Journal of Immunological Methods

UNLABELLED: Cytotoxicity studies determining hemolytic properties of antimicrobial peptides or other drugs are an important step in the development of novel therapeutics for clinical use. Hemolysis is an affordable, accessible, and rapid method for initial assessment of cellular toxicity for all drugs under development. However, variability in species of red blood cells and protocols used may result in significant differences in results. AMPs generally possess higher selectivity for bacterial cells but can have toxicity against host cells at high concentrations...

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Because of their superior properties for certain biological applications small antibody derivatives like fragment of antigen binding (Fab) have found widespread use in basic research and as therapeutics. However, generation of Fab-fragments is still a rather complex matter, reflected by the fact that a variety of methods and purification techniques are necessary for the production of all the different classes of Fab-fragments (kappa/lambda light chains, type of species). Here we demonstrate that Fab-fragments derived from six different antibodies of human or murine origin produced by transient expression in HEK cells can be purified in a single step to a high degree of purity by standard protein G affinity chromatography...

OBJECTIVE: The clinical value of Serum amyloid A (SAA) and Lactoferrin (LTF) has received significant attention, but their detection methods are inadequate, which limits their application. This study aims to develop a dual detection method based on stable element labeling strategies and inductively coupled plasma mass spectrometry (ICP-MS) for SAA/LTF and to assess whether it can be widely used in clinical practice. METHODS: A duplex immunoassay system based on sandwich method was constructed...

Complement plays a critical role in the immune response toward nanomaterials. The complement attack on a foreign surface results in the deposition of C3, assembly of C3 convertases, the release of anaphylatoxins C3a and C5a, and finally, the formation of membrane attack complex C5b-9. Various technologies can measure complement activation markers in the fluid phase, but measurements of surface C3 deposition are less common. Previously, we developed an ultracentrifugation-based dot blot immunoassay (DBI) to measure the deposition of C3 and other protein corona components on nanoparticles...

Chimeric antigen receptor (CAR) redirected T cells are successfully employed in the combat against several hematological malignancies, however, are often compromised by low transduction rates making refinement of the CAR T cell products necessary. Here, we report a broadly applicable enrichment protocol relying on marking CAR T cells with an anti-glycine4 -serine (G4S) linker antibody followed by magnetic activated cell sorting (MACS). The protocol is broadly applicable since the G4S peptide is an integral part of the vast majority of CARs as it links the VH and VL recognition domains...

On March 13, 2021, Tunisia started a widespread immunization program against SARS-CoV-2 utilizing different vaccinations that had been given emergency approval. Herein, we followed prospectively a cohort of participant who received COVID-19 vaccine (Pfizer BioNTech and Sputnik-Gameleya V). The goal of this follow-up was to define the humoral and cellular immunological profile after immunization by assessing neutralizing antibodies and IFN- γ release. 26 vaccinated health care workers by Pfizer BioNTech (n = 12) and Sputnik-Gameleya V (n = 14) were enrolled from June to December 2021 in Military hospital of Tunis...

CD3ζ is part of the T cell receptor (TCR)/CD3 complex that plays a critical role in antigen recognition and subsequent T cell activation. Understanding the mechanisms that regulate CD3ζ can provide new insights into the T cell-mediated immune responses. However, it is challenging to deliver exogenous genes into T cells for functional and mechanistic analyses. To this end, we established a non-T cell transfection system based on HEK293FT cells to screen for candidate regulatory proteins. The transfection was optimized using relatively high confluent cultures and the transfection reagent PolyJet™...

Development of assays to reliably identify and characterize anti-drug antibodies (ADAs) depends on positive control anti-idiotype (anti-id) reagents, which are used to demonstrate that the standards recommended by regulatory authorities are met. This work employs a set of therapeutic antibodies under clinical development and their corresponding anti-ids to investigate how different positive control reagent properties impact ADA assay development. Positive controls exhibited different response profiles and apparent assay analytical sensitivity values depending on assay format...

Streptococcus pyogenes, commonly referred to as Group A Streptococcus (Strep A), causes a spectrum of diseases, with the potential to progress into life-threatening illnesses and autoimmune complications. The escalating threat of antimicrobial resistance, stemming from the prevalent reliance on antibiotic therapies to manage Strep A infections, underscores the critical need for the development of disease control strategies centred around vaccination. Phagocytes play a critical role in controlling Strep A infections, and phagocytosis-replicating assays are essential for vaccine development...

Graves' disease is a type of autoimmune hyperthyroidism caused by thyroid-stimulating antibodies (TSAb).1 The combination of a porcine thyroid cell bioassay and cyclic adenosine monophosphate (cAMP) immunoassay (TSAb-enzyme immunoassay; EIA) is a clinically approved TSAb measurement method. Due to the requirement of multiple procedures and a long assay time of 6 h in the TSAb-EIA, a simplified and rapid assay is desired. Herein, we developed a rapid homogeneous TSAb bioassay (rapid-TSAb assay) using the human embryonic kidney cell line (HEK293), engineered to express the human thyroid-stimulating hormone receptor (TSHR), along with a cAMP-dependent luminescence biosensor...

