Commercial Antigen Research Articles

Development of immunochromatographic and homogeneous assay based on quantum dot-functionalized polystyrene nanoprobes for the qualitative and quantitative screening of respiratory viruses

Accurately differentiating respiratory diseases caused by viruses is challenging because of the similarity in their early or clinical symptoms. Moreover, different infection sources require different treatments. However, the current diagnostic methods have limited differentiating efficiency and sensitivity. We developed a dual-system immunosensor with a bilayer fluorescent label as a signal amplifier for the on-site, sensitive, and accurate identification of multiple respiratory viruses (RVs). The nanomaterial, comprising a polystyrene (PS) nanosphere core encapsulated by two layers of CdSe@ZnS-COOH quantum dots (QDs), outperforms the conventional color and fluorescent labels in RV detection. The dual-system detection platform, comprising a PS@DQD-based lateral flow immunoassay (LFIA) and a PS@DQD-based homogeneous sensor, enables qualitative and quantitative screening of multiple respiratory viruses within 10 and 30 min, respectively, depending on the specific detection requirements for different application scenarios. This remarkable method provides 51.2 to 1000 times sensitivity improvement over commercial antigen detection kits and greater than 12.5 to 100 times improvement over QD-based immunosensors. Furthermore, we comprehensively evaluated the specificity, reproducibility, and stability of the integrated dual-system detection platform, demonstrating its reliability. Remarkably, the respiratory viral testing was validated using biological samples, thus illustrating its promise and convenience in the detection of respiratory viruses.

Overcoming Limited Access to Virus Infection Rapid Testing: Development of a Lateral Flow Test for SARS-CoV-2 with Locally Available Resources.

The COVID-19 pandemic highlighted testing inequities in developing countries. Lack of lateral flow test (LFT) manufacturing capacity was a major COVID-19 response bottleneck in low- and middle-income regions. Here we report the development of an open-access LFT for SARS-CoV-2 detection comparable to commercial tests that requires only locally available supplies. The main critical resource is a locally developed horse polyclonal antibody (pAb) whose sensitivity and selectivity are greatly enhanced by affinity purification. We demonstrate that these Abs can perform similarly to commercial monoclonal antibodies (mAbs), as well as mAbs and other pAbs developed against the same antigen. We report a workflow for test optimization using nasopharyngeal swabs collected for RT-qPCR, spiked with the inactivated virus to determine analytical performance characteristics as the limit of detection, among others. Our final prototype showed a performance similar to available tests (sensitivity of 83.3% compared to RT-qPCR, and 90.9% compared to commercial antigen tests). Finally, we discuss the possibility and the challenges of utilizing affinity-purified pAbs as an alternative for the local development of antigen tests in an outbreak context and as a tool to address inequalities in access to rapid tests.

Investigation of canine parvovirus occurrence in cats with clinical signs of feline panleukopenia in Slovakia - pilot study.

Feline panleukopenia is a contagious viral disease caused by the feline panleukopenia virus (FPV). A closely related pathogen is canine parvovirus (CPV), and amino acid substitutions in this virus allow it to acquire a feline host range. In feline hosts, the disease induced by CPV manifests with similar symptoms to those caused by FPV or milder ones, leading to its underdiagnosis. The aim of this study was to determine the presence of CPV type 2 (CPV-2) in cats with clinical symptoms of panleukopenia and to assess the use of commercial CPV antigen tests for the clinical diagnosis of FPV. Samples from 59 cats from central Slovakia were included in the study. Rectal swabs were collected and clinically tested for parvovirus infection using a commercial antigen test. Antigen-positive samples were confirmed by PCR targeting the viral VP2 gene. The sequences of the PCR products were established with the Sanger method. Of 59 samples, 23 were revealed to be positive for parvovirus infection by both antigen and PCR test (38.9%). Analysis with the National Center for Biotechnology Information BLASTn application showed 99.78-100% pairwise identity with FPV. The mortality rate of parvovirus-infected cats included in this study was 8.69% (2/23). Although feline disease with CPV-2 was not confirmed, the CPV antigen test was able to detect FPV infection.

Commercial Synovial Antigen Testing is Inferior to Traditional Culture for the Diagnosis of Periprosthetic Joint Infection in Patients Undergoing Revision Total Knee Arthroplasty

