Zika virus (ZIKV) has become a global public health issue due to its teratogenicity and ability to cause Guillain-Barré syndrome in adults. Although anti-ZIKV envelope protein neutralizing antibodies correlate with protection, the non-neutralizing function of ZIKV antibodies including antibody-dependent cell-mediated cytotoxicity (ADCC) is incompletely understood. To study the role of ADCC antibodies

Flow cytometry and fluorescence-activated cell sorting have become invaluable tools to analyze and isolate specific cell populations in a wide range of biomedical research and clinical applications. In countless approaches worldwide, scientists are using single cell analyses to better understand the significance and variation within different cellular populations, and fluorescence-activated cell sorting

T cell immunotherapies have shown great promise in patients with advanced cancer disease, revolutionizing treatment. T cell cytotoxicity is crucial in its efficacy, therefore developing ex vivo methods testing tumor and T cell interactions is pivotal. Increasing efforts have been made in developing co-culture assays with sophisticated materials and platforms aiming to mimic the tumor microenvironment

Detection of anti-drug antibodies (ADA) that have a neutralizing capacity is an important aspect of immunogenicity evaluation during development of biotherapeutics, but developing and validating neutralizing antibody (NAb) assays that show direct interference of a biologic function is a challenging and resource-intensive activity. In particular, the need for adequate drug and target tolerance often

Background ABO subgroups or weaker variants of A or B are group A or B subjects whose erythrocytes give a weak or negative reaction serologically with anti-A or Anti – B antisera respectively. Occurrence of these subgroups may lead to an ABO discrepancy which often puts transfusion services in a quandary. ABO subgroups which present as ABO discrepancies can be missed if reverse grouping is not performed

Among the new biomarkers to propose therapeutic decisions in patients suffering from multiple sclerosis (MS) are the IgM oligoclonal bands (OCBs) in cerebrospinal fluid (CSF). At the current time, however, IgM OCBs are detected in laboratories at investigation level and not in the routine practice due to their complexity. For this, we have applied a semi-automated method based on an isoelectrofocusing

Microglia and astrocytes, the two innate cells in CNS, are thought to protect and remodel of synapses for proper maintenance and plasticity of neuronal circuits. The two types of cells are the major responders by producing and releasing inflammatory mediators. Isolation of microglia and astrocytes from CNS tissue provides a powerful tool to study basic cell biology and examine the effects of in vivo

There is growing evidence that inflammation underpins many common diseases. Inflammatory/immunomodulatory/immune mediators, such as cytokines, are key modulators of inflammation and mediate both immune cell recruitment and complex intracellular signalling pathways. Ovine models of disease are increasingly utilized in pre-clinical research, however existing methods for measuring cytokine levels are

We introduce a new method for the analysis of enzyme-linked immunosorbent assay (ELISA) data. The new method can use data near the asymptotes and does not give undue weight to responses on the flatter parts of the dose-response curve. We apply it to simulated data and to two real-world assays and show it is more accurate and more precise than the traditional interpolation method. In particular, the

Coronavirus Disease 2019 (COVID-19) convalescent plasma (CCP) was approved by the FDA for use in severe cases of COVID-19 under an emergency Investigational New Drug (IND) protocol. Eligibility criteria for CCP donors includes documentation of evidence of COVID-19 either by viral RNA detection at the time of illness or positive SARS-CoV-2 IgG after recovery if diagnostic testing for COVID-19 was not

Background Assessment of pure polysaccharide response to the 23-valent pneumococcal polysaccharide vaccine (PPV23) can be biased by previous exposure to the conjugate vaccine (PCV). We applied pre-analytical modification to the existing ELISA by pre-incubating serum with PCV. Methods PCV-adsorbed and non-adsorbed sera were prepared before measuring the concentration of anti-pneumococcal capsular polysaccharide

The screening for IgG subclass donor-specific antibodies (DSAs) in allograft recipients uses IgG1-4 subclass-specific monoclonal antibodies (mAbs) that should be mono-specific. The cross-reactivity discrepancies reported for IgG subclass-specific mAbs warranted a critical cross-reactivity pattern analysis of the IgG subclass-specific mAbs most commonly used to detect DSAs. We tested the reactivity

Japanese encephalitis (JE) is a mosquito-borne flaviviral zoonotic disease and is one of the major causes of encephalitis in children. Swine, being an amplifier host of Japanese encephalitis virus (JEV), play an important role in its epidemiology. Therefore, early detection of either JEV or antibodies against JEV in swine is a feasible alternative for initiating necessary measures to prevent the spread

