (PVC) wells of a high-binding-capacity ELISA plate are first coated with an affinity-purified rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are “captured” on the coated PVC surface and detected by screening with biotin- or histidine (His)–tagged antigen. The antigen can be labeled to a high specific activity and thus very little antigen is required for this procedure.

Molecular Characterization and Pathogenicity of Trichoderma Isolates to Meloidogyne javanica

Nematodes are considered a serious problem for agriculture. Nematodes of the Meloidogyne genus can attack a wide range of plants, needing different management methods to decrease its population. Fungi from the Trichoderma genus has been related to have potential as biological control agents. However, before an organism is used as biological control agent, first it is necessary to prospect, characterize and test its potential as biocontrol agent, so the objective of this work was to characterize and test fungi isolates of the Trichoderma genus to control M. javanica. We obtained forty isolate to carry out this experiment. We extracted the DNA of each isolate to discover which species we were testing, by doing a PCR and sequencing. We tested in vitro their parasitism effect using ELISA plate. Also, we extracted their filtrate to see if their metabolites have potential to reduce nematode population by showing a high mortality or inhibiting hatching. The results confirmed the high potential of the fungi of Trichoderma genus as a biological agent to control Meloidogyne javanica.

ANTI BACTERIAL AND BIOFILM INHIBITORY ACTIVITIES OF AEGLE MARMELOS METHANOL LEAF EXTRACT

The Aegle marmelos commonly known as BAEL belongs to family Rutaceae plays a role in traditional culture and medication from ancient periods. This plant lacks sufficient evidences regarding the values and components it has. Therefore, we framed out our studies to evaluate the phytochemical analysis, antibacterial activity, antibiofilm activity. These studies are evaluated using different solvents like methanol, acetone, chloroform, toluene leaf extracts of Aegle marmelos. We evaluated the potency of different solvents leaf extracts using Agar well diffusion method. Antibacterial activity was also evaluated using ELISA plate assay. The potency of different solvents extracts to inhibit biofilm of selected microbial strains. In accordance to results, the leaf extracts revealed the presence of several biologically active phytochemicals with highest quantities of carbohydrates, phenols, alkaloids, flavonoids, tannins, saponins, steroids, aminoacids etc. The antibacterial activity was found significant against microbial strains of both gram positive and gram negative bacteria. These strains showed susceptibility nature towards the different solvents extracts with zone of inhibitions (mm). On the other hand, the inhibition of biofilm was also significant at all tested concentrations. The biofilm inhibition of microbial strains was found significant at 1 XMIC, 2 XMIC, 3 XMIC. Based on our studies here we conclude that the different solvents leaf extracts possessed inhibitory activity against selected human pathogenic organisms.

Factor H Is Bound by Outer Membrane-Displayed Carbohydrate Metabolism Enzymes of Extraintestinal Pathogenic Escherichia coli and Contributes to Opsonophagocytosis Resistance in Bacteria

Extraintestinal pathogenic Escherichia coli (ExPEC) causes bloodstream infections in humans and animals. Complement escape is a prerequisite for bacteria to survive in the bloodstream. Factor H (FH) is an important regulatory protein of the complement system. In this study, ExPEC was found to bind FH from serum. However, the mechanisms of ExPEC binding to FH and then resistance to complement-mediated attacks remain unclear. Here, a method that combined desthiobiotin pull-down and liquid chromatography-tandem mass spectrometry was used to identify the FH-binding membrane proteins of ExPEC. Seven identified proteins, which all were carbohydrate metabolic enzymes (CMEs), including acetate kinase, fructose-bisphosphate aldolase, fumarate reductase flavoprotein subunit, L-lactate dehydrogenase, dihydrolipoamide dehydrogenase, phosphoenolpyruvate synthase, and pyruvate dehydrogenase, were verified to recruit FH from serum using GST pull-down and ELISA plate binding assay. The ELISA plate binding assay determined that these seven proteins bind to FH in a dose-dependent manner. Magnetic beads coupled with any one of seven proteins significantly reduced the FH recruitment of ExPEC (p < 0.05) Subsequently, immunofluorescence, colony blotting, and Western blotting targeting outer membrane proteins determined that these seven CMEs were located on the outer membrane of ExPEC. Furthermore, the FH recruitment levels and C3b deposition levels on bacteria were significantly increased and decreased in an FH-concentration-dependent manner, respectively (p < 0.05). The FH recruitment significantly enhanced the ability of ExPEC to resist the opsonophagocytosis of human macrophage THP-1 in an FH-concentration-dependent manner (p < 0.05), which revealed a new mechanism for ExPEC to escape complement-mediated killing. The identification of novel outer membrane-displayed CMEs which played a role in the FH recruitment contributes to the elucidation of the molecular mechanism of ExPEC pathogenicity.