BackgroundDespite its limitations, a culture remains the “gold standard” for pathogen identification in patients who have periprosthetic joint infection (PJI). Recently, a synovial fluid antigen test has been introduced by a commercial entity. The purpose of this multicenter study was to determine the accuracy of the antigen test in the diagnosis of PJI. MethodsThis retrospective study identified 613 patients undergoing revision total knee arthroplasty who had undergone preoperative synovial fluid analysis. A PJI was defined using the 2018 International Consensus Meeting (ICM) criteria. Patients who had an extended period (> 180 days) from aspiration to revision procedure (n = 62), those presenting within 90 days of their index arthroplasty procedure (n = 17), and patients who had an inconclusive ICM score (n = 8) were excluded. Using receiver operator characteristic curve analyses, we examined the utility of the microbial identification (MID) antigen test and any positive culture (either preoperative or intraoperative) in the diagnosis of PJI. ResultsA total of 526 patients were included. Of these, 125 (23.8%) were ICM positive and 401 (76.2%) were ICM negative. Culture demonstrated an area under the curve (AUC) of 0.864, sensitivity of 75.2%, and specificity of 97.5%. On the other hand, the MID test exhibited an AUC of 0.802, sensitivity of 61.6%, and specificity of 98.8%. The AUC of culture was significantly higher than that of the MID test (P = .037). The MID test was positive in 41.9% of culture-negative PJI cases. We also observed a high rate of discordance (29.7%) when both culture and the MID test were positive in the ICM-positive group. ConclusionsSynovial fluid antigen testing does not provide additional clinical benefit when compared to traditional cultures for the diagnosis of PJI. The antigen test had low sensitivity in the diagnosis of PJI and a relatively high rate of discordance with culture. Level of EvidenceLevel III.

Development of a new immunochromatographic strip mediated by colloidal Gold-MAb nanoparticles for rapid detection of subgroup K Avian leukemia virus

K subgroup of Avian leukemia virus (ALV-K), as the latest reported exogenous ALV subgroup, can cause severe immunosuppression and tumorigenesis in infected chickens and bring serious threats to the poultry. Culling the positive chickens from breeder flocks is the most effective measurement for controling its infection at present. To setup a specific and rapid method for detecting ALV-K, an immunochromatographic strip (ICS) based on colloidal gold nanoparticles and double monoclonal antibodies (MAbs) immunochemical reaction was successfully developed. The solution containing colloidal gold particles (colloidal 20 nm) was prepared by sodium citrate reduction, and the ascites MAbs against ALV-K were purified and identified. The antibody concentration of the colloidal gold-MAb complex reaction system was 9.6 μg/mL, and the optimum pH was 7.5. The optimum coating concentrations of the captured antibody and quality control antibody on the NC membrane were 1.2 mg/mL and 0.6 mg/mL, respectively. The results showed that the prepared ICS could specifically detect ALV-K and had no cross reaction with exogenous ALV-A/B/J strains. Its sensitivity was 64 TCID50/mL against ALV-K and 0.25 μg/mL against ALV-K gp85 protein. The ICS stored at 4 °C for 180 d, at 25 °C for 45 d, and at 37 °C for 15 d could stably detect ALV-K in the samples. In comparision detection of 128 clinical samples, the coincidence rates in cloacal swab samples, serum samples, egg albumen samples and tissue homogenate samples between the ICS and the commercial ALV p27 antigen ELISA kit were 100 %, 100 %, 96.7 % and 96.9 %, respectively; moreover, the prepared ICS in operations was simpler and more convenient. This study supplies a new specific and rapid method for detecting ALV-K in clinics. This is the first report on the application of nano-colloidal gold ICS in ALV-K differential detection.

Plasmon-Enhanced Digital Fluoroimmunoassay for Subfemtomolar Detection of Protein Biomarkers.

Rapid and accurate quantification of low-abundance protein biomarkers in biofluids can transform the diagnosis of a range of pathologies, including infectious diseases. Here, we harness ultrabright plasmonic fluors as "digital nanolabels" and demonstrate the detection and quantification of subfemtomolar concentrations of human IL-6 and SARS-CoV-2 alpha and variant proteins in clinical nasopharyngeal swab and saliva samples from COVID-19 patients. The resulting digital plasmonic fluor-linked immunosorbent assay (digital p-FLISA) enables detection of SARS-CoV-2 nucleocapsid protein, both in solution and in live virions. Digital p-FLISA outperforms the "gold standard" enzyme-linked immunosorbent assay (ELISA), having a nearly 7000-fold lower limit-of-detection, and outperforms a commercial antigen test, having over 5000-fold improvement in analytical sensitivity. Detection and quantification of very low concentrations of target proteins holds potential for early detection of pathological conditions, treatment monitoring, and personalized medicine.

Antibody-labelled gold nanoparticles synthesized by laser ablation to detect SARS-CoV-2 antigen spike.

Rapid detection test via lateral flow immunoassay (LFIA) is employed as an alternate method to detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Gold nanoparticles (AuNPs), a vital component of LFIA, can be synthesized by laser ablation technique. This intense laser radiation may result in monodisperse gold nanoclusters, which are impurity-free and demonstrate innovative biocompatible surface chemistry. In this current research, laser-ablated AuNPs are produced and coupled with an anti-spike SARS-CoV-2 monoclonal antibody (mAb) generated in our prior study. The AuNPs from 30,000 shots of laser ablation exhibited a robust red color with a maximum absorbance peak at 520 nm. The performance of AuNPs-mAb conjugates as a signal reporter was then evaluated in half-stick LFIA. The size distribution of AuNPs shows a relatively monodisperse and unimodal distribution with average particle diameters of