Concept: Secondary antibody
In resource-constrained countries, affordable methodologies for the detection of disease biomarkers at ultralow concentrations can potentially improve the standard of living. However, current strategies for ultrasensitive detection often require sophisticated instruments that may not be available in laboratories with fewer resources. Here, we circumvent this problem by introducing a signal generation mechanism for biosensing that enables the detection of a few molecules of analyte with the naked eye. The enzyme label of an enzyme-linked immunosorbent assay (ELISA) controls the growth of gold nanoparticles and generates coloured solutions with distinct tonality when the analyte is present. Prostate specific antigen (PSA) and HIV-1 capsid antigen p24 were detected in whole serum at the ultralow concentration of 1 × 10(-18) g ml(-1). p24 was also detected with the naked eye in the sera of HIV-infected patients showing viral loads undetectable by a gold standard nucleic acid-based test.
The recombinant viral protein based indirect enzyme-linked immunosorbent assay (ELISA) is cheap, safe, specific and rapid tool to diagnose the viral infection. Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. The N protein was selected based on its immuno dominance and conservation among different NiV strains. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for swine sera was optimized using the recombinant NiV-N protein as an antigen along with negative and positive controls. The Background reading was blocked using skim milk powder and chicken serum. A total number of 1709 swine serum samples from various states of India were tested with indirect ELISA and Western blot. The test was considered positive only when its total reactivity reading was higher than 0.2 cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Since, specificity is high for western blotting it was used as standard test for comparison of results of indirect ELISA. Sensitivity and Specificity of indirect ELISA was 100% and 98.7% respectively in comparison with western blotting. Recombinant N protein based ELISA can be used in screening large number of serum samples for epidemiological investigations in developing countries where high containment laboratories are not available to handle this zoonotic virus.
Microglia-mediated neuroinflammation induced by α-synuclein in the substantianigra likely either initiates or aggravates nigral neuro degeneration in Parkinson’s disease (PD). We aimed to explore the effects of α-mangostin (α-M), a polyphenolicxanthone derivative from mangosteen on α-synuclein-stimulated DA neurodegeneration. Primary microglia, mesencephalic neuron, mesencephalic neuron-glianeuronal cultures, and transwell co-cultures were prepared separately. Liquid scintillation counting was used to determine the radioactivity in DA uptake. Enzyme-linked immunosorbent assay (ELISA) was performed in the IL-1β, IL-6, and TNF-α assay. The expression of proteins was analyzed by Western blot. α-M inhibited the increased levels of pro-inflammatory cytokines, NO, and ROS in α-synuclein-stimulated primary microglia. Mechanistic study revealed that α-M functioned by inhibition of nuclear factor kappa B (NF-κB) and NADPH oxidase. Further, α-M protected α-synuclein-induced microglial and direct neurotoxicity. Although detailed mechanisms remain to be defined, our observations suggest a potential compound, which inhibits microglial activation induced by α-synuclein by targeting NADPH oxidase, might be a therapeutic possibility in preventing PD progression.
Progranulin (PGRN) is a crucial secreted growth factor involved in various kinds of physiologic and disease processes and often has a protective role in inflammatory diseases. This study was designed to investigate the protective effects of PGRN on endotoxic shock in a mouse model of PGRN deficiency. After lipopolysaccharide (LPS) injection to induce endotoxic shock in mice, PGRN levels were induced in wild-type (WT) mice at 6 and 24 hrs. Survival rate analysis, haematoxylin and eosin staining, immunohistochemical staining, enzyme-linked immunosorbent assay and in situ terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick-end labelling assay were used to reveal the susceptibility, lung injury, inflammatory cell infiltration, production of inflammatory mediators and lung cell death in mice after LPS injection. PGRN-deficient (Grn(-/-) ) mice were highly susceptible to LPS-induced endotoxic shock, with decreased survival, severe lung injury, increased production of pro-inflammatory mediators, and inflammatory cell infiltration and apoptotic death in the lung. Additionally, recombinant PGRN (rPGRN) administration before LPS stimulation ameliorated the survival of and abnormalities in both WT and Grn(-/-) mice. Altogether, these findings indicate that PGRN may be a novel biologic agent with therapeutic potential for endotoxic shock probably by inhibiting LPS-induced systemic and local inflammation in mice for treating endotoxic shock.
Antibodies Biotinylated Using a Synthetic Z-domain from Protein A Provide Stringent In Situ Protein Detection
- The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society
- Published over 4 years ago
Antibody-based protein profiling on a global scale using immunohistochemistry constitutes an emerging strategy for mapping of the human proteome, which is crucial for an increased understanding of biological processes in the cell. Immunohistochemistry is often performed indirectly using secondary antibodies for detection, with the benefit of signal amplification. Direct immunohistochemistry instead brings the advantage of multiplexing; however, it requires labeling of the primary antibody. Many antibody-labeling kits do not specifically target IgG and may therefore cause labeling of stabilizing proteins present in the antibody solution. A new conjugation method has been developed that utilizes a modified Z-domain of protein A (ZBPA) to specifically target the Fc part of antibodies. The aim of the present study was to compare the ZBPA conjugation method and a commercially available labeling kit, Lightning-Link, for in situ protein detection. Fourteen antibodies were biotinylated with each method and stained using immunohistochemistry. For all antibodies tested, ZBPA biotinylation resulted in distinct immunoreactivity without off-target staining, regardless of the presence of stabilizing proteins in the buffer, whereas the majority of the Lightning-Link biotinylated antibodies displayed a characteristic pattern of nonspecific staining. We conclude that biotinylated ZBPA domain provides a stringent method for antibody biotinylation, advantageous for in situ protein detection in tissues.
