Avian coronavirus, also known as infectious bronchitis virus (IBV), poses a significant threat to the poultry industry, with the IBV QX variant strain now endemic in multiple countries. Traditional vaccines have demonstrated limited effectiveness in providing robust immune protection against this pathogen. In this study, we developed two IBV QX strain S1-mRNA vaccines based on different antigen domains and evaluated their safety and immune efficacy. The S1A mRNA-LNP and S1B mRNA-LNP vaccines were successfully produced based on epitope prediction of the S1 protein of the IBV QX strain. Both developed mRNA vaccines, along with a conventional inactivated vaccine, were capable of stimulating serum antibody production. ELISPOT assay results showed that the mRNA vaccines were more potent in stimulating lymphocytes to secrete interferon-γ (IFN-γ) than the inactivated vaccine. This enhanced immunostimulatory capacity translated into a significant reduction in viral load within the kidneys. Moreover, the S1A mRNA vaccine conferred immune protection earlier than the inactivated vaccine. In conclusion, the IBV S1-mRNA vaccines developed in this study can protect against IBV infection by inducing cellular and humoral immune responses. Selecting different S1 regions also affects the immunogenic efficacy of the mRNA vaccine.

Developed a rapid, sensitive, and homogeneous immunoassay to characterize specific immunoglobulin E (sIgE) against House Dust Mite (HDM) components.

Modern mass spectrometry-based methods for the isotyping and quantitation of monoclonal proteins (M-proteins) are now supplanting traditional gel-based methods such as serum protein electrophoresis (SPEP). The resultant improvement in resolution poses new signal processing challenges in the separation of M-protein from normal variations in polyclonal immunoglobulin background. We model normal variations in this background by means of a singular value decomposition and apply a coupled alternating-projection algorithm to separate normal and abnormal spectral components, both of which are variable. Ion simulation results guide our algorithm details and highlight limitations that can exist when testing M-proteins of high concentration. We show experimental quantitation results on clinical submitted sera with acceptable results. Finally, we outline enhancements to the fundamental method which have resulted in an enhanced analytical measuring range for clinical care of patients with plasma cell disorders.

Heavy water (DO) labeling is the state-of-the-art technique to track the dynamics of circulating cells in vivo. DO labels dividing cells through incorporation of deuterium into newly synthesized DNA, which is measured using GC/MS. The labeling rate depends on (1) the level of body water enrichment, (2) cell kinetics, and (3) an amplification factor quantifying the deoxyribose enrichment relative to the body water enrichment. This amplification factor is typically estimated using a reference population undergoing rapid turnover (such as granulocytes), and is larger than one because deoxyribose contains seven hydrogens that can be replaced by deuterium. In a meta-analysis, we found that individuals differ markedly in this amplification factor. Since the amplification factor also depends on the level of body water enrichment, we use conventional binomial expressions to describe the fractions of deoxyribose incorporating zero, one, two, or more deuterium atoms. We extend this classic binomial model with a new parameter, 0<γ<1, describing the relative contribution of hydrogens from body water during deoxyribose synthesis. While for most studies, our 'novel Binomial' model reasonably explains the slope with which the amplification factor declines with the level of body water enrichment, we find that some individual amplification factors differ considerably from their expected values Re-fitting deuterium labeling data of granulocytes with the Binomial model reveals that the actual decrease is steeper than expected. We speculate that this residual variation depends on differences in diet, metabolism, and/or life style, which apparently correlate with daily fluid intake.

This study presents the automation of the pooled donor basophil activation test (PD-BAT) for chronic urticaria, transitioning from a previously published manual assay in tubes to an automated 96-well plate format using the Hamilton Microlab STAR system. The primary aim is to enhance throughput and robustness in a commercial lab environment while maintaining assay accuracy and reproducibility. Donor screening included 36 individuals, of which 16 were selected for pooled assays. A total of 90 sera were analyzed in parallel by manual and automated methods. The automated PD-BAT demonstrated 93.3 % qualitative agreement (r > 0.97) with the manual assay. Throughput improved through automation, a 66 % reduction in hands-on time was observed between the manual (9 h/batch) and automated (3 h/batch) methods. Discordant results (6.7 %) occurred only near the technical cutoff, highlighting both the robustness and limitations of the method. This automation represents a valuable advancement over the manual assay, optimizing laboratory workflows.