Antioxidant Activity and Nutraceutical Potential of Selected Nepalese Wild Edible Fruits

Wild edible fruits play an important role in the nutrition of rural people especially in the hilly and mountainous region, where the wild fruits could be the only source to consume. Though wild edible fruits are widely utilized throughout the country, little works have been done in Nepal on their nutritional and phytochemical analysis. The main objective of this study was to evaluate the antioxidant activity and nutraceutical potential of the selected wild edible fruits. The fruit samples were extracted in appropriate solvents and all the analyses were done in triplicates using 96 well ELISA plate reader. Nutritionally, Rubus acuminatus was found to be rich in Vitamin C (0.78 ± 0.01 mg/g) over other fruits. Protein content was found to be high in Berberis napaulensis (2.26 ± 0.71 %) and R. ellipticus showed greater lipid (0.15 ± 0.01 %) and β-carotene content (1.08 ± 0.01 mg/100mg). R. acuminatus was found to have high flavonoid content (9.26 ± 0.40 mg QE/g) and exhibited higher antioxidant activity while B. angulosa (29.67 ± 2.28 mg GAE/g) had the highest phenolic content.

A commercial ELISA for detection of interferon gamma in white rhinoceros

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is endemic in Kruger National Park, South Africa, home to the largest population of white rhinoceros ( Ceratotherium simum) in the world. In 2016, the first cases of naturally occurring bTB were reported in white rhinoceros; however, there is a lack of understanding of infection and disease process in this species. Prevention and control of transmission depends on the availability of accurate tools to detect M. bovis infection. Interferon gamma (IFN-γ) assays are a reliable detection method for TB in other animal species, and studies have indicated that these tests can be used in white rhinoceros. We sought to screen and optimize a commercial IFN-γ enzyme-linked immunosorbent assay (ELISA) to detect endogenous white rhinoceros IFN-γ in mitogen-stimulated whole blood as a basis for developing a test for M. bovis infection. Optimizations included identifying ELISA antibodies and determining the effect of sample matrix, ELISA plate incubation temperature, ELISA linearity, assay reproducibility, and the assay’s limit of quantification. The optimized assay employed an equine IFN-γ antibody pair that was used to create a commercial ELISA kit. This ELISA had a linear response to recombinant equine and endogenous rhinoceros IFN-γ (range: 7.8–125 pg/mL). When incubated at 37°C, the ELISA was highly reproducible, with an optimal recovery and a low limit of quantification, indicating that the Mabtech equine IFN-γ ELISAPRO kit is a robust assay for measuring white rhinoceros IFN-γ.

Prediction of haematocrit in dried blood spots from the measurement of haemoglobin using commercially available sodium lauryl sulphate

Background When preparing dried blood spots (DBSs), haematocrit (Hct) can affect the ability of the blood to spread through the filter paper, thus resulting in varying quantities of sample being measured when fixed subpunches of the DBSs are taken. It may be important to predict the sample Hct to correct volume differences. Methods Blood (10 µL) was applied to Perkin Elmer 226® paper. The samples ( n = 165) were allowed to dry for 24 h, and the entire blood spots were cut out. Subpunch analysis was also performed on blood spots prepared from 75 µL EDTA blood, taking 6 mm subpunches centrally and peripherally from the spots ( n = 59). The spots were eluted with 100 µL water, and a 10 µL aliquot of lysate was added to sulfolyser reagent (80 µL) in a microtitre plate. Hb was measured at 550 nm using an ELISA plate reader. DBS samples were compared against blood samples measured on a routine Sysmex XN-9000 analyser. Results The Passing and Bablock regression showed Hct (DBS-predicted) = 0.99 Hct (Sysmex) −0.02, R2 = 0.87. Intra-assay imprecision measured at Hct values of 0.27, 0.40 and 0.52, gave CVs of 4.1%, 2.8% and 4.2%, respectively. Inter-assay imprecision showed CVs of 6.2%, 5.2% and 4.2%, respectively. DBS samples were stable for up to two days at 60℃, one month at room temperature and six months at 4℃. Conclusion This method provides a simple and fast estimation of predicted Hct in dried blood spots.

