Journal of immunological methods最新文献

Background

The measurement of antigen-specific serum IgE is common in clinical assessments of type I allergies. However, the interaction between antigens and IgE won't invariably trigger mast cell activation. We previously developed the IgE crosslinking-induced luciferase expression (EXiLE) method using the RS-ATL8 mast cell line; however, the method may not be sensitive enough in some cases.

Methods

In this study, we introduced an NF-AT-regulated luciferase reporter gene into the RBL-2H3 rat mast cell line and expressed a chimeric high-affinity IgE receptor (FcεRI) α chain gene, comprising an extracellular domain from humans and transmembrane/intracellular domains from rats.

Results

We generated multiple clones expressing the chimeric receptor. Based on their responsiveness and proliferation, we selected the HuRa-40 clone. This cell line exhibited significantly elevated human α chain expression compared to RS-ATL8 cells, demonstrating a 10-fold enhancement of antigen-specific reactivity. Reproducibility across different batches and operators was excellent. Moreover, we observed a detectable response inhibition by an anti-allergy drugs (omalizumab and cyclosporin A).

Conclusions

HuRa-40 cells—which carry the human-rat chimeric IgE receptor—comprise a valuable reporter cell line for the EXiLE method. Their versatility extends to various applications and facilitates high-throughput screening of anti-allergy drugs.

Aim

Quality control testing of the vaccine for lot release is of paramount importance in public health. A recent pandemic caused by the SARS-CoV-2 virus brought together all spheres of vaccine to combat the virus. The scientific advancement in the development of vaccines facilitated the scientists to develop the vaccine against SARS-CoV-2 in a record time. Thus, these vaccines should be stringently monitored for their safety and efficacy as per the latest WHO and national regulatory guidelines, and quality control evaluation of the product should be done at national control laboratories before releasing the product into the market as it assures the quality and safety of the vaccine.

Methods

The SARS-CoV-2 exploited the ACE2 (Angiotensin Converting Enzyme 2) receptor, a surface protein on mammalian cells to gain entry into the host cells. The viral surface protein that interacted with the ACE2 receptor is the Spike protein of SARS-CoV-2. Thus, in the development of the vaccine and assessing its quality, the Spike protein of SARS-CoV-2 became an attractive immunodominant antigen. In National Institute of Biologicals, an apex body in the testing of biologicals in India, received the Adenovector (Adenovirus + vector) based COVID-19 vaccine, a finished product for quality evaluation. Due to the lack of a pharmacopeial monograph, the testing of the vaccine was done as per the manufacturer's specifications and methods. The routine assays of identification employed by the manufacturer do not reflect the expression of Spike protein which is required for the immune system to get activated. In this report, we showed the determination of Spike protein expression by immunoblotting and immunofluorescence for identification parameters in the quality testing of the COVID-19 vaccine. We determined the translation of the SARS-CoV-2 Spike gene cloned into an Adenovector.

Results

The results from these experiments indicated the expression of Spike protein upon infection of mammalian cells with viral particles suggested that the expression of immunodominant Spike protein of SARS-CoV-2 may be employed by quality control laboratories as a parameter for identification.

Conclusion

The study suggested that the determination of the expression of Spike protein is pertinent to identifying the Adenovector based vaccines against COVID-19.

Cerebrospinal fluid (CSF) is a critical body fluid to examine in attempts to discover potential biomarkers for neuroinflammatory and other disorders of the central nervous system (CNS). Serum and/or plasma cytokine levels have been associated with a variety of inflammatory conditions, and some have been shown to be actionable therapeutic targets. Less is known, however, about cytokine levels in CSF. Serum and plasma cytokine testing is widely available in clinical and research laboratories, but cytokine testing in CSF is extremely limited and if performed, accompanied by a disclaimer that it is an unvalidated specimen type. In this study, we validate CSF as a suitable specimen type and determine normal reference intervals for multiple cytokines as well as a soluble cytokine receptor. CSF was validated as a specimen type for testing using a laboratory developed multiplexed cytokine assay previously validated to measure 13 cytokines/markers in serum and plasma. Performance parameters including specimen dilution, specimen interference, linearity and precision were examined. Reference intervals were established using 197 normal and control CSF specimens by non-parametric quantile-based methods. CSF cytokine analysis demonstrated within and between run precision of <10% and < 20% CV, respectively and linearity of ±15% for all analytes throughout the analytical measurement range of the assay. Reference intervals for the 13 cytokines/markers were established from 197 normal and control CSF specimens (78 Male; mean 44.8 y ± 21.7 SD, 119 Female; mean 42.8 y ± 20.3 SD). Cytokine concentrations in CSF from normal donors and controls were less than the lower limit of quantitation of our assay for 6 of the 13 measured cytokines/markers. The chemokine IL8 demonstrated the highest concentration of all analytes measured. CSF demonstrated acceptable performance as a specimen type in our multiplexed cytokine assay. By validating CSF as a specimen type and establishing normal reference intervals for cytokine concentrations in CSF, their potential as biomarkers for infectious, autoimmune and other inflammatory CNS disorders can be more appropriately investigated.

