Journal of Immunological Methods

GP101 is a single chain diabody composed of Fv regions of antibodies directed to CD3 and CD19. It is designed to mimic commercial blinatumomab (Blincyto(R)) that simultaneously co-engages patient T lymphocytes and CD19 positive B cell leukemias and lymphomas, facilitating their targeted destruction. This study details the purification of GP101 protein and development of a highly sensitive ELISA for its detection, achieving a sensitivity of 15pg/mL in 25% mouse serum. This ELISA has been validated according to GLP standards.

BackgroundFor kidney transplant recipients, assessing non-invasively the individualized risk of acute rejection is one of the most unmet need. Urinary chemokines are one of the short-term most promising biomarkers, because of their simple and low-cost analytical method in easily accessible samples, and their high diagnostic performance consistently assessed over the last decade. In this study, we aimed at confronting all practical issues of routine implementation of kidney allograft rejection monitoring using urine CXCL9 and CXCL10. MethodsThe next-generation immunoassay Ella(R) was investigated as feasible quantification platform, from sample collection to render of the results. Urine CXCL9/10 levels were measured using Ella(R) microfluidic cartridges, across preanalytical (sample preparation, storage conditions, freeze-thaw cycles) and analytical (linearity, ranges, intra/inter-assay variability) performances studies, and diagnostic accuracy was assessed in comparison to the ELISA reference method, in urine samples from previously published cohorts. ResultsUpon assay preparation, Ella(R) appeared very efficient with a minimal workflow (urine sample thaw> centrifugation>1:2 dilution>loading) and a time to result of 90 minutes. Preanalytical studies showed high stability of urine CXCL9/10 levels across various temperatures (4{degrees} vs 25{degrees}C) and time (24/48/72H) before storage and over 5 freeze-thaw cycles. How complex urine matrix, analytical studies confirmed excellent linearity of dosage, as well as intra-assay ([&le;]5%) and inter-assay precision. Across 1024 samples, Ella(R) results were highly correlated to those quantified by ELISA (P<0.0001), and further entered into our previously validated urine chemokine model. 268 out of the 1024 samples were collected at time of acute rejection (26.2%). In this cohort, accuracy was 0.85 for acute rejection diagnosis. ConclusionElla(R) fulfils all prerequisites for clinical implementation of urinary chemokines monitoring. It has proven a robust, easy-to-use platform with unprecedented validation to quantify urine chemokines.

Tacrolimus is an immunosuppressant drug commonly used in transplantation. Although multiple studies have demonstrated that polymorphisms in the CYP3A5 gene impact the metabolism of tacrolimus, routine pre-transplant testing for these markers is still not broadly implemented. TacroType - a new laboratory developed test implemented by One Lambda Laboratories - utilizes a qPCR-based six-plex assay for CYP3A5 genotyping and detects the three most common genetic variants (*3, *6 and *7) associated with loss of CYP3A5 protein function and reduced tacrolimus metabolism. TacroType was optimized to address known sources of protocol, technical or sample variability to achieve accurate and reproduceable genotyping results. An analytical performance study was completed following CLSI guidelines. Accuracy was confirmed for each possible CYP3A5 genotype involving 6 target alleles using 32 well-characterized reference samples. TacroType exhibited accurate performance within a broad range of DNA concentrations and quality. Precision studies indicated consistent genotyping results across 4 operators, 2 instrument types and 5 lots of reagents. Accurate and reproducible assay performance was demonstrated using whole blood from 100 and buccal swabs from 70 donors. The analytical performance of TacroType was evaluated in 4014 total qPCR reactions, with a report rate of 99.8% and genotyping accuracy of 100% (95% confidence interval of 99.9%).

