Performance of two modified two-tier algorithms for the serologic diagnosis of Lyme disease.

We compared the performance of a new modified two-tier testing (MTTT) platform, the Diasorin Liaison chemiluminescent immunoassay (CLIA), to the Zeus enzyme-linked immunoassay (ELISA) MTTT and to Zeus ELISA/Viramed immunoblot standard two-tier testing (STTT) algorithm. Of 537 samples included in this study, 91 (16.9%) were positive or equivocal by one or more screening tests. Among these 91 samples, only 57 samples were concordant positive by first-tier screening tests, and only 19 of 57 were concordant by the three second-tier methods. For IgM results, positive percent agreement (PPA) was 68.1% for Diasorin versus 89.4% for Zeus compared to immunoblot. By contrast, the PPA for IgG for both Diasorin and Zeus was 100%. Using a 2-out-of-3 consensus reference standard, the PPAs for IgM were 75.6%, 97.8%, and 95.6% for Diasorin, Zeus, and immunoblot, respectively. The difference between Zeus MTTT and Diasorin MTTT for IgM detection was significant (P = 0.0094). PPA for both Diasorin and Zeus MTTT IgG assays was 100% but only 65.9% for immunoblot STTT (P = 0.0005). In total, second-tier positive IgM and/or IgG results were reported for 57 samples by Diasorin MTTT, 63 by Zeus MTTT, and 54 by Viramed STTT. While Diasorin CLIA MTTT had a much more rapid, automated, and efficient workflow, Diasorin MTTT was less sensitive for the detection of IgM than Zeus MTTT and STTT including in 5 early Lyme cases that were IgM negative but IgG positive. IMPORTANCE: The laboratory diagnosis of Lyme disease relies upon the detection of antibodies to Borrelia species. Standard two tier testing (STTT) methods rely upon immunoblots which have clinical and technical limitations. Modified two-tier testing (MTTT) methods have recently become available and are being widely adopted. There are limited independent data available assessing the performance of MTTT and STTT methods.

Recognition of Immunoreactive Proteins in Leishmania infantum Amastigote-Like and Promastigote Using Sera of Visceral Leishmaniasis Patients: a Preliminary Study.

PURPOSE: Visceral leishmaniasis (VL) is a systemic and parasitic disease that is usually fatal if left untreated. VL is endemic in different parts of Iran and is caused mainly by Leishmania infantum. This study aimed to recognition immunoreactive proteins in amastigote-like and promastigote stages of L. infantum (Iranian strain) by antibodies present in the sera of VL patients. METHODS: Total protein extract from amastigote-like and promastigote cells was separated by two-dimensional electrophoresis (2DE). To detect the immunoreactive proteins, 2DE immunoblotting method was performed using different pools of VL patients' sera. RESULTS: Approximately 390 and 430 protein spots could be separated in 2DE profiles of L. infantum amastigote-like and promastigote stages, respectively. In immunoblotting method, approximately 295 and 135 immunoreactive proteins of amastigotes-like reacted with high antibody titer serum pool and low antibody titer serum pool, respectively. Approximately 120 and 85 immunoreactive proteins of promastigote extract were recognized using the high antibody titer sera pool and low antibody titer sera, respectively. CONCLUSION: The present study has recognized a number of antigenic diversity proteins based on the molecular weight and pH in amastigote-like and promastigote stages of L. infantum. These results provide us a new concept for further analysis development in the field of diagnosis biomarkers and vaccine targets.

Immunoblot Analysis from Single Cells Using Milo™ Single-Cell Western Platform.

In this new era of precision medicine, characterization of single-cell subpopulations to better understand disease etiology is paramount. It is thus an opportune time to explore techniques that allow molecular analysis of single cells and to better understand the basis of pathogenesis of diseases like cancer. Single-cell western blotting is one such method that allows analysis of single cells at the protein level. In contrast to traditional western blotting, which relies heavily on bulk analysis of lysates generated from tissues and is often indicative of the population average, this technique allows analysis of lysates from single-cell subpopulations thereby providing a glimpse into cell heterogeneity. The method entails the use of a chip containing 30 µm thick photoactivated polyacrylamide gel spotted with nearly 6400 microwells. Single cells loaded on the chip are captured in the microwells by passive gravity and are then lysed and electrophoresed using the MILO™ single-cell western platform. This method forgoes the use of transfer of proteins on a PVDF and a nitrocellulose membrane, as performed in traditional western blotting, and all other steps including probing of primary and fluorescent secondary antibodies against the protein of interest are performed directly on the chip. The proteins of interest can then be visualized by scanning a chip with the use of a microarray scanner. The entire procedure can be performed in as less as 4-6 h, and thus this method provides several advantages over traditional western blotting.

