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.
BACKGROUND Low levels of 1-25-dihydroxyvitamin D3 [1,25(OH)2D3] in serum may be a risk factor for several tumor types. Also, high cathelicidin antimicrobial peptide (CAMP) expression is regarded to be important against tumor progression. We evaluated the potential importance of 1,25(OH)2D3 in the diagnosis and treatment of papillary thyroid cancer (PTC). MATERIAL AND METHODS The preoperative serum level of 1,25(OH)2D3 was measured using a double-antibody sandwich enzyme-linked immunosorbent assay. Vitamin D3 receptor (VDR) expression was detected by streptavidin-peroxidase immunohistochemical staining in PTC specimens. Receiver operating characteristic (ROC) curves were created to assess the diagnostic value of 1,25(OH)2D3. The effect of 1,25(OH)2D3 on the proliferation and apoptosis of PTC cell lines were studied by Cell Counting Kit (CCK)-8 assay and Annexin V/propidium iodide staining, respectively. CAMP expression was measured by qRT-PCR and western blotting. Short interfering RNAs were used to reduce CAMP expression in PTC cell lines. RESULTS The preoperative serum level of 1,25(OH)2D3 in PTC was obviously lower than that in nodular goiter (NG) (P<0.05). The ROC curve suggested that 1,25(OH)2D3 might serve as a potential diagnostic value at a cutoff of 20.13 pg/mL, The VDR showed higher expression in PTC than in paired adjacent non-cancerous tissue. 1,25(OH)2D3 inhibited the proliferation and induced the apoptosis of PTC cells, and increased CAMP expression significantly, whereas CAMP knockdown demonstrated opposite effects. CONCLUSIONS 1,25(OH)2D3 may be a new, potential biomarker for the identification of PTC and NG. It may also become 1,25(OH)2D3 may a potential target for drug action to treat PTC through CAMP.
Tissue antigenicity represents the main limitation for the use of xenografts in clinical practice. To eliminate xenoantigens and avoid graft rejection in human, decellularization is often used to remove all immunoreactive components from the extracellular matrix (ECM). After decellularization, acellular scaffolds are required to be investigated regarding the presence of antigens, but commonly used detection methods solely focus on known xenoantigens such as alpha Gal (Galα1,3-Galβ1-4GlcNAc-R) or major histocompatibility complex-I (MHC-I) However, there are unknown xenoantigens, which escape the standard methods. In order to evaluate the complete immunological potential of xenogenic tissues, new in vitro methods need to be developed. Therefore, we established a novel human serum based approach including Dot Blot, Western Blot, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA). With these methods, we analyzed protein extracts and tissue samples of native and decellularized bovine carotid arteries. All methods verified an effective removal of potential immunogens from the ECM through decellularization and relative quantification with ELISA showed that 99.9 % (p<0.01) of antigenic components were successfully eliminated. We compared our human serum based methods with commonly used assays for the detection of alpha Gal and MHC-I. Our results showed highly increased sensitivity for xenoantigens using the human serum antibody pool. This novel in vitro detection system allows the direct determination of the immunogenic potential of xenografts and is a vast improvement in comparison to the methods used so far. That way, it is possible to optimize the decellularization process to prevent hyperacute graft rejection in patients.
Almost all immunological approaches [immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), Western blot], that are used to quantitate specific proteins have had to address high backgrounds due to non-specific reactivity. We report here for the first time a quantitative comparison of methods for reduction of background of commercial biotinylated antibodies using the Python-based ELISA_QC program. This is demonstrated using a recombinant humanized anti-cocaine monoclonal antibody. Several approaches, such as adjustment of the incubation time and the concentration of blocking agent, as also the dilution of secondary antibodies, have been explored to address this issue. In this report, systematic comparisons of two different methods, contrasted with other more traditional methods to address this problem are provided. Addition of heparin (HP) at 1 μg/ml to the wash buffer prior to addition of the secondary biotinylated antibody reduced the elevated background absorbance values (from a mean of 0.313 ± 0.015 to 0.137 ± 0.002). A novel immunodepletion (ID) method also reduced the background (from a mean of 0.331 ± 0.010 to 0.146 ± 0.013). Overall, the ID method generated more similar results at each concentration of the ELISA standard curve to that using the standard lot 1 than the HP method, as analyzed by the Python-based ELISA-QC program. We conclude that the ID method, while more laborious, provides the best solution to resolve the high background seen with specific lots of biotinylated secondary antibody.
A Community-Based Study to Estimate the Seroprevalence of Trichinellosis and Echinococcosis in the Roma and Non-Roma Population of Slovakia
- International journal of environmental research and public health
- Published about 2 months ago
Trichinellosis and cystic and alveolar echinococcosis are serious parasitic diseases transmissible between animals and humans. Moreover, alveolar echinococcosis is considered one of the most dangerous of human helminthoses. Roma communities are particularly numerous in Central and Eastern Europe. They are often concentrated in economically undeveloped regions and live in segregated localities with unsatisfactory housing and sanitary conditions. The study aimed to find out the seroprevalence of Trichinella and Echinococcus infections in the Roma population of segregated settlements and to compare it with the seropositivity of the non-Roma population of eastern Slovakia. Out of 823 samples, three sera showed seropositivity to Trichinella in the ELISA (Enzyme-linked immunosorbent assay) test. Subsequent Western blot reaction (WB) confirmed seropositivity in two Roma women. ELISA seropositivity to E. multilocularis was recorded in six persons (0.73%), and five (0.61%) respondents were seropositive to E. granulosus, but WB confirmed the presence of antibodies to Echinococcus spp. in one Roma participant. Positive persons suffered from unspecific clinical symptoms; Trichinella-positive persons reported headache, cough, fatigue, and muscle pain. The Echinococcus-positive participant suffered from headache and back pain. The study showed that the worse living conditions of the Roma community did not significantly influence the occurrence of Trichinella and Echinococcus infections in this minority.
The present study aimed to investigate the mechanism by which the Janus kinase (JAK)2/signal transducer and activator of transcription (STAT)3 signaling pathway mediates cerebral ischemia and the efficacy of pharmaceutical intervention. The rat model of middle cerebral artery occlusion (MCAO) was established and confirmed via assessment of changes in the expression of phosphorylated (p)‑JAK2, p‑STAT3, high‑mobility group box 1 (HMGB1), and inflammatory factors using ELISA and western blot analysis. The effects of JAK2/STAT3 inhibitor and curcumin on the expression of p‑JAK2, p‑STAT3, HMGB1, and inflammatory factors after cerebral ischemia were observed with ELISA, western blotting and immunohistochemical staining. The concentrations of tumor necrosis factor (TNF)‑α and HMGB1 in brain tissue homogenate of MCAO group were significantly higher than in the sham group (P<0.01). The concentration of p‑JAK2/JAK2 and p‑STAT3/STAT3 in the brain tissue homogenate of MCAO group was significantly higher than in the sham group (P<0.05). The concentrations of TNF‑α, interleukin (IL)‑1β, IL‑6, and HMGB1 in the group treated with STAT3 inhibitor (MCAO + rapamycin), JAK2 inhibitor (MCAO + AG490), and MCAO + curcumin were significantly lower than in the MCAO group (P<0.01), as well as the relative content of p‑JAK2/JAK2 and p‑STAT3/STAT3 (P<0.05). Inhibition of the JAK2/STAT3 signaling pathway, such as curcumin can reduce the expression of HMGB1 in brain tissue after cerebral ischemia, which can significantly reduce the inflammatory response after cerebral ischemia.