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An Introduction to the Immune System and Vaccines
Published in Patricia G. Melloy, Viruses and Society, 2023
There are a few ways to test for a viral infection. Scientists have found that antibody-antigen reactions can work in vivo (in the body) or in vitro (in a test tube). The interaction itself may cause a precipitation or clumping of the reacting material out of solution, which can be picked up by several tests. Scientists often do a type of test known as an enzyme-linked immunosorbent assay (ELISA), in which an antigen/antibody interaction can be quantified (Coico and Sunshine 2015). The test relies on a fluorescent tag on the antibody or an enzymatic reaction with a substrate that changes color to detect the antigen-antibody interaction. In an ELISA test, an interaction between an antigen (foreign material) and an antibody (protein made in response to a particular antigen) is detected using a plastic welled plate to perform the binding reactions. Plates can be examined by eye for a positive test or can be read in a machine to quantify the strength of the antigen-antibody interaction. Another more labor-intensive test, known as a western blot, involves separating proteins using agarose gel electrophoresis, transferring them to a membrane, and then probing for a particular antigen using an antibody (Coico and Sunshine 2015; Alberts et al. 2019). Antibody testing has become a powerful laboratory tool for the detection of viral infection.
The Viruses
Published in Julius P. Kreier, Infection, Resistance, and Immunity, 2022
The immunoblot assay also termed “Western blot” is performed by first solubilizing a viral preparation such as purified virus particles or virus-infected cells and separation of the mixture of molecules by gel electrophoresis and transfer of viral proteins to membranes. The membrane containing the viral proteins is then treated with antiserum followed by detection of bound antibody using radiolabeled or enzyme-labeled antispecies antibodies. The test allows the detection of antibodies to specific proteins of the virion and is useful to discriminate between infections by closely related viruses and is often used to confirm the results of ELISA tests.
Introduction to Retro Virology and Retroviral Testing
Published in Niel T. Constantine, Johnny D. Callahan, Douglas M. Watts, Retroviral Testing, 2020
Niel T. Constantine, Johnny D. Callahan, Douglas M. Watts
Presently, well over 100 different diagnostic assays from more than 40 commercial companies are available to detect antibodies to the retroviruses (Appendix A). Although the enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) assay are still the most popular, alternatives exist which offer a choice for different testing situations. However, local regulations may permit only certain tests to be used; for example, in the U.S. only FDA-licensed screening tests can be used. The tests currently in use in most countries are sensitive and specific, but are not free from false-positive and -negative results. Therefore, supplemental, confirmatory, or alternative tests must be used. However, even with the best tests, the quality of results critically depends on the proficiency of the individuals who perform the tests.
Suppression of prostate cancer and amelioration of the immunosuppressive tumor microenvironment through selective immunoproteasome inhibition
Published in OncoImmunology, 2023
Julia Koerner, Dennis Horvath, Franziska Oliveri, Jun Li, Michael Basler
Cells were lysed in RIPA buffer (50 mM Tris-buffered HCl, pH 7.5 containing 150 mM NaCl, 1% NP-40, 0.5% SDS) and tissue fractions were lysed in RIPA buffer supplemented with 2 mM Na3VO4, 10 mM NaF, 1 mM PMSF and complete protease inhibitor cocktail (Roche Pharmaceuticals) for 30 minutes on ice. Total protein in the supernatants of the cell extracts was quantified by the bicinchoninc acid method (Pierce BCA Protein Assay Kit, Thermo Scientific). Equal amounts of protein lysates (25 µg) were resolved under reducing conditions on 15% SDS-polyacrylamide gels for electrophoresis and transferred onto nitrocellulose-FL membranes (0.45 µm) (GE healthcare). Membranes were blocked in Odysee® blocking buffer (LI-COR Biosciences) for 1 h at RT and incubated using primary antibody in 1x Roti®-Block/TBS-T followed by fluorescently labeled IRDye® secondary anti-rabbit or anti-mouse Ig antibody (IRDye® 680, IRDye® 800, LI-COR Biosciences). A list of Western Blot antibodies can be found in the Supplementary Information. Protein bands were detected by NIR fluorescence using the Odyssey® Fc Infrared Imaging system (LI-COR Biosciences). Quantification of band intensities was performed with the aid of the Image Studio Lite software (LI-COR, Biosciences). To ascertain equivalent loading of the lanes and to normalize respective protein levels to internal loading controls, β-actin (1:5000, Cell Signaling) was used.
Kaempferol attenuates diabetic nephropathy in streptozotocin-induced diabetic rats by a hypoglycaemic effect and concomitant activation of the Nrf-2/Ho-1/antioxidants axis
Published in Archives of Physiology and Biochemistry, 2023
The western blot protocol is well-established in our laboratories. Total, cytoplasmic, and nuclear proteins (50 µg/well) were separated by SDS page, transferred onto nitrocellulose membranes, and then incubated with the primary antibodies against β-actin (Cat. No. 3700, 45 kDa), Cleaved Caspase-3 (Asp175) (Cat# 9664, 17/19 kDa), cytochrome-c (Cat# 11940, 14 kDa), Bcl2 (Cat# 8276, 28 kDa), Bax (Cat# 5023; 20 kDa), NF-κB p65 Antibody (Cat# 3034, 65 kDa), Nrf2 (Cat# 17212, 100 kDa), HO-1 (Cat# 5061, 28 kDa) (Cell Signalling Technology, USA), JNK1/2 (Cat. No. sc-137019, 46/54 kDa), p-JNK1/2 (Thr 183/Tyr 185) (Cat# sc-293136, 46/54 kDa), Lamin A (a nuclear marker) (Cat# sc-293162, 69 kDa), p38 MAPK (Cat# sc-81621, 38 kDa), p-p38 MAPK kDa (Tyr182) (Cat# sc-166182, 38 kDa), α-tubulin (a cytoplasmic marker) (Cat# sc-5286, 55 kDa) (Santa Cruze Biotechnology). Membranes were then incubated with the proper secondary HRP-conjugated antibody. Band detection was performed by enhanced Chemiluminescence and then scanned and analysed using the C-Di Git blot scanner (LI-COR, NE, USA). A single membrane was stripped up to four times.
Echinatin mitigates sevoflurane-induced hippocampal neurotoxicity and cognitive deficits through mitigation of iron overload and oxidative stress
Published in Pharmaceutical Biology, 2022
Zilong Xu, Yanqiu You, Qiuqin Tang, Hui Zeng, Tianshou Zhao, Juan Wang, Fujun Li
Then, we investigated the effects of Ech on oxidative stress and ferroptosis of hippocampal neurons. Results showed that the levels of ROS (Figure 2(A,B)) and MDA (Figure 2(C)) in hippocampal neurons were increased after Sev treatment, but this consequence was weakened after Ech treatment. On the contrary, the GSH level was decreased after Sev treatment, whereas this influence was attenuated by Ech treatment in hippocampal neurons (Figure 2(D)). On the other hand, the iron level in hippocampal neurons was increased by Sev treatment, however this elevation was attenuated after Ech treatment (Figure 2(E)). Subsequently, western blot was performed to detect the levels of related proteins. The ferritin level was increased but GPX4 level was decreased in hippocampal neurons with Sev treatment, but these changes were weakened after Ech treatment (Figure 2(F)). Therefore, these findings indicated that Ech inhibited oxidative stress and ferroptosis in hippocampal neurons with Sev treatment.