Protocol to analyze the role of various metabolites in impacting global RNA 5-methylcytosine levels in cultured cells by dot blot.

RNA 5-methylcytosine (m5C) modification critically impacts many biological processes. Here, we provide a protocol to analyze the role of various metabolites in impacting global RNA m5C levels in cultured cells by dot blot. We describe steps for treating cultured cells with various metabolites; extracting, quantifying, and denaturing RNA samples; and performing dot blot to detect global RNA m5C levels in cultured cells. We then detail procedures to verify the input loading by methylene blue staining and quantify using ImageJ. For complete details on the use and execution of this protocol, please refer to Chen et al.1.

Protocol for detection of in vitro R-loop formation using dot blots.

Dot-blot analysis is a technique that allows for fast and convenient detection and identification of nucleic acids and proteins. Here, we provide a guide for nucleic acid isolation from eukaryotic cells and sample processing to detect RNA/DNA hybrids. We then provide detailed steps to quantify dot signal intensity. This protocol can be adapted for screening conditions that result in the accumulation of R-loops. For complete details on the use and execution of this protocol, please refer to Smith et al.1.

Rapid Dot-Blot Immunoassay for Detecting Multiple Salmonella enterica Serotypes.

Salmonella, a major contributor to foodborne infections, typically causes self-limiting gastroenteritis. However, it is frequently invasive and disseminates across the intestinal epithelium, leading to deadly bacteremia. Although the genus is subdivided into >2,600 serotypes based on their antigenic determinants, only few serotypes are responsible for most human infections. In this study, a rapid dot-blot immunoassay was developed to diagnose multiple Salmonella enterica serotypes with high incidence rates in humans. The feasibility of 10 commercial antibodies (four polyclonal and six monoclonal antibodies) was tested using the 18 serotypes associated with 67.5% Salmonella infection cases in the United States of America (U.S.A) in 2016. Ab 3 (polyclonal; eight of 18 serotypes), Ab 8 (monoclonal; 13 of 18 serotypes), and Ab 9 (monoclonal; 10 of 18 serotypes) antibodies exhibited high detection rates in western blotting and combinations of two antibodies (Ab 3+8, Ab 3+9, and Ab 8+9) were applied to dot-blot assays. The combination of Ab 3+8 identified 15 of the tested 18 serotypes in 3 h, i.e., S. Enteritidis, S. Typhimurium, S. Javiana, S. I 4,[5],12:i:-, S. Infantis, S. Montevideo, S. Braenderup, S. Thompson, S. Saintpaul, S. Heidelberg, S. Oranienburg, S. Bareilly, S. Berta, S. Agona, and S. Anatum, which were responsible for 53.7% Salmonella infections in the U.S. in 2016. This cost-effective and rapid method can be utilized as an on-site colorimetric method for Salmonella detection.

Isolation and characterization of small extracellular vesicles from murine primary mammary tumor.

Tumor-derived small extracellular vesicles (TEVs) play a pivotal role in cancer progression by transferring functional biomolecules between the parental and recipient cells. Here, we present a protocol to isolate TEVs directly from murine primary mammary tumor using differential centrifugation. We describe steps for tissue dissociation, enzymatic digestion, and centrifugation. We then detail procedures for characterization of TEVs through transmission electron microscopy, immunoblotting, and nano-flow cytometry. This protocol can be used to extract EVs from other solid tumor types. For complete details on the use and execution of this protocol, please refer to Li, Mei-Xin et al. (2023).1.

Protocol for in situ visualization of mitochondrial ROS and apoptosis in spatially confined cells and sample preparation for biochemical analysis.

Locomotion through spatially confining spaces is an important in vivo migration mode. Here, we present a protocol for in situ visualization of mitochondrial reactive oxygen species and apoptosis in cancer cells during confined migration. We then detail sample preparation of confined cells for transcriptome and immunoblotting analysis by using transwell chambers. This approach allows in situ evaluation of a variety of cellular functions during confined migration and preparation of the samples of confined cells for further biochemical analysis. For complete details on the use and execution of this protocol, please refer to Cai et al.1.

Protocol to study human mitochondrial ribosome using quantitative density gradient analysis by mass spectrometry and complexome profiling analysis.

