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A Comprehensive HLA-DRB, -DQB, and -DPB Oligotyping Procedure by Hybridization with Sequence-Specific Oligonucleotide Probes
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
Recently, on the bases of the reverse dot blot technology40 and of the sandwich hybridization, this DR generic typing procedure has been further simplified by using oligonucleotide-coated microtiter plates for a 1 h-hybridization step at 37°C, followed by automated washing and colorimetric reading of the plates.49 The use of 20 oligonucleotides and of a single DR generic PCR allows the discrimination of 30 DRB specificities in a single microtiter plate assay.
Screening Tests for HIV-1 Infection
Published in Niel T. Constantine, Johnny D. Callahan, Douglas M. Watts, Retroviral Testing, 2020
Niel T. Constantine, Johnny D. Callahan, Douglas M. Watts
Not long after the HIV ELISAs were marketed, other techniques became available. Latex, red cell, and gelatin particle agglutination tests were introduced in order to offer alternatives for ease of performance and for cost savings. Subsequently, the simple-to-perform dot-blot assays were introduced.
Paracoccidioides Complex
Published in Rossana de Aguiar Cordeiro, Pocket Guide to Mycological Diagnosis, 2019
Zoilo Pires de Camargo, Anderson Messias Rodrigues
Tests for the detection of antibodies against P. brasiliensis are useful and were first used as a diagnostic aid in the 1960s (Fava-Netto, 1961), although after decades of studies, the establishment of an ideal assay for PCM presenting high sensitivity and specificity was not found. The existence of cross-reacting antigens among the dimorphic fungi, due to the complexity of the fungal cell wall, limits the specificity of the serological reactions. Furthermore, antigenic preparations obtained from P. brasiliensis currently used were developed empirically. Exceptionally, the immunodiffusion test for PCM has proven very useful in routine laboratory so far. Most antigenic preparations used for testing are derived from crude culture filtrates containing a range of metabolic products (de Camargo et al., 1988). In addition, purified antigens such as glycoprotein of 43,000 daltons (gp43) and other purified molecules have also been used for serological diagnosis. Other assays such as ELISA (Albuquerque et al., 2005), dot blot (Taborda & Camargo, 1994), and Western blot (Camargo et al., 1989) may be used as adjuncts for diagnosis. More recently, a simple slide agglutination test using latex particles was proposed for diagnostic purposes, for detection of antigens and antibodies circulating in biological fluids of patients, with excellent results (Dos Santos et al., 2015). The serological tests above are not limited to diagnosis but are also useful to monitor the patient response to therapy (Marques-da-Silva et al., 2004; da Silva et al., 2004).
Characterization of DNA hydroxymethylation profile in cervical cancer
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Jing Wang, Yi Su, Yongju Tian, Yan Ding, Xiuli Wang
Dot blot assay was performed as previously described [16]. In brief, the patients’ cervicitis or CSCC tissues were extracted by genomic DNA using QIAquick Gel Extraction Kit (Qiagen, Dusseldorf, Germany). After quantifying and diluting the equal DNA concentration, each sample (2 μl) was dropped on nitrocellulose (NC) membranes with double proportion dilution (0, 5, 10, 20 and 40 ng) for dot blot assay. NC membranes were naturally dried at room temperature (RT) and incubated with 5hmC (1:500 dilution, Abcam, ab106918, Cambridge, UK) or 5mC antibodies (1:500 dilution, Abcam, ab10805) in TBST 4 °C overnight, followed by washing by TBST three times for 10 min in room temperature. followed by appropriate secondary antibodies (1:5000 dilution, Beyotime, Shanghai, China) in 10 ml of TBST for 1 h at room temperature with gentle shaking and washed again for three times and washed again for three times. Finally, the spots on NC membranes were developed with 3 ml of DAB substrate/chromogen for 5 min in darkness and analyzed the gray values.
Tetraspanin-decorated extracellular vesicle-mimetics as a novel adaptable reference material
Published in Journal of Extracellular Vesicles, 2019
Estefanía Lozano-Andrés, Sten F. Libregts, Victor Toribio, Félix Royo, Sara Morales, Soraya López-Martín, Mar Valés-Gómez, Hugh T. Reyburn, Juan Manuel Falcón-Pérez, Marca H. Wauben, Manuel Soto, María Yáñez-Mó
The requirement for suitable standards for EV isolation, characterisation and analysis methods in the fast developing EV-field is obvious [38], particularly since the techniques for EV isolation and characterisation are constantly evolving, and their applicability is exponentially increasing. Appealing features such as size distribution, morphology and particle concentration make these bioengineered nanovesicles suitable for different EV isolation and detection procedures. We have chosen to initiate the development of EVMs with tetraspanin LELs, but the technology that we describe here is adaptable to the incorporation of different protein markers. By taking advantage of the high-affinity interaction between (strept)avidin and biotin molecules it is possible to perform surface-decoration with any potential protein of interest, which make these nanovesicles tuneable. In addition, they can be generically stained with fluorescent dyes. Since the here-described surface-decorated niosomes display similar biophysical properties as naturally occurring EVs, and are suited for staining with antibodies directed against the most widely used tetraspanins within the EV-field, they can be powerful intrinsic controls for EV characterisation platforms like NTA, Electron Microscopy and FC. Additionally, they are detectable by different bulk-based analysis techniques, such as dot blot, western blot and bead-based FC. Moreover, because of their specific density properties, they could be easily adapted as spike-ins for robust assessment in all these techniques.
Levels of Anti-Retinal Antibodies in Retinal Detachment and Proliferative Vitreoretinopathy
Published in Current Eye Research, 2018
Reo Ichinohasama, Koji M Nishiguchi, Kosuke Fujita, Naoko Aizawa, Takashi Inoue, Erika Sasaki, Hiroshi Kunikata, Toru Nakazawa
The dot-blot assay represents a simplification of the western blot methods in which the retinal protein to be detected is not separated by electrophoresis. Although it offers no information on the size of the interacting retinal protein, it is useful for judging the presence or absence of anti-retinal antibodies and quantifying their total amount. The dot-blot assay to measure the levels of anti-retinal IgG antibodies used marmoset retinal cells. An isolated marmoset retina was lysed in RIPA buffer containing a protease inhibitor cocktail (Roche, Basel, Switzerland) and sonicated twice for 30 s on ice. TP concentration in the retina was measured using a Pierce BCA protein assay kit (Thermo Fisher). TP from the retinal lysate (1 and 0.3 µg) was blotted onto Amersham Protran 0.45 NC nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). The membranes were air-dried, rinsed once in PBS-T, blocked in 5% non-fat milk/PBS-T for 30 min, and then incubated with patient aqueous humor or vitreous at 1/100 dilution for 1 h. Membranes were washed three times with PBS-T (5 min/wash) and then incubated with HRP-conjugated antihuman IgG antibodies (1/50 000; Merck Millipore, Billerica, MA) for 0.5 h. The signal was detected with ECL prime (GE Healthcare) and analyzed with ImageJ software. When there was an obvious discrepancy between the intensity of the 1- and 0.3-µg dots, the serum was retested. Measurements of the 0-µg dots were subtracted from corresponding 1-µg dots to determine the level of anti-retinal antibodies.