Polyethylene glycol (PEG) represents an effective strategy to improve the pharmacokinetic profile of a molecule as it extends the biotherapeutic's half-life, masks immunogenic epitopes or modifies its distribution. The addition of one or multiple PEG moieties, in either linear or branched form, is known to carry the risk of potentially inducing an immunogenic response against PEG. The importance of

In human autoimmune diseases, low plasma levels of complement factors C3 and C4 are commonly used as a proxy for complement activation. The measurements of C3 and C4 concentrations (the result of synthesis and consumption) however, show low sensitivity in patient follow-up. We find that the estimation of the C3dg fragment released during complement activation is a better parameter for complement activation

One of the most abundant coagulation proteins is β2-glycoprotein I (β2GPI) that is present in humans at a concentration of around 200 mg/L. Its physiological role is only partially understood, but it adopts several different structural forms the majority of which are the open and closed forms. We isolated native (circular) β2GPI and converted it into an open conformation. The effectiveness of these

Here we show that an approach of in-vitro transcribed mRNA nucleofection expands the range of transfection of primary human T cells. It represents a reproducible and time-efficient technology, and is thus an ideal tool in basic research involving highly controlled in-vitro experiments with a gene of interest aiming at identifying its biological human T cell function.

The expression of Kv1.3 and KCa channels in human T cells is essential for maintaining cell activation, proliferation and migration during an inflammatory response. Recently, an additional residual current, sensitive to anandamide and A293, compounds specifically inhibiting currents mediated by TASK channels, was observed after complete pharmacological blockade of Kv1.3 and KCa channels. This finding

Genus Flavivirus, which includes 53 virus species, is the leading cause of arthropod-borne diseases in humans. Diagnosis of these viral diseases is complicated by their overlapping epidemiology and clinical manifestations, and the fact that cross-reactive antibody responses are frequently elicited by individuals in response to infection. We developed a bead-based immunoassay to concomitantly profile

As a category A toxic, the botulinum toxin(BoNT) is responsible for human botulism with an estimated lethal dose of 1 ng/kg which greatly increases the potential risk of use as bioweapons. Therefore, the development of anti-BoNT antibodies is urgent. In this paper, the HC domain of BoNT/A was purified and immunized with Balb/c mice. Monoclonal antibodies were screened against BoNT/A from 55 stable

The presence of (1 → 3)-β-D-glucan in human plasma is a marker for fungal infections. Currently, the Limulus amebocyte lysate (LAL)-based assay is widely used for the quantification of plasma (1 → 3)-β-D-glucan. However, it has limitations in clinical use, such as an unstable supply of natural resources, complicated manufacturing process, and low-throughput of the reagents. Alternative assays exploiting

This report covers the methodology for generation of stable heterohybridoma clones producing Foot-and-mouth disease virus (FMDV) reactive porcine monoclonal antibodies (mAbs). Swine received five inoculations of an inactivated O1 Manisa FMDV vaccine prior to the harvest of splenocytes. Due to the lack of a species-specific hybridoma fusion partner, the Sp2/0 murine myeloma cell line was utilized for

Recombinant envelope protein-1 (E1) and E2 of Chikungunya virus (CHIKV) has been shown to elicit neutralizing antibodies and a balanced Th1/Th2 response in mice however with limited protection. Recently reported CHIK virus-like particles showed augmented immunity and protection in adult mice in comparison to E1 and E2, however exacerbated the disease in aged subjects. In order to improve the overall

The skin prick test is used to diagnose patients' sensitization to antigens through a mediated IgE response. It is a practical and quick exam, but its diagnosis depends on instruments for measuring the allergic response and observer's interpretation. The conventional method for inferring about the allergic reaction is performed from the dimensions of the wheals, which are measured using a ruler or

Objectives In the diagnostic work up of autoimmune gastritis several immunological methods are available for the detection of antibodies against Intrinsic Factor (IF) and Parietal Cells (PC). However, there are no recent reports directly comparing all the available assays and methods. The objective of this study was to compare the performance of several commercially available anti-IF and anti-PC antibody