Dynamic macromolecular complexes containing a large number of components are often difficult to study using conventional approaches, such as immunoblotting. Here, we present a protocol for the analysis of macromolecular complexes in near-native conditions using a flexible setup to suit different cellular targets. We describe analysis of human mitochondrial ribosome, composed of 82 proteins, in a standardized way using density gradient ultracentrifugation coupled to quantitative mass spectrometry and subsequent analysis of the generated data (ComPrAn). For complete details on the use and execution of this protocol, please refer to Páleníková et al.1 and Rebelo-Guiomar et al.2.

A redox-based characterization of human immune cell subsets by polychromatic flow cytometry.

Cellular redox state determinants are traditionally studied using fluorescent microscopy and immunoblot analysis; however, no procedure has been developed for simultaneous measurement in various immune cell subsets. Here, we present a flow cytometry assay for measuring antioxidant defense systems and reactive oxygen species simultaneously in T, B, and natural killer lymphocytes. We describe steps for preparing and treating peripheral blood mononuclear cells, surface and dye staining, cell fixation/permeabilization, and intracellular staining. We then detail machine standardization, acquisition, and analysis.

Immunochemical Characterization of Salix alba (S. alba) Pollen Allergens and Evaluation of the Cross-Reactivity Pattern with Common Allergenic Pollen Grains.

BACKGROUND: Pollens, particularly tree and plant pollens, are one of the major causes of allergic respiratory diseases worldwide. Allergy to pollens of different species of Salix trees has been reported in various regions of the world. The most common type of Salix tree in Iran is white willow (Salix alba). OBJECTIVES: This study aimed to identify and determine the immunochemical characteristics of allergenic proteins in S. alba tree pollen extract using SDS-PAGE and IgE- immunoblotting methods. Moreover, the cross-reaction pattern of the specific IgE antibody of S. alba tree pollen proteins with pollen allergens of common allergenic trees, i.e., Populus nigra (P. nigra), Cupressus sempervirens (C. sempervirens), Pinus brutia (P. brutia) and Platanus orientalis (P. orientalis) in the region was investigated. METHODS: The reaction of allergenic proteins in S. alba pollen extract with specific IgE antibodies in patients' sera was investigated using SDS-PAGE and IgE-immunoblotting methods. The cross-reaction of specific IgE antibodies of the proteins present in S. alba pollen extract with pollen allergens of common allergenic trees in the region was investigated using ELISA and immunoblotting inhibition methods. In silico methods such as phylogenetic tree drawing and alignment of amino acid sequences were used to examine the evolutionary relationship and homology structure of common allergenic proteins (Panallergens) responsible for cross reactions. RESULTS: More than 11 protein bands binding to specific IgE antibodies in patients' sera with a molecular weight between 13 and 95 kDa were identified in the S. alba tree pollen extract. ELISA and immunoblotting inhibition results showed that P. nigra extract could inhibit the binding of IgE antibodies to S. alba pollen extract proteins to a greater extent than C. sempervirens, P. brutia, and P. orientalis tree extracts. In silico methods investigated the results of ELISA and immunoblotting inhibition methods. Moreover, a high structural homology and evolutionary relationship were observed between S. alba and P. nigra tree pollen panallergens. CONCLUSION: In this study, it was found that more than 80 % of the sensitive patients who were examined had specific IgE antibodies reacting with the approximately a 15 kDa-protein present in the S. alba pollen extract. Furthermore, the specific IgE-binding proteins found in the pollens of S. alba and P. nigra trees had relative structural homology, and it is likely that if recombinant forms are produced, they can be used for diagnostic and therapeutic purposes for both of the trees.

Examining PI3K-signaling-dependent regulation of lens organelle free zone formation via immunolocalization and immunoblotting in chick embryos.

The elimination of lens organelles during development, required for mature lens function, is an autophagy-dependent mechanism induced through suppression of PI3K signaling. Here, we present a protocol for investigating the signaling pathways responsible for induction of the formation of this lens organelle free zone. We describe steps for preparation of lens organ culture and use of signaling pathway inhibitors. We then detail procedures for analyzing their impact using both confocal microscopy imaging of immunolabeled lens cryosections and immunoblot approaches. For complete details on the use and execution of this protocol, please refer to Gheyas et al. (2022).1.

A simple and highly reproducible method for the detection of erythrocyte membrane ZIP metal transporters by immunoblotting.

Manganese is one of the essential trace elements found in erythrocytes. Metal transporters situated on the plasma membrane generally facilitate the movement of manganese into and out of cells. This study aims at determining whether two recently discovered manganese importers, ZIP8 and ZIP14, are located in the erythrocyte membrane. We outline a simple, effective and repeatable method for the isolation of erythrocyte membrane from a minimum of 50 µL mouse blood, followed by the identification of ZIP metal transporters using immunoblotting. Our results revealed that ZIP8 is expressed within the erythrocyte membrane, in contrast to ZIP14 which is not identified using immunoblotting approach. A direct measurement of the ZIP8 protein expression in erythrocyte membranes could provide valuable information for further analyzing its biological function.