Heavy water (DO) labeling can be used to track the dynamics of circulating cells in vivo. DO is administered via the drinking water, dividing cells become labeled by incorporating deuterium in their newly formed DNA, and the accrual of labeled deoxyribose molecules in the DNA is measured by gas-chromatography mass-spectrometry (GC-MS). Deuterium labeling studies need to correct for a background enrichment that is due to several naturally occurring isotopes, but the procedure for performing this correction differs between studies. We show that a background correction that is based upon subtracting the background enriched fraction (i.e., the atom percent excess, APE) underestimates the true enrichment, whereas one that is based upon subtracting the background enriched ratio (i.e., the tracer-to-tracee ratio, TTR) provides an enrichment that correctly describes the additional effect of the deuterium labeling. This difference is reflected in the so-called 'amplification factor', which quantifies the relative enrichment of deoxyribose in a fast reference population of cells to the enrichment in the body water. Using mechanistic binomials we reanalyze deuterium labeling data and show that previous studies based upon an APE-based background correction have underestimated this amplification factor by about 25%, which explains most of the variation among studies in their average amplification factor. We propose a novel model in which the amplification factor is defined by a binomial expression allowing for metabolic differences between volunteers in the contribution of body water to the hydrogens incorporated into deoxyribose during the synthesis of DNA.

The oral administration of the Bacillus Calmette-Guérin (BCG) vaccine aims to replicate the earliest inoculation route. We investigated the impact of this method by analyzing major Peyer's Patches (PP) cell lineages in mice. Oral BCG altered T/CD3, B/B220, double-labeled CD3-B220, double-labeled IgMB220, and triple-labeled CD49b-CD3-B220 cells. Both immunized (IL) and non-immunized (NIL) lactating mice served as controls in the flow cytometry analyses. The findings suggest that oral BCG vaccination induces changes in specific cell lineages, which may have implications for the development of novel vaccination strategies. Since population percentages vary according to source, frequencies based on fluorescence intensity were calculated according to percentiles, and subpopulations were subsequently analyzed per percentile. A robust statistical analysis assessed whether immunization influenced either the percentage of B220-labeled cells or the cellular expression of B220 per bin. The Thy1.2-depleted population revealed B220 as a receptor shared by both B and T lymphocytes. Statistical evaluation by receptor type and cell difference distance revealed subpopulations in transitional phases and showed that, among polychromatic receptor cells such as B220-IgM (CD11b), oral BCG selectively increased B220 expression. Oral BCG also induced CD3 cell activation, evidenced by a decrease in CD5 within the double-labeled CD3-CD5 cluster, despite an overall increase in CD3 receptors. Receptor analysis in regularly turning-over populations, such as PP lymphoid tissue, highlighted the double-labeled B220-CD3 cells. Notably, oral BCG influenced the development of double-labeled B220-CD3 cells in NKT-like populations.

RAW 264.7, a well-established cell line, is widely used in research; however, it is easily polarized upon subtle changes in environmental conditions or repeated passages. As the effects of cell storage conditions and duration on their phenotypic changes remain unknown, we investigated the effects of long-term storage at -80 °C on M2 marker expression in RAW 264.7 cells after stimulation with IL-4. Cells, stored in a - 80 °C deep freezer for a few years (defined as "long-stored cells") and newly purchased cells (referred to as "new cells"), were compared through flow cytometry for CD68-positive naïve macrophages (M0) and the expression of three types of M2 markers, CD206-FITC, CD206-PE, and Arginase-1-Alexa Fluor 488, in the M2 state. CD68 positivity was similar between the M0 macrophage types. After stimulation with IL-4, the proportion of CD206-FITC-positive cells was 5.0 ± 0.7 % and 61.5 ± 3.1 % in long-stored cells and new cells, respectively (p < 0.0001), while that of CD206-PE-positive cells was 9.2 ± 0.2 % and 69.8 ± 0.6 %. Similarly, the proportion of Arginase-1- Alexa Fluor 488-positive cells was 3.0 ± 0.1 and 9.0 ± 9.6 %, respectively (p = 0.0006). Overall, these results demonstrate that long-term storage of RAW 264.7 in a deep freezer affects their M2 marker expression. These results highlight the need to consider the effect of cell storage conditions and duration on experimental results.