The type II autoimmune subtype of Chronic Spontaneous Urticaria (CSU) is characterized by the presence of IgG autoantibodies targeting IgE or the IgE high-affinity receptor (FcεRI) on mast cells and basophils. In evaluation of CSU patients, indirect basophil activation testing (BAT), has been utilized, involving the mixing of patient serum with heterologous peripheral blood donors, followed by flow cytometric assessment of basophil markers. However, the reliability of the indirect BAT results hinges on the quality of the donor basophils utilized. In this study, we introduce an innovative approach where multiple potential basophil donors undergo rigorous BAT characterization alongside control samples. By selecting and pooling donors with optimal performance, we significantly enhance the inter-assay reproducibility of the indirect BAT test.

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. Knowing the hemolytic activity of the peptides we are developing contributes to our understanding of their potential toxicity. Computational approaches for predicting hemolytic activity of AMPs exist and were tested head-to-head with our experimental results.

Results

Starting with an observation of high hemolytic activity of LL-37 peptide against human red blood cells that were collected in EDTA, we explored alternative approaches to develop a more robust, accurate and simple hemolysis assay using defibrinated human blood. We found significant differences between the sensitivity of defibrinated red blood cells and EDTA treated red blood cells.

Significance

Accurately determining the hemolytic activity using human red blood cells will allow for a more robust calculation of the therapeutic index of our potential antimicrobial compounds, a critical measure in their pre-clinical development.

Conclusion

We introduce a standardized, more accurate protocol for assessing hemolytic activity using defibrinated human red blood cells. This approach, facilitated by the increased commercial availability of de-identified human blood and defibrination methods, offers a robust tool for evaluating toxicity of emerging drug compounds, especially AMPs.

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. This is most likely due to alternative contact sites for protein G located in the CH1 domain of the Fab heavy chain. Our data demonstrate that protein G affinity chromatography as for whole antibodies is a robust method for the purification of tag-less Fab-fragments independent of species, significantly simplifying the process of Fab-fragment purification.

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. After optimization, methodological evaluation was performed with the guidelines of Clinical Laboratory Standards Institute (CLSI). Finally, 131 plasma samples were collected to analyze whether the new method is suitable for clinical detection.

Results

The LoB, LLoQ, ULoQ, and linear range of the assay were 1.09 ng/mL, 3 ng/mL, 1500 ng/mL, 3–1500 ng/mL for SAA and 0.85 ng/mL, 2 ng/mL, 1200 ng/mL, 2–1200 ng/mL for LTF respectively. The recovery rates were 95.01% to 106.26%, the intra-batch precision of low, intermediate, and high-level samples was <8%, and the inter-batch of them was <11%, the deviation of interference test results was less than±10%. The Area Under the Curve (AUC) was 0.9809 for SAA, 0.8599 for LTF, and 0.9986 for combination.

Conclusion

The quantitative duplex immunoassay for SAA/LTF has high accuracy, good precision, and high specificity, which meets the clinical testing requirements and can be widely used in clinical practice.

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. Here, we validate the repeatability of the DBI and its correlation with pathway-specific and common fluid phase markers. Moreover, we discuss the advantages of DBI, such as cost-effectiveness and versatility, while addressing potential limitations. This study provides insights into complement activation at the nanosurface level, offering a valuable tool for nanomedicine researchers in the field.

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. We demonstrate the feasibility by using the canonical second generation CARs specific for CEA and Her2, respectively, obtaining highly purified CAR T cell products in a one-step procedure without impairing cell viability. The protocol is also applicable to a dual specific CAR (tandem CAR). Except for CD39, T cell activation/exhaustion markers were not upregulated after separation. Purified CAR T cells retained their functionality with respect to antigen-specific cytokine secretion, cytotoxicity, and the capacity to proliferate and eliminate cognate tumor cells upon repetitive stimulation. Collectively, the one-step protocol for purifying CAR T cells extends the toolbox for preclinical research and specifically for clinical CAR T cell manufacturing.