Knockout of c-Cbl/Cbl-b slows c-Met trafficking resulting in enhanced signaling in corneal epithelial cells.

In many cell types, the E3 ubiquitin ligases c-Cbl and Cbl-b induce ligand-dependent ubiquitylation of the hepatocyte growth factor (HGF)-stimulated c-Met receptor and target it for lysosomal degradation. This study determines whether c-Cbl/Cbl-b are negative regulators of c-Met in the corneal epithelium (CE) and if their inhibition can augment c-Met-mediated CE homeostasis. Immortalized human corneal epithelial cells were transfected with Cas9 only (Cas9, control cells) or with Cas9 and c-Cbl/Cbl-b guide RNAs to knockout each gene singularly (-c-Cbl or -Cbl-b cells) or both genes (double KO [DKO] cells) and monitored for their responses to HGF. Cells were assessed for ligand-dependent c-Met ubiquitylation via immunoprecipitation, magnitude, and duration of c-Met receptor signaling via immunoblot and receptor trafficking by immunofluorescence. Single KO cells displayed a decrease in receptor ubiquitylation and an increase in phosphorylation compared to control. DKO cells had no detectable ubiquitylation, had delayed receptor trafficking, and a 2.3-fold increase in c-Met phosphorylation. Based on the observed changes in receptor trafficking and signaling, we examined HGF-dependent in vitro wound healing via live-cell time-lapse microscopy in control and DKO cells. HGF-treated DKO cells healed at approximately twice the rate of untreated cells. From these data, we have generated a model in which c-Cbl/Cbl-b mediate the ubiquitylation of c-Met, which targets the receptor through the endocytic pathway toward lysosomal degradation. In the absence of ubiquitylation, the stimulated receptor stays phosphorylated longer and enhances in vitro wound healing. We propose that c-Cbl and Cbl-b are promising pharmacologic targets for enhancing c-Met-mediated CE re-epithelialization.

Immunogenic mapping of rDyn-1 and rKDDR-plus proteins and selection of oligopeptides by immunoblotting for the diagnosis of Leishmania infantum-infected dogs.

Endemic in Brazil, visceral leishmaniasis (VL) is a zoonotic infection that is among the most important parasitic diseases transmitted by vectors. Dogs are the main reservoirs of canine leishmaniasis (CanL) and their identification is used in some countries as part of disease prevention and control measures in the canine and human population. In this context, serological tests are necessary, composed of antigens capable of correctly identifying infected dogs, minimizing the number of false-negative cases. This study aimed to identify more immunoreactive peptides derived from two previously described whole proteins (rDyn-1 and rKDDR-plus) and compare their performance to the control antigens rK39 and the crude extract for the detection of dogs infected with L. infantum, especially the asymptomatic ones. The three selected peptides and a mixture of them, along with the rDyn-1, rKDDR-plus, rK39, and crude extract antigens were evaluated using indirect ELISA with sera samples from 186 dogs with CanL, being asymptomatic (n = 50), symptomatic (n = 50), co-infected (n = 19), infected with Babesia sp. (n = 7), Ehrlichia sp. (n = 6), T. cruzi (n = 20) and uninfected (n = 34). The results showed that the rDyn-1 protein and the peptide mixture had the highest sensitivity (100% and 98.32%, respectively) and specificity (97.01 and 98.51, respectively). A high degree of kappa agreement was found for rDyn-1 protein (0.977), mixed peptides (0.965), rKDDR-plus protein (0.953), K-plus peptide 1 (0.930) and Dyn-1 peptide (0.893). The mixture of peptides showed the highest likelihood (65.87). The ELISA using the mixture of peptides and the rDyn-1 protein showed high performance for CanL serodiagnosis. More mix combinations of the peptides and additional extended field tests with a larger sample size are recommended.

Best practices in assessing cardiac protein O-GlcNAcylation by immunoblot.