Antibodies against human platelets cause a variety of thrombocytopenic disorders, which lead to potentially fatal haemorrhage. Therefore, their prompt detection is mandatory for successful patient treatment. Solid phase red cell adherence (SPRCA) assay allows for platelet antibody detection widely. However, preparation of fresh platelets with HLA-I and human platelet antigens (HPA)1–5,15 genotyped

Since the FDA approval of two Chimeric Antigen Receptor (CAR) T cell therapies against CD19+ malignancies, there has been significant interest in adapting CAR technology to other diseases. As such, the ability to simultaneously monitor manufacturing criteria and functional characteristics of multiple CAR T cell products by a single instrument would likely accelerate the development of candidate therapies

Neoantigen-based cancer immunotherapies hold the promise of being a truly personalized, effective treatment for diverse cancer types. ELISPOT assays, as a powerful experimental technique, can verify the existence of antigen specific T cells to support basic clinical research and monitor clinical trials. However, despite the high sensitivity of ELISPOT assays, detecting immune responses of neoantigen

Critical to managing the spread of COVID-19 is the ability to diagnose infection and define the acquired immune response across the population. While genomic tests for the novel Several Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) detect the presence of viral RNA for a limited time frame, when the virus is shed in the upper respiratory tract, tests able to define exposure and infection beyond

IgA antibodies are key immune effectors against invading pathogens but also possess essential immunoregulatory functions. Detecting and quantifying human IgA+ B-cell subsets and secreted IgA molecules is needed for investigating the protective, modulatory and pathophysiologic roles of IgAs. Here, we produced a recombinant tagged trimeric form of the streptococcal IgA-binding peptide (SAP) by transient

Objective To evaluate the analytical performance of our previously developed chemiluminescence immunoassay (CLIA) kit for the detection of procalcitonin (PCT) and compare with the results obtained using the Vidas B.R.A.H.M.S. PCT™ test (PCT-V). Design and methods Our laboratory previously designed a novel CLIA kit and supporting instrument (AE-180) for the detection of PCT. We analyzed the clinical

A commonly employed method to determine the function of a particular cell population and to assess its contribution to the overall system in vivo is to selectively deplete that population and observe the effects. Using monoclonal antibodies to deliver toxins to target cells can achieve this with a high degree of efficiency. Here, we describe an in vivo model combining the use of immunotoxins and multidrug

Researchers routinely use antibodies to assess the expression levels of proteins on the surface or intracellularly in a variety of different cell types. In this current study we highlight the importance of careful validation of antibodies for analysis of protein expression by flow cytometry and how failure to do so can significantly impact the interpretation of the data generated leading to false-positive

The ingestion of apoptotic corpses by macrophages, a process called efferocytosis, is a crucial step in inflammation resolution, since it alters macrophage phenotype toward a pro-resolving profile to foil inflammation and to favor tissue repair. Up to now, the resolving macrophages remain poorly characterized, especially in humans. Global investigations, like RNA sequencing, would be very helpful to

The cut point is an important parameter for immunogenicity assay validation and critical to immunogenicity assessment in clinical trials. FDA (2019) recommends using a statistical approach to derive cut point, with an appropriate outlier removal procedure. In general, the industry follows the methods described in Shankar et al. (2008) and Zhang et al. (2013) among others to determine cut point. Outlier

Covalent immobilization of antibodies to protein G beads is a basic molecular biology method, although the beads present poor recovery results. Our aim was to reuse the immobilized antibody-protein G complex on a very small scale, therefore we optimized the crosslinking procedure to be used on the wells of a standard 96-well microplate. The method used involves the affinity binding of the antibody

Due to the technical innovations in generating bispecific antibodies (BsAbs) in recent years, BsAbs have become important reagents for diagnostic and therapeutic applications. However, the difficulty of producing a heterodimer consisting of two different arms with high yield and purity constituted a major limitation for their application in academic and clinical settings. Here, we describe a novel

High priority stereospecific targeting (SST) featuring selective production of conformation-specific monoclonal antibodies was directed against a native receptor, EphA2 (ephrin type-A receptor 2). A critical point for this technology is selection of sensitized B lymphocytes by antigen-expressing myeloma cells through their B-cell receptors (BCRs). The essential point is that antigens expressed on myeloma

Monoclonal antibodies (mAb) are unique tools in therapeutics and immunodiagnostics applications but many of these applications rely on conjugated mAbs. Whether conjugating drugs or tracers, the conjugation process, frequently taking advantage of primary amines on lysine residues, may affect the binding activity of the antibodies. Furthermore, due to the sticky nature of many mAbs, unfavorable interactions