Cytokines are important mediators of immunity in the female genital tract, and their levels may be associated with various reproductive health outcomes. However, the measurement of cytokines and chemokines in vaginal fluid samples may be influenced by a variety of factors, each with the potential to affect the sensitivity and accuracy of the assay, including the interpretation and comparison of data. We measured and compared cytokine milieu in samples collected via Softcup® menstrual cup versus vulvovaginal swabs...

Epitope mapping provides critical insight into antibody-antigen interactions. Epitope mapping of autoantibodies from patients with autoimmune diseases can help elucidate disease immunogenesis and guide the development of antigen-specific therapies. Similarly, epitope mapping of commercial antibodies targeting known autoantigens enables the use of those antibodies to test specific hypotheses. Anti-Neutrophil Cytoplasmic Autoantibody (ANCA) vasculitis results from the formation of autoantibodies to multiple autoantigens, including myeloperoxidase (MPO), proteinase-3 (PR3), plasminogen (PLG), and peroxidasin (PXDN)...

A fluorescent immunochromatographic test (FM-ICT) was developed for rapid detection of anti-Orientia tsutsugamushi antibodies in serum samples. The FM-ICT was constructed based on the dual-antigen sandwich method. Truncated 56 kDa outer membrane protein of O. tsutsugamushi strain SJ, was expressed in E. coli and mixed with those of Ptan and Gillam strains. A thin line of the protein mixture was precisely sprayed across a nitrocellulose membrane making this the "Test" line. Polyclonal antibodies (pAbs) to O...

Current methodologies for assessing vaccine effectiveness and longevity primarily center on measuring vaccine-induced neutralizing antibodies in serum or plasma. However, these methods overlook additional parameters such as the presence of memory B cells, even as antibody levels wane, and the pivotal role played by memory T cells in shaping antigen-specific memory B cell responses. Several studies have employed a combination of polyclonal activators, such as CpG and R848, along with various cytokines to provoke the recall of memory B cells from peripheral blood mononuclear cells (PBMCs) into antibody-secreting cells (ASCs)...

Premature lymphocytes develop into non-autoreactive, mature naïve CD4+ or CD8+ T cells in the thymus before entering the circulation. However, in-depth characterization of human thymocyte development remains challenging due to limited availability of human thymus samples and the fragile nature of thymocyte populations. Thymocytes often do not survive cryopreservation and thawing procedures, especially the fragile CD4+ CD8+ double positive population. It is generally recommended to use fresh human thymus tissue on the day of excision to avoid any biases in thymocyte composition...

While the single-platform flow cytometric CD34+ cell counting method is the preferred choice to predict the yield of mobilized peripheral blood stem cells, most flow cytometers lack the ability of hematology counter analyzers to perform volumetric counting. However, one of the problems using reference microbeads is the vanishing counting bead phenomenon. This phenomenon results in a drop in microbeads concentration and reduces the total and relative number of beads in calibration procedures. In the last years, flow cytometers including a volumetric system to quantify cells have been developed and may represent a promising alternative to enumerate CD34+ cells avoiding the use of beads...

Tumor necrosis factor-alpha, TNF-α, a cytokine recognized as a key regulator of inflammatory responses, is primarily produced by activated monocytes and macrophages. Measuring TNF-α levels serves as a valuable indicator for tracking several diseases and pathological states. Gold nanotechnology has been identified as a highly effective catalyst with unique properties for measuring inflammatory cytokines. This study aimed to synthesize gold nanoclusters (AuNCs) and the AuNCs-streptavidin system, along with their characterizations and spherical morphology...

Mass cytometry and full spectrum flow cytometry have recently emerged as new promising single cell proteomic analysis tools that can be exploited to decipher the extensive diversity of immune cell repertoires and their implication in human diseases. In this study, we evaluated the performance of mass cytometry against full spectrum flow cytometry using an identical 33-color antibody panel on four healthy individuals. Our data revealed an overall high concordance in the quantification of major immune cell populations between the two platforms using a semi-automated clustering approach...

The importance of structural genetic variants, such as copy number variations (CNVs), in modulating human disease is being increasingly recognized. Several clinical conditions require investigation of human neutrophil antigen (HNA-1), which is encoded by the Fc gamma receptor IIIb gene (FCGR3B), including suspicion of neutropenia, infections, and proactive testing of blood component donors to reduce the potential risk in transfusion. In this study, we compared real-time quantitative polymerase chain reaction (qPCR) with two digital PCR (dPCR) platforms, namely droplet digital PCR and an array-based platform, to determine CNV in FCGR3B...