Dried blood spots (DBS) and oral fluids (OF) are easily attainable biospecimen types that have enabled population scale antibody monitoring for SARS-CoV-2 exposure and vaccination. However, the degree to which the two different biospecimen types can be used interchangeably remains unclear. To begin to address this question, we generated contrived DBS (cDBS) and OF (cOF) from serum panels from SARS-CoV-2 infected, vaccinated, and uninfected individuals. The contrived samples were evaluated using SARS-CoV-2 multiplexed microsphere immunoassays (MIAs) at two different institutions. Intra-laboratory tests revealed near perfect agreement between cDBS and cOF for N and S antigens, as evidenced by {kappa} = 0.97-1 and 98%-100% agreement. Inter-laboratory comparisons were equally robust for both N ({kappa} = 0.94-0.96; 97.5%-98 % agreement) and S ({kappa} = 0.98 -1.0; 99.0%-100%). Furthermore, assays were transferred between labs, including methods and reagents, and a subset of cDBS and cOF samples (n = 52) were tested. Qualitative concordance remained high ({kappa} = 0.94-1.0; 97.5%-100% agreement), confirming that integrity of the assays is retained upon transfer. In summary, our results provide evidence that DBS and OF can be used interchangeably across laboratories and institutions for the qualitative assessment of SARS-CoV-2 antibody determinations.

Efficient pathogen detection is essential for the successful treatment and prevention of infectious disease; however, current methods are often too time intensive to be clinically relevant in cases requiring immediate intervention. We have developed a Surface Programmable Activation Receptor (SPAR) diagnostic platform comprised of universal biosensor cells engineered for use in combination with custom or commercial antibodies to achieve rapid and sensitive pathogen detection. SPAR cells are stably transfected Jurkat T cells designed to constitutively express a modified T cell mouse Fc{gamma}RI receptor on the cell surface and a high level of the luminescent reporter protein aequorin in the cytoplasm. The modified mFc{gamma}RI-CD3{zeta} receptor protein binds with high affinity to the Fc region of any full-length mouse IgG2a and some IgG2 antibodies: this allows customized target detection via the selection of specific antibodies. T-cell receptor aggregation in response to target antigen binding results in signal transduction which, when amplified via the endogenous T cell signal cascade, triggers the rapid intracellular release of calcium. Increased Ca2+ concentrations activate the expressed reporter protein aequorin resulting in the immediate emission of detectable light. Testing demonstrates the accurate and specific detection of numerous targets including P. aeruginosa, E. coli O111, and E. coli O157. We report that the SPAR biosensor cell platform is a reliable pathogen detection method that enables the rapid identification of bacterial causative agents using standard laboratory instrumentation. The technology lends itself to the development of efficient point-of-care testing and may aid in the implementation of effective and pathogen-specific clinical therapies.

Purpose and appropriate sample typesThis multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional ({gamma}{delta}-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naive (TNaive), central memory (TCM), effector memory (TEM) and terminal effector (TTE) T cells. Activated cattle T cells (TAV) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-colour, 10-parameter flow cytometry panel simultaneously identifies cattle TNaive, TAV, TCM, TEM, TTE and {gamma}{delta}-TCR cells. This panel will improve our ability to examine T cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimised for other bovid species as many of these reagents are likely to cross react.

Several secretion capture assays have been developed to measure a secreted protein of interest, using paired monoclonal antibodies of different specificities that bind simultaneously to a cell surface antigen and to the secreted protein of interest. Here we have developed a novel method to measure antibody secretion on B cells, which, by capitalising on the bivalent nature of an antibody molecule, utilises an anti-immunoglobulin (Ig) antibody to bind on one side to the membrane-bound Ig (in the B cell receptor), and then capture any secreted Ig with the second binding site. Binding of the secreted cargo is then detected through a second application of anti-Ig, this time fluorophore-labelled. We demonstrate that this can effectively measure single cell Ig secretion in a population of B cells undergoing differentiation to plasma cells. This offers a reliable and accessible tool for quantifying antibody secretion dynamics at the single-cell level with potential applications in immunological studies, antibody discovery and functional profiling of B cell responses.

Magnetic particles are commonly used to isolate specific cell types from blood samples. Residual particles present in the genomic DNA extracted from these cells can interfere with concentration measurements by UV absorbance spectrophotometry. In this study, we determined the degree of inaccuracy of DNA quantification by UV spectrophotometry, in the context of the lineage-specific chimerism analysis workflow. We showed that the presence of residual magnetic particles and RNA leads to an overestimation of the DNA concentration.