The modification of serine and threonine amino acids of proteins by O-linked N-acetylglucosamine (O-GlcNAc) regulates the activity, stability, function, and subcellular localization of proteins. Dysregulation of O-GlcNAc homeostasis is well established as a hallmark of various cardiac diseases, including cardiac hypertrophy, heart failure, complications associated with diabetes, and responses to acute injuries such as oxidative stress and ischemia-reperfusion. Given the limited availability of site-specific O-GlcNAc antibodies, studies of changes in O-GlcNAcylation in the heart frequently use pan-O-GlcNAc antibodies for semiquantitative evaluation of overall O-GlcNAc levels. However, there is a high degree of variability in many published cardiac O-GlcNAc blots. For example, many blots often have regions that lack O-GlcNAc positive staining of proteins either below 50 or above 100 kDa. In some O-GlcNAc blots, only a few protein bands are detected, while in others, intense bands around 75 kDa dominate the gel due to nonspecific IgM band staining, making it difficult to visualize less intense bands. Therefore, the goal of this study was to develop a modifiable protocol that optimizes O-GlcNAc positive banding of proteins in cardiac tissue extracts. We showed that O-GlcNAc blots using CTD110.6 antibody of proteins ranging from <30 to ∼450 kDa could be obtained while also limiting nonspecific staining. We also show that some myofilament proteins are recognized by the CTD110.6 antibody. Therefore, by protocol optimization using the widely available CTD110.6 antibody, we found that it is possible to obtain pan-O-GlcNAc blots of cardiac tissue, which minimizes common limitations associated with this technique.NEW & NOTEWORTHY The post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is recognized as mediating cardiac pathophysiology. However, there is considerable variability in the quality of O-GlcNAc immunoblots used to evaluate changes in cardiac O-GlcNAc levels. Here we show that with relatively minor changes to a commonly used protocol it is possible to minimize the intensity of nonspecific bands while also reproducibly generating O-GlcNAc immunoblots covering a range of molecular weights from <30 to ∼450 kDa.

Inflammasome Activation in Human Macrophages: IL-1ß Cleavage Detection by Fully Automated Capillary-Based Immunoassay.

Interleukin (IL)-1ß is a key mediator of inflammation and activates via pattern recognition receptors (PRR) of the inflammasome family by proteolytic maturation. Proteolysis is driven by proteases such as caspase-1 (also known as IL-1 converting enzyme, ICE) and converts the intact pro-IL-1ß ~31 kDa pro-peptide into a mature, ~17 kDa form that can exit cells through nanomolecular pores or via microvesicles. Whereas pro-IL-1ß fails to trigger IL-1 receptor (IL-1R) activation, mature IL-1ß, upon release from the cell, triggers pleiotropic downstream effects, establishing an inflammatory state. Hence, being able to detect IL-1ß conversion is physiologically relevant for measuring inflammation, but it cannot be easily accomplished by conventional ELISA or flow cytometry as most commercially available antibodies do not discriminate mature and pro-form. Furthermore, unlike for other cytokines, the mere induction and translation of IL1B mRNA cannot serve as a proxy of inflammasome PRR activation. Rather the cleavage of IL-1ß needs to be verified. Hence, conventional immunoblotting has emerged as the gold standard for demonstrating inflammasome activation as the difference in molecular weight between pro- and mature form can easily be detected. However, conventional immunoblotting suffers from poor standardization, quantification, and reproducibility, may require sample concentration, and is also not suitable for medium to high throughput. Some of these shortcomings are prohibitive for analysis of human primary samples but can be overcome by fully automated capillary-based immunoassay as we outline here. We here provide a practical guide to quantify pro- vs mature IL-1ß directly from unconcentrated supernatants of human monocyte-derived macrophages. The assay may be useful for more standardized and medium-throughput analysis in these cells or other biospecimen.

On-Line Dual-Active Valves Based Centrifugal Microfluidic Chip for Fully Automated Point-of-Care Immunoassay.

There remains an unmet need for a fully integrated microfluidic platform that can automatically perform multistep and multireagent immunoassays. Here, we proposed a novel online dual-active valve-based centrifugal microfluidic chip, termed DAVM, for fully automatic point-of-care immunoassay. Practically, the puncture valve, one of the dual active valves, is capable of achieving precise, on-demand, sequential release of prestored reagents, while the other valve-reversible active valve enables controlled retention and drainage of the reaction solutions. Thereby, our technology mitigates the challenges of hydrophilic/hydrophobic modifications and unstable valve control performance commonly observed in passive valve controls. As a proof of concept, the indirect enzymatic immunoblotting technique was employed on DAVM for fully automated immunological analysis of eight targets, yielding outcomes within an hour. Furthermore, we conducted a comparative analysis of 28 clinical samples with autoimmune diseases. According to 224 clinical data, the sample testing concordance rate between DAVM and the traditional instrument was 82%, with a target compliance rate of 97%. Therefore, our DAVM system has powerful potential for fully automated immunoassays.