A cytotoxicity assay was developed to measure the potency of GP101, a single chain diabody. GP101 is comprised of two linked Fv domains, with one that binds CD3 (expressed on T cells), and the other that binds CD19 (expressed on B cells). GP101 directs CD3 positive T lymphocytes to CD19 positive B lymphocytes. This immunotherapy redirects CD3+ T cells to CD19-expressing B-cell malignancies, enabling T-cell-mediated tumor cell lysis. We developed a GMP cell-based potency assay to satisfy the FDA requirements. The potency assay uses a cytotoxic T cell line, and a B cell lymphoma cell line as the target. The target lymphoma B cell line is prelabeled with a fluorescent dye, and upon T cell mediated killing, the fluorescence dye is released and detected using a fluorometer. The emitted fluorescence is proportional to the dose of GP101. Greater than 90 % of the target B cells were killed within two hours exposure in vitro with the lowest amount of detectable killing at 60 pg/mL GP101. The assay is suitable for measuring purified GP101, or GP101 expressed by cells transduced by GP101 plasmid or AAV preparations, and bioactivity in animal or human blood. This novel assay met GMP/GLP compliance, allowing a quantifiable and reproducible measure of efficacy, ensuring batch-to-batch consistency, and met safety and effectiveness regulatory requirements. This potency assay may be applicable for testing other CD3-CD19 T cell engagers and suitable for developing other diabody mediated potency assays with appropriate antigens.

Placental malaria due to Plasmodium falciparum (Pf) is associated with adverse pregnancy outcomes. Infected erythrocytes (IEs) bind to placental chondroitin sulfate A (CSA) and antibodies that inhibit this adhesion are a potential correlate of protection. We developed a simplified adhesion inhibition assay that uses diaminofluorene to measure haemoglobin release from IEs bound to CSA. Using hyperimmune plasma, the assay demonstrated concentration-dependent inhibition of CSA adhesion. Assay performance was consistent over time with strong reproducibility (r = 0.82), and results correlated with a published assay (r = 0.53). In 466 Malawian pregnant women (321 P. falciparum-infected and 145 uninfected at first antenatal visit), adhesion inhibitory antibodies were significantly higher in mid-pregnancy in infected multigravidae (46.3 % IQR 23.2 %, 74.8 %) compared to infected primigravidae (9.7 % IQR 0 %, 29.3 %, p < 0.001) and in uninfected multigravidae (29.9 % IQR 8.6 %, 54.5 %) than uninfected primigravidae (15.6 % IQR 0 %, 36.4 %, p = 0.04). Similar, significant gravidity-dependent differences were observed at delivery in both infected and uninfected women. Between enrolment and delivery, changes in antibodies were similar in infected and uninfected women. Adhesion inhibitory antibodies protected against placental malaria. Of 162 women with infection at mid-pregnancy, 88 with no placental malaria had more adhesion inhibitory antibody (33.8 %, IQR 1.9 %, 63.2 %) than 74 with past placental malaria (12.5 %, IQR 0 %, 37.8 %; p = 0.002). This low cost, reproducible, and rapid high-throughput adhesion inhibition assay shows promise as a correlate of protection against placental malaria.