BackgroundNatural killer (NK) cell degranulation is a key immune defense mechanism where exposure to tumor or virus-infected cells triggers the fusion of cytoplasmic granules containing apoptotic proteins, perforin, and granzyme with the cell membrane. This process transiently expresses CD107a on the NK cell surface, and measuring CD107a is a standard method to assess NK cell activity. MethodsWe compared two stimulation protocols differing only in duration (6-hour vs. 18-hour) using K562 target cells to induce NK cell degranulation. Isolated PBMCs without stimulation served as controls to assess spontaneous degranulation. Anti-CD107a-PE antibody was present throughout stimulation in both test and control samples. After stimulation, cells were stained with anti-CD45, anti-CD3, and anti-CD56 and analyzed by flow cytometry. ResultsFor 6 of 7 healthy controls, results from both methods fell within 2 standard deviations. Notably, longer (18-hour) stimulation resulted in lower CD107a expression than the 6-hour assay. Interlaboratory comparisons of two samples showed no significant difference (p>0.05). In a suspected hemophagocytic lymphohistiocytosis (HLH) case, two labs reported similarly reduced CD107a expression (9% and 7%). Inter-day variability was observed in a donor across both time points. The 6-hour assay showed higher sensitivity and specificity than the 18-hour assay. A resting period before ex vivo PBMC assays was found necessary. ConclusionStimulation periods beyond 6 hours are unsuitable for clinical NK degranulation assays. Screening for HLH should include multiple stimulants to improve assay reliability.

Bovine dendritic-cell subsets can be readily identified in flow cytometry by staining for Flt3, CD4 and CD13. While this gating strategy could be confirmed by bulk RNA-seq of subsets sorted from peripheral blood mononuclear cells, it remains to be determined if the same gating strategy can be applied to gate on cDC1, cDC2 and pDC within lymphoid and non-lymphoid tissues. With this preprint we aim to inform on the current status of phenotyping experiments performed on Flt3+ cells in mesenteric lymph nodes, spleen and lung of cattle. Distinct DC subsets can be gated in all tissues based on CD4 and CD13, with high frequencies of Flt3+CD13+ cells (putative cDC1) detected in the lung. Further phenotyping performed on lymph node cells (in particular CADM1, CD26 and CD123) support the subset classification analogous to blood, with pDC being Flt3+CD4+CD13- and cDC1 being Flt3+CD4-CD13+. Heterogeneous expression of CADM1 and CD26 on Flt3+CD4-CD13- putative cDC2 warrants further investigations.

Among dendritic cells (DC) and monocytes in blood of cattle we can distinguish conventional and plasmacytoid DC (cDC1, cDC2, pDC) and classical, intermediate and nonclassical monocytes (cM, intM, ncM), respectively. Phenotypic definitions of subsets rely on combinations of only a few markers such as CD13 and CD4 for Flt3+ dendritic cells, and CD14 and CD16 for CD172ahigh monocytes. Additional reagents for flow cytometry are urgently needed to refine these phenotypic classifications and account for heterogeneity of these cells, in particular cDC2 and intM. In the current study we have investigated expression of CX3CR1 and ADGRE1 on bovine blood DC and monocyte subsets, and have tested two newly generated bovine recombinant proteins (XCL1 and CSF1) for staining of XCR1 and CSF1R. Staining patterns obtained by multicolor flow cytometry of peripheral blood mononuclear cells from two cows were largely in line with gene expression data available from a previous study (bulk RNA-seq of sorted DC and monocyte subsets from bovine blood).