Pengaruh variansi konsentrasi ekstrak kulit batang jambu mete terhadap sitotoksikitas sel fibroblas

The influence of cashew stembark extract on citotoxicity fibroblast. The aim of this study was to determine the effect of variation in the concentration of cashew stem bark extract as the base material of mouthwash of the cytotoxic effect on fibroblast cells. The material used in this study was cashew stem bark extracted by maceration method using 70% of ethanol. A total of 15 samples were grouped into 5, each of which consisted of 3 samples (ISO 10993-5). Concentrations used were 1.6%, 0.8%, 0.4%, 0.2% and 0.05%. Cytotoxicity test used the MTT method by comparing the optical density (ELISA plate reader) between treated groups with control groups. Cell viability was obtained by comparing the treated groups with control groups. Cell viability data was analyzed using one-way ANOVA and LSD. The results showed that cashew stem bark has an anticardia acid. Cytotoxicity test used the mean of fibroblast cell viability due to various cashew stem bark extracts successively from concentrations 1.6%, 0.8%, 0.4%, 0.2% and 0.05% with the mean of 15.35 ± 0.443%, 30.84% ± 1.59, 47.78 ± 8.09%, 65.74% ± 3.20, 74.95 ± 7.26%. ANOVA showed a significant influence of various cashew stem bark on cell viability (p<0,05). The results of LSD showed a significant difference between treated groups except between concentrations 0.95% and 0.2%. In conclusion, Cashew extract have anacardic acid and there was influence on various cashew stem bark extract concentrations on the cytotoxicity of fibroblast cell. The concentration of 2% was not cytotoxic.ABSTRAKTujuan penelitian ini adalah untuk mengetahui kandungan ekstrak kulit batang jambu mete dan pengaruh variasi konsentrasi terhadap sitotosisitas sel fibroblas. Penelitian ini menggunakan bahan kulit batang jambu mete (Mojolegi) yang diindentifikasi dan diekstrak menggunakan metode maserasi dengan pelarut etanol 70%. Ekstrak diuji kandungannya menggunakan metode KLT (Kromatografi Lapis Tipis). Uji sitotoksikistas menggunakan sampel sejumlah 15 dikelompokkan menjadi 5, masing-masing kelompok 3 (ISO-10993-5). Variasi konsentrasi adalah 1,6%, 0,8%, 0,4%, 0,2% dan 0,05%. Uji sitotoksikitas menggunakan metode MTT dengan cara membandingkan optical density (ELISA plate reader) antar kelompok perlakuan dengan kelompok kontrol. Viabilitas sel didapatkan dengan membandingkan nilai optical density pada kelompok perlakuan dan kelompok kontrol. Data viabilitas sel dianalisis menggunakan ANAVA satu jalur dan LSD. Hasil penelitian menunjukkan rerata ekstrak mengandung senyawa asam anakardat dan asam galat. Uji sitotoksikistas sel fibroblas akibat variasi ekstrak kulit batang jambu mete secara berturut-turut dari konsentrasi 1,6%, 0,8%, 0,4%, 0,2% dan 0,05% dengan rerata sebesar 15,35% ± 0,443, 30,84% ± 1,59, 47,78% ± 8,09, 65,74% ± 3,20, 74,95% ± 7,26. Uji ANAVA menunjukkan adanya pengaruh variasi konsentrasi ekstrak kulit batang jambu mete bermakna terhadap viabilitas sel (p<0,05). Hasil uji LSD menunjukkan bahwa terdapat perbedaan yang bermakna (p<0,05) antar kelompok perlakuan, kecuali antara konsentrasi 0,05% dengan konsentrasi 0,2%. Kesimpulan Ekstrak kulit batang jambu mete mengandung asam anakardat dan asam galat dan terdapat pengaruh variasi konsentrasi ekstrak kulit batang jambu mete terhadap sitotoksikitas sel fibroblas. Konsentrasi 0,2% merupakan konsentrasi yang tidak toksis terhadap sel fibroblas secara in vitro.

Use of Heavy Water (D2O) in Developing Thermostable Recombinant p26 Protein Based Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Equine Infectious Anemia Virus Infection

Thermostabilizing effect of heavy water (D2O) or deuterium oxide has been demonstrated previously on several enzymes and vaccines like oral poliovirus vaccine and influenza virus vaccine. In view of the above observations, effect of heavy water onin situthermostabilization of recombinant p26 protein on enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of equine infectious anemia virus (EIAV) infection was investigated in the present study. The carbonate-bicarbonate coating buffer was prepared in 60% and 80% D2O for coating the p26 protein in 96-well ELISA plate and thermal stability was examined at 4°C, 37°C, 42°C, and 45°C over a storage time from 2 weeks to 10 months. A set of positive serum (n=12) consisting of strong, medium, and weak titer strength (4 samples in each category) and negative serum (n=30) were assessed in ELISA during the study period. At each time point, ELISA results were compared with fresh plate to assess thermal protective effect of D2O. Gradual increase in the stabilizing effect of 80% D2O at elevated temperature (37°C < 42°C < 45°C) was observed. The 80% D2O provides the thermal protection to rp26 protein in ELISA plate up to 2 months of incubation at 45°C. The findings of the present study have the future implication of adopting cost effective strategies for generating more heat tolerable ELISA reagents with extended shelf life.