Monoclonal antibodies are currently essential biological molecules for immunotherapy and diagnosis. The adaptability of an antibody to humans is crucial for ensuring safety and achieving high therapeutic efficacy as a medicinal drug. A considerable amount of effort has been devoted to predicting the human likeness of recombinant antibody molecules to assess their suitability for clinical use. Many previous studies have utilized a database of antibody sequences. However, a rapid assessment of adaptability is also helpful in the early stage of high-affinity antibody molecular design to a target antigen. To characterize the amino acid sequences of human antibodies, we statistically analyzed 742 antigen-antibody complex structures extracted from the Protein Data Bank. The frequency and position of the appearances of the respective residues are surveyed, and their probabilities were obtained for three complementarity-determining regions of heavy and light chains. In particular, the populations were examined from the viewpoint of which positions the respective residues were likely to appear in each complementary determining region. Based on a statistical analysis, an arithmetic index was proposed to evaluate sequence compatibility with humans and assess antibody models in computational molecular design. An examination using a collection of neutralizing antibodies for viral infectious diseases suggested that the index can distinguish human antibodies from those of mice. An examination of a collection of approved antibody drugs showed a certain degree of correlation between the calculated index and the immunogenicity of the antibody drugs. These evaluations demonstrated the feasibility of the proposed index as a rapid method for evaluating molecular adaptability to medicinal antibodies.

During the COVID-19 Pandemic in 2020, the need for highly-specific, wide-spread, and rapid serological testing surged. In this study, we tested the utility of the Volumetric Absorptive Microsampling (VAMS) using Mitra home sampling kits by comparing SARS CoV2 spike antibody levels from serum or Mitra dried blood samples from the same individuals with or without prior COVID 19 infection. We showed a very strong correlation between venous blood collection and capillary VAMS for the detection of anti-SARS CoV2 IgGs using a sensitive, semi-quantitative ELISA protocol. Furthermore, we found that the antibody levels detected from the Mitra blood samples were stable at room temperature for several months. This study demonstrates the utility of using at-home, patient-centric testing to enhance the sero-surveillance methods and large scale community testing for viral tracking, monitoring and vaccine studies.

This study explores the value of the Dihydrorhodamine-123 (DHR-123) flow cytometry assay to rapidly measure donor neutrophil engraftment (chimerism) in patients with Chronic Granulomatous Disease (CGD) following hematopoietic stem cell transplant (HSCT). Flow cytometry-based chimerism testing was compared with traditional molecular methods including sequence tandem repeats (STR), Quantitative-PCR DNA fingerprinting, and next-generation sequencing (NGS). Results comparing the two technologies in a cohort of CGD patients and healthy control subjects demonstrated that the DHR-123 based flow cytometry chimerism assay is an accurate, inexpensive, and rapid method for assessing functional donor neutrophil engraftment post-transplant, and which demonstrated significant correlation with the results of the molecular chimerism assays (R = 0.9965), making it a valuable tool for ongoing chimerism monitoring post-transplant. The flow cytometry-based assay offers a more rapid and inexpensive alternative to traditional molecular chimerism techniques.

Dengue, caused by the dengue virus (DENV), is a rapidly spreading infectious disease that poses a significant global health challenge. Rapid and accurate diagnosis is critical for effective disease management and epidemic control, as dengue is a kind of vector-borne disease and lacks definitive medical treatments. Current antibodies and antigens for DENV immunoassay, including immunoglobulin G (IgG), immunoglobulin M (IgM), and non-structural protein 1 (NS1), have drawbacks like cross-reaction and false-negative results in the late stages of primary infection or secondary infection. Envelope domain III (EDIII) protein, which is immunogenic and is a recognition region and binding site for DENV receptors, can also be the potential detection target of DENV infection. However, few studies of EDIII protein as a detection target have been reported. Therefore, this study developed a sensitive and specific ELISA for DENV-2_EDIII protein detection. Specific polyclonal antibodies (pAbs) against the DENV-2_EDIII protein were generated by immunizing the New Zealand White rabbits with the recombinant DENV-2_EDIII protein. Using these pAbs, a highly sensitive and specific ELISA method was developed. The assay demonstrated a wide detection range (7.8 ng/mL to 500 ng/mL) and a low limit of detection (LOD) of 0.156 ng/mL and showed no cross-reactivity with other orthoflaviviruses. These findings may provide a new methodological framework and research direction for the diagnosis of dengue after further clinical verifications.