Intravenous Immunoglobulins (IVIGs) are prepared from thousands of donor plasmas and as a result comprise an extreme broad mix and depth of Antibody (Ab) specificities. IVIG formulations available in Australia are from both local and overseas donor sources and extracted using a variety of purification methods and immunoglobulin purities, with the Australia-derived and prepared IVIG listed at >98% and the overseas-derived preparations at [&ge;] 95%. Because of these differences it was predicted that the formulations might individually vary in composition and that together with obvious genetic and geographic antigenic (Ag) environment differences may result in notable variability between formulations. Hence a focussed comparative proteomic profiling of IVIG formulations was undertaken to identify notable similarities and differences across products. Comparisons between formulations did reveal marked qualitative differences in 2D-gel Antibody profiling that included parameters of isoelectric charge (pI), as well as immunoglobulin (Ig) monomer to dimer ratio variability between products, including high molecular weight (MW) immunoglobulin multimers for some. These notable differences were in part quite likely a product of the respective purification methods used, and capacity to select (or de-select) for antibodies of such different properties. Furthermore, for identification of non-Ig proteins carried over from plasma through purifications Mass spectrometry was performed. This identified a few such ancillary proteins, and their identities, in general, differed between formulations. Proteins detected included the most abundant protein of plasma, albumin, as well as other mostly large and abundant proteins; RAG1 - V(D)J recombination activating protein1, gelsolin, complement Factor-B, serotransferrin, tetranectin, NADH ubiquinone oxidoreductase, caspase 3 and VEGFR1. An alternate strategy used commercial Multiplex xMAP assay to detect cytokines, which are small and present in plasma at trace but highly active quantities. This revealed various different cytokine profiles across the formulations studied. The identification of additional proteins, and especially cytokines in IVIGs, is particularly notable, and the positive, negative or null biological relevance for clinical use, needs resolution. Collectively these findings reveal marked differences between Australian and overseas-derived (non-Australian) IVIGs in immunoglobulin composition and biochemical characteristics, and presence of additional carry-over proteins from plasma. These findings prompt the need for further evaluation of the micro-compositions of individual formulations. Perhaps detailed mining and improved comparative understanding of each IVIG formulation, may enable highly tailored and strategic clinical use of certain formulations that are personalised best-fit treatments for particular conditions. Such as in treatment of a neuropathy, as compared to another formulation, more suited for treating a particular infectious disease. The most salient and overarching study conclusion is need for caution in attributing equivalence across IVIGs.

Comparing experimental results across different instruments and laboratories is challenging due to variations in instrument sensitivity and resolution, particularly for analyzing nano-sized and dimly fluorescing particles like viruses and extracellular vesicles. To address this challenge, we conducted a study using fluorescent DNA calibrator beads to test, calibrate, and compare the sensitivity of five different flow cytometers. Our approach involves DNA beads produced through a bottom-up DNA origami method. These beads contain a defined number of fluorophores (0-220) and exhibit no autofluorescence. Fluorophores are precisely conjugated onto the beads in a controlled array, minimizing dye interactions. This allows triggering on one fluorophore and calibration based on another, facilitating precise and accurate calibration in absolute fluorophore numbers. We tested five flow cytometers--FACSAria III, NovoCyte 3000YBG, NovoCyte Quanteon 4500, LSRFortessa, and CytoFlex S--using Cy5 and FAM DNA calibrator beads. We investigated various instrument parameters, including count rate, trigger and calibration gain, gain amplification linearity, and fluorescence trigger sensitivity. Using fluorescent triggering, we demonstrated the capability to detect dimly fluorescent particles, enabling instrument calibration and fluorescence sensitivity comparison using DNA calibrators. In conclusion, our study offers a robust method for addressing instrument fluorescence channel resolution in absolute fluorophore numbers in the analysis of nano-sized and dimly fluorescent particles.