Three different porcine enteric coronaviruses (PECs), i.e., porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine Deltacoronavirus (PDCoV) are currently circulating in U.S. commercial swine herds. Differential diagnosis of PECs relies on laboratory methods. This study describes the development of an ELISA-like multiplex planar immunoassay based on virus-specific

Anti-Staphylococcal Enterotoxin B single domain antibodies were engineered to include the N-terminal peptide sequence of the major outer membrane lipoprotein from Escherichia coli, which directs the N-terminal addition of lipid to the single domain antibody. We produced and purified two different single domain antibodies as well as a variant and dimer construct of one of the two, all with and without

Background Numerous studies have demonstrated the capabilities of the basophil activation test (BAT) but various parameters such as a lack of standardization and a time consuming and labor intensive workflow continue to hinder the field to fully leverage the capabilities of this technique. When pediatric patients have to be considered, an additional limitation is related to blood volume consumption

Whole-blood fixation provides a rapid and simplified method for cell preservation compared to isolation of peripheral blood mononuclear cells (PBMCs). This can be especially important for sample acquisition and storage in resource-limited settings. However, some caveats have been reported, such as reduced cell marker recognition. Here, we evaluated the whole-blood proteomic stabilizer PROT1 and compared

The surface of the mucosa is the biggest path through which pathogens enter the human body. We need an understanding of mucosal immune systems to use vaccines that generate protective mucosal and systemic immunity to regulate the outbreak of various infectious diseases. The better impact of the mucosal vaccine over traditional injectable vaccines are that not only do they induce efficient immune reactions

We describe an adaptation of conventional ELISA methods to an ELISA-Array format using non-contact Piezo printing of up to 30 spots of purified recombinant viral fusion proteins and vaccine on 96 well high-protein binding plates. Antigens were printed in 1 nanoliter volumes of protein stabilizing buffer using as little as 0.25 nanograms of protein, 2000-fold less than conventional ELISA. The performance

Alarms periodically emerge for viral pneumonia infections due to coronavirus. In all cases, these are zoonoses passing the barrier between species and infect humans. The legitimate concern of the international community is due to the fact that the new identified coronavirus, named SARS-CoV-2 (previously called 2019-nCoV), has a quite high mortality rate, around 2%, and a strong ability to spread, with

The recent identification of human monoclonal antibodies with broad and potent neutralizing activity against HIV-1 (bnAbs) has resulted in substantial efforts to develop these molecules for clinical use in the prevention and treatment of HIV-1 infection. As with any protein therapeutic drug product, it is imperative to have qualified assays that can accurately detect and quantify anti-drug antibodies

The ability to detect, quantify, and interrogate the properties of immune responses raised against biological therapeutics is not only important to our understanding of these molecules, but also to their success in the clinic. A tiered assay approach to identify the presence, specificity, and titer of anti-drug antibody (ADA) responses has been adopted as a gold standard by industry leaders, the FDA

Drug allergies pose a great deal of danger for the patients. It hinders effective treatment procedures in hospitalized patients. Moreover, it complicates the symptoms due to the allergic reactions of the immune system. Allergic reactions may arise against any medication including antibiotics and chemotherapeutics. Therefore, it is crucial to assess the sensitization pattern of the patients to culprit

Gene silencing using small interfering ribonucleic acids (siRNA) is a powerful method to interfere with gene expression, allowing for the functional exploration of specific genes. siRNA interference can be applied in both cell lines, as well as in primary, non-dividing cell types like dendritic cells. However, the efficacy in different cell types is variable and requires optimization. Here, we showed

Monitoring recency of infection helps to identify current transmission in vulnerable populations for effective disease control. We have established an in-house avidity based hepatitis C virus (HCV) recency assay based on the Monolisa Anti-HCV PLUS Version 3 ELISA kit for use of dried serum/plasma spots (DS/PS) in order to distinguish recent and long-term infections. A first panel of DS/PS (n = 218;

The specific recognition between a monoclonal antibody (mAb) and its epitope can be used in a tag system that has proved valuable in a wide range of biological applications. Herein, we describe a novel tag called RA-tag that is composed of a seven amino acid sequence (DIDLSRI) and recognized by a highly specific mAb, 47RA, against the bacterial toxin Vibrio vulnificus RtxA1/MARTXVv. By using recombinant