The production of carbapenemase is the primary mechanism of bacterial resistance to carbapenem antibiotics. This resistance seriously compromises the efficacy of carbapenem antibiotics in treating infections and poses a significant challenge to clinical anti-infective therapy. Oxacillinase-48 (OXA-48) is a class D carbapenemase, whose genes are usually located on transferable elements and can spread between different strains and genera. Therefore, from the perspective of infection control, effective monitoring and prevention of OXA-48 are imperative. In this study, CdSe/ZnS quantum dots (QDs) were coupled with an anti-OXA-48 monoclonal antibody (mAb) as a fluorescent signal probe to establish a quantum dot-based fluorescent immunochromatographic strip. The test strip contains a Test line (T-line) coated with an anti-OXA-48 monoclonal antibody and a Control line (C-line) coated with Staphylococcus protein A (SPA). Based on a double-antibody sandwich detection mode, it can complete highly sensitive detection of OXA-48 within 10 min. The visual detection limit of this test strip for OXA-48 recombinant protein was 3.13 ng/mL, and there was no cross-reaction with other common carbapenemases (IMP-1, NDM-1, KPC-2, VIM-2, OXA-23). It can be positioned as a rapid and reliable OXA-48 detection tool, providing a novel method for the rapid identification of OXA-48-producing resistant bacterial strains in clinical practice.

Here we describe a magnetic bead-based indirect ELISA that enables the rapid and scalable detection of human IgM and IgG antibodies reactive to dengue virus (DENV). The assay can be performed in 12 min in the chromogenic format to IgM or IgG detection. The system can also handle simultaneous detection of IgM and IgG within less than 8 min when combined with fluorescent-labelled secondary conjugated IgM and IgG detection antibodies. The sensitivity and specificity of the assay are in the same range as described for commercially available ELISA kits. This simple and ultrafast assay for dengue seroconversion detection is an efficient alternative for the rapid track dengue cases.

Rabies serology is most importantly done to evaluate immune response following vaccine administration in patients who have been exposed to rabies or are highly likely to be. When detecting antibodies after vaccination it is best to target rabies neutralizing antibodies specifically. Clinical laboratories performing this testing are required to uphold a strict set of standards laid out by federal and state guidelines. Sample integrity throughout the process of testing, from collection to results is of the utmost importance. Samples may experience a wide range of temperature variation depending on how each sample is shipped or handled by the submitter. In order to uphold quality clinical testing it is important to study the stability of rabies neutralizing antibodies across a wide range of temperatures.

Ionizing radiation induced DNA DSBs can cause large deletions in the human genome. This property may be utilized for gene disruption to create knockout mutants. The X-linked hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene is commonly used as a reporter to evaluate the frequency and molecular nature of somatic mutations in the human population. Mutations at the HPRT locus are measured through a T lymphocyte cloning assay. This assay relies on lymphoblast feeder cells that support T lymphocyte proliferation via cell-to-cell contact and by helping in the expression of functional interleukin-2 receptors. However, the feeder cells must be HPRT-depleted to prevent feeder cell DNA from serving as substrate during molecular analysis of T lymphocyte mutations. To the best of our knowledge, the human lymphoblastoid TK6 cell line available in Indian cell repositories has an intact HPRT gene (HPRT+). In this study, the parent TK6 cell line was subjected to the clastogenic effects of a high dose gamma radiation to isolate TK6 cells with a total deletion of the HPRT locus. The HPRT-depleted cell line (TK6) was characterized by mPCR for the loss of exons and the extent of the deletion by flanking STS markers analysis. Further, the feeder cell function of TK6 was evaluated by estimating the cloning efficiency of human peripheral blood lymphocytes. Thus, the study demonstrates the potential of low LET gamma radiation to generate gene knockout cell line. The method described here can be applied to various cell types to produce functionally null cell lines for gene-specific studies.