TCR- and BCR-sequencing (TCR/BCR-seq) are two important technologies in studying the immune repertoire of samples such as PBMCs or tumors. In their most common form, these assays combine multiplex PCR of the repertoire using primers targeting regions of the V(D)J and the constant region with next-generation sequencing (NGS). The data produced by this assay provide a slew of information regarding immune repertoire(s) including the presence critical clonotypes, repertoire diversity, variable (V) gene usage, analysis of public clonotypes, etc. One issue that can arise during generation of the TCR/BCR-seq data is sequence bias during the PCR or NGS steps. To combat this, unique molecular identifiers (UMIs) have been used to identify and eliminate sequence bias. However, UMI fragments can be long and very diverse, resulting in the UMI sequences interfering with any of the multitude of primers during multiplex PCR. Here, we introduce Validator Barcodes (VBCs), a set of eight short barcodes (6-9 nucleotides in length). This compact set of barcodes improves PCR efficiency and facilitates PCR primer designs. Also, like UMIs, the VBCs may be used to estimate the number of template molecules (RNA or DNA). Using VBC-labeled primers for TCR and BCR repertoire profiling from PBMCs produces highly comparable results and similarly template values to those obtained through UMI-based assay counts. Overall, VBCs are a useful and simpler alternative to UMIs in assaying TCR and BCR repertoires.

The human papillomavirus (HPV) 16 genome encodes two oncoproteins, E6 and E7, which are essential for viral carcinogenesis. While E7 promotes cell proliferation, E6 abolishes the resulting p53-dependent apoptotic response. Due to this specific function, E6 is considered a suitable target for the development of a variety of therapeutic agents such as antibodies. Here, we review anti-E6 antibodies/antibody fragments generated by us and others, as well as present our latest results with Camelidae-derived single-domain antibodies (sdAbs). We had previously isolated a pool of anti-E6 sdAbs to identify E6 binders with the potential to be used clinically and in research. While our previous work has focused on recombinant E6 proteins, here we evaluated these sdAbs binding capacity to the endogenous E6 protein using co-immunoprecipitation and immunofluorescence. We obtained reproducible results in these applications with two sdAbs, filling a gap in HPV research. Despite their apparent E6 binding ability, these sdAbs do not raise p53 levels or induce apoptosis. Thus, while these reagents are valuable diagnostic and detection tools, identifying their therapeutic potential will require further development and testing.

High-throughput customizable CBA automated script-based analysisThe utility of high-throughput systems to evaluate antigen-specific antibody (Ab) has been highlighted by the SARS-CoV-2 pandemic. Pathogen-specific Ab levels are often used to assess protection following vaccination and, in the case of novel pathogens, an indication of prior exposure. Several platforms exist to visualize antigen-specific Ab, however most are not quantitative and are difficult to scale for population level studies. Additionally, the sensitivity across platforms differs making direct comparisons between studies difficult. Cytometric bead arrays (CBA) are an attractive platform for antigen-specific Ab measurements as they can be used to assess Ab reactivity against several antigens and of several isotypes to be performed simultaneously. Additionally, CBAs exhibit high sensitivity and can be designed to provide quantitative measurements. Using commercially available particles, a biotin-Streptavidin bead loading strategy, and the inclusion of indirect standards, we describe a flexible system that can be modified to include a variety of antigens. Here we describe two arrays, focused on antigens derived from either {beta}-coronaviruses or influenza virus. To support the high-throughput capacity of this system, we developed a suit of automated software tools, the CBA Toolbox, to process raw data into antigen-reactive IgM, IgA, and IgG concentrations. We describe quality control requirements, assay performance, and normalizations to accurately quantitate antigen-specific Ig.

Inflammation of the mammary gland, or mastitis, is a common disease of dairy cows with important consequences on the economy of dairy farms, well-being of cows and antibiotic usage. Monitoring of the inflammatory status of mammary gland currently relies on milk somatic cell counting. In order to improve this monitoring, methods aiming at differentiating cell types among milk cells are being developed. The objective of the present study was to explore the use of gene expression profiling from raw milk samples to define the inflammatory status of udder quarters. A pilot study allowed us to select 36 genes among 95 whose expression were associated with one of three milk cell types, i.e. neutrophils, macrophages or T lymphocytes, purified by cell sorting. Based on expression data measured by RT-qPCR for these genes, we were able to identify four different types of milk samples, each with specific properties. One cluster was characterized by increased expression of genes typifying neutrophils, a second one was identified by increased expression of genes typifying macrophages while the two others were distinguished based on the expression of genes associated with T lymphocytes. Clustering of samples based on gene expression was shown to be comparable to that obtained by flow cytometry analysis. Our results highlight the potential of profiling of milk samples based on gene expression for the analysis of the inflammatory status of udders.