- Biochemical and biophysical research communications
- Published 23 days ago
The steroidal saponin DT-13 (25(R,S)-ruscogenin-1-O-[β-d-glucopyranosyl-(1 → 2)][β-d-xylopyranosyl-(1 → 3)]-β-d-fucopyranoside), one of the major active compounds of the herb Liriope muscari (Decne.), exhibits significant anti-inflammatory, anti-tumour and cardioprotective effects. This study aimed to explore the protective effect of DT-13 on endothelium through regulating of nitric oxide production induced by TNF-α. The results demonstrated that DT-13 inhibits inflammatory cell infiltration and thus plays a protective effect, as shown by H&E staining and immunohistochemical staining. ELISA results demonstrated that DT-13 could suppress the TNF-α-induced upregulation of ROS, TNFR, IL-8, MCP-1 and nitric oxide in vivo in a dose-dependent manner. Moreover, it suppressed nitric oxide production in vitro as shown by DAF-FMDA. Western blotting results indicated that DT-13 could down-regulate phosphorylation of eNOS significantly in TNF-α-induced HUVECs. Taken together, we speculate that DT-13 inhibits endothelium vascular inflammation through regulating nitric oxide production and the expression of ROS, TNFR, IL-8, MCP-1, which are associated with inflammation.
This study is aimed to investigate the potential role of interleukin (IL)-33 in transplanted kidney interstitial fibrosis and the associated mechanism. Serum IL-33 levels were detected using an enzyme-linked immunosorbent assay (ELISA) in healthy volunteers, stable kidney transplantation recipients (KTRs) (stable), KTRs with acute rejection (AR), and KTRs with chronic allograft dysfunction (CAD) (CAD). Immunohistochemical (IHC) staining, Western blotting, and quantitative real-time PCR (qRT-PCR) were used to detect the expression of IL-33 in human kidney tissues obtained from control and CAD patients. In addition, human kidney (HK)-2 cells were treated with human IL-33 at different doses or intervals, and the markers of epithelial to mesenchymal transition (EMT) were assessed by the presence of proteins and mRNA extracted from these cells using Western blotting and qRT-PCR. Cell motility and migration were evaluated with a cell motility and migration assay. The mechanism involved in EMT induced by IL-33 was investigated by Western blot. Finally, fibronectin, E-cadherin, and α-SMA expression, as well as the level of activity in the MAPK signaling pathway in the kidney tissues from the control and CAD group were also detected using a Western blot and an IHC staining assay. The intensity of fibrosis was substantially higher in the CAD group. IL-33 was significantly upregulated in the CAD patients compared to the control group. In vitro, IL-33 could induce EMT in a dose-dependent and time-dependent manner and promoted both the cellular motility and migration capabilities of HK-2 cells. Moreover, the p38 MAPK signaling pathway might be involved in the pathogenesis of EMT induced by IL-33, which was consistent with the in vivo results of the kidney specimens from the control and CAD patients. IL-33 was upregulated in CAD patients and could promote EMT of HK-2 cells.
Developing an enzyme-free, non-amplification strategy for biomarker detection with universality and easy implementation is of central importance in clinical diagnosis and therapeutic monitoring. Herein, we report for the first time a universal and enzyme-free magnetic bead-based sandwich-format immunoassay platform for biomarker detection by combining secondary antibody functionalized AuNPs and automatic AuNP counting readout. For the prostate specific antigen (PSA), the detection limit is found to be 1 ng mL(-1), and the spike recoveries (n = 3) with 10% fetal bovine serum are 113.5% for 2 ng mL(-1) and 107.7% for 10 ng mL(-1). The assay also presents reasonable repeatability as indicated by the coefficient of variance of 13.1% with 5 measurements in 60 days. This strategy has been successfully applied to the determination of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), demonstrating the universality of this strategy. Our proposed non-amplification platform presents sensitivity comparable to that of the enzyme-linked immunosorbent assay (ELISA) with better repeatability; and more importantly, our method has better simplicity than most of the amplification-based methods, and thus is more suitable for routine analysis. The highlights of our work suggest that it is a promising method and would be potentially an alternative for ELISA in laboratories where routine analyses are intensively performed.
- Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology
- Published about 2 months ago
Standard diagnostic testing for HIV infection has traditionally relied on a high sensitivity HIV antibody screening test using an enzyme-linked immunosorbent assay (ELISA) followed by a high specificity antibody confirmatory test such as a Western Blot. Recently several of the screening assays have been enhanced with an ability to identify p24 antigen thereby narrowing the diagnostic window.
Fasudil, a selective rho kinase (ROCK) inhibitor, has been reported to play a beneficial role in systemic?inflammation?in acute?lung injury, but its mechanism for ameliorating pulmonary edema and inflammation remains unclear. Using hematoxylin-and-eosin (HE) staining, immunohistochemistry, enzyme-linked immunosorbent assay, quantitative real time PCR and Western blotting, we found that fasudil attenuated LPS-induced lung injury, decreased lung edema, and suppressed inflammatory responses including leukocyte infiltration and IL-6 production. Further, fasudil upregulated LPS-induced aquaporin 5 reduction and inhibited NF-kB activation in the lungs of mice. Our results suggest that fasudil could restore the expression of aquaporin 5 to eliminate LPS-induced lung edema and prevent LPS-induced pulmonary inflammation by blocking the inflammatory pathway. Collectively, blockade of the ROCK pathway by fasudil may be a potential strategy for the treatment of acute lung injury.