Chimeric Antigen Receptor T-cells, commonly known as CAR-T cells, have represented a major scientific breakthrough in cancer cell therapy revolutionizing the treatment of haematological disorders. Pharmacokinetic studies highlighted the importance of peripheral blood CAR-T cell monitoring to predict therapeutic outcomes and manage side effects. This study aimed to establish and evaluate on three different flow cytometers (DxFLEX, Navios and Navios EX) the performances of a dual-platform protocol for CAR-T quantitation method using flow cytometry in a context of hospital routine use. Fresh whole blood was stained using a biotinylated CD19 or BCMA recombinant protein and a PE-coupled anti-biotin antibody. CAR-T cells were identified among CD45+ CD3+ cells after doublets elimination, using a control tube to define CAR-T positive expression threshold among T lymphocytes. Results were expressed as percentages of CAR T cells among CD3+ T lymphocytes. Optilyse C and Versalyse red blood cell lysis reagents (Beckman Coulter) gave comparable results in terms of CAR T cell percentage among T lymphocytes as well as using 0.5 μL CAR detection reagent compared to the 2 μL recommended by the manufacturer. Using this protocol, we did not observe any cross-reactivity between CD19 and BCMA CAR detection reagents or decreased signal even up to >3000 circulating CAR-T cells/μL. The coefficient of variations for repeatability and intermediate precision were systematically below 10 % whatever the flow cytometer, the type of CAR reagent detection used and the proportion of circulating CAR-T cells. All three flow cytometers returned correlated and comparable results. Finally, concerning whole blood sample and stained cell storage duration, we observed a significant decreased of CAR-T percentages when staining was delayed for more than one day after sampling, and after three days of stained cells storage at 4 °C. In conclusion, we propose a dual-platform protocol for CAR-T cell enumeration which demonstrated consistent and reliable performances for BCMA and CD19 CAR-T cell quantification.

Immunostaining and cytology, commonly used to detect cervical cancer, are time-consuming and pathologist dependent. In this work, for the first time, we have developed an ELISA assay for the extraction and co-measurement of p16 and Ki-67 in both vaginal and cervical samples for cervical cancer screening. Novel custom-made antibodies were developed, and a competitive ELISA assay was developed and optimized based on the concentration and incubation time of different steps to improve the sensitivity and specificity of the final assay in assessing cervical samples. The as-developed ELISA assay shows high sensitivity and selectivity in different collection media. The LoD of the test is 0.00004 and 0.0058 μg/mL for p16 and Ki-67, respectively. The as-developed assay can be used in future studies to determine protein thresholds for different lesion grades of cervical cancer for samples collected in different settings. These assays can revolutionize the cervical cancer screening management process in the near future.

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate Toll-like receptor 9 (TLR9), thereby triggering a diverse range of immunomodulatory responses. Although the immune-stimulating properties of CpG-ODNs have been extensively studied, their potential application as apoptosis-inducing tools remains underexplored, particularly for probiotic-derived sequences. In this study, we established a method for inducing extrinsic apoptosis in mouse splenocytes using MsST, a CpG-ODN identified from the lacZ gene of Streptococcus thermophilus ATCC19258. In genomic DNA fragmentation assays, MsST induced moderate but significant internucleosomal DNA fragmentation. Flow cytometric analysis using annexin V/7-aminoactinomycin D staining confirmed a time-dependent increase in the apoptotic cell population following MsST treatment. Furthermore, Western blot analysis revealed elevated levels of cleaved caspase-8, thus verifying activation of the extrinsic apoptosis pathway. Compared to a representative CpG-ODN and the apoptosis inducer actinomycin D, MsST exhibited distinct apoptosis-inducing characteristics. These findings highlight MsST as a potential tool for controlled apoptosis induction, providing a methodological basis for further exploration of CpG-ODNs in immunological and therapeutic applications, including cancer immunotherapy and the treatment of autoimmune diseases and inflammatory disorders.