Spotted Fever Group (SFG) Rickettsia species maintain an s-layer with two major outer membrane proteins (OmpA and OmpB) aligned within a lipopolysaccharide (LPS) matrix external to the bacterial outer membrane. While the two typhus group species (R. typhi and R. prowazekii) maintain a similar s-layer containing only OmpB with its own specific form of LPS. A major component of these two types of OmpB is a specific and highly conserved beta peptide ({beta}-peptide) non-covalently attached to the respective OmpB passenger domain. This {beta}-peptide is initially translated as the c-terminus of the 168 kDa polypeptide and initially forms a membrane pore to allow the larger passenger domain to exit the bacterial cytoplasm through the outer membrane. Prior to the full exit of this passenger domain from the cytoplasm the long polypeptide chain is cleaved by a peptidase, leaving the c-terminus remaining as a beta-folded membrane pore and the N-terminal passenger domain exiting to the space between the bacterial membrane and the s-layer. The fate of this pore structure and the attachment of {beta}-peptide to the OmpB continues to be examined and will be discussed as another protective component of the Rickettsia. An EIA IgM assay has been developed to utilize this antigen for clinical diagnostic use to accurately detect acute rickettsial infection in testing labs and lead to the availability of rapid test (lateral flow) formats for more immediate testing.

Polymerase Chain Reaction (PCR)-based assays were widely deployed during the SARS-CoV-2 pandemic for population-scale testing. High-throughput molecular diagnostic labora-tories required a high degree of process automation to cope with huge testing demand, fast turn-around times and quality requirements. However, the critical step of preparing a PCR MasterMix has often been neglected by process developers and optimisers, and is largely dependent upon operator skill for the manual pipetting of reagents to construct the PCR MasterMix. Dependence on manual procedures introduces variation, inconsistency, wastage and potentially risks data integrity. To address this issue, we developed a liquid-handler based solution for automated, traceable and compliant PCR MasterMix preparation. Here, we show that a fully automated PCR MasterMix protocol can substitute manual pipetting, without affecting clinical calling, accuracy or precision. Ultimately, this method reduced cost-per-test at a high-throughput laboratory by eliminating operator-induced wastage while improving the quality of results.

Serological tests are important for understanding the physiopathology and following the evolution of the Covid-19 pandemic. Assays based on flow cytometry (FACS) of tissue culture cells expressing the spike (S) protein of SARS-CoV-2 have repeatedly proven to perform slightly better than the plate-based assays ELISA and CLIA (chemiluminescent immuno-assay), and markedly better than lateral flow immuno-assays (LFIA). Here, we describe an optimized and very simple FACS assay based on staining a mix of two Jurkat cell lines, expressing either high levels of the S protein (Jurkat-S) or a fluorescent protein (Jurkat-R expressing m-Cherry, or Jurkat-G, expressing GFP, which serve as an internal negative control). We show that the Jurkat-S&R-flow test has a much broader dynamic range than a commercial ELISA test and performs at least as well in terms of sensitivity and specificity. Also, it is more sensitive and quantitative than the hemagglutination-based test HAT, which we described recently. The Jurkat-flow test requires only a few microliters of blood; thus, it can be used to quantify various Ig isotypes in capillary blood collected from a finger prick. It can be used also to evaluate serological responses in mice, hamsters, cats and dogs. Whilst the Jurkat-flow test is ill-suited and not intended for clinical use, it offers a very attractive solution for laboratories with access to tissue culture and flow cytometry who want to monitor serological responses in humans or in animals, and how these relate to susceptibility to infection, or re-infection, by the virus, and to protection against Covid-19. NoteThis manuscript has been refereed by Review Commons, and modified thanks to the comments and suggestions from two referees. Those comments, and our replies, are provided at the end of the manuscripts pdf, and can also be accessed by clicking on the box with a little green number found just above the "Abstract " tab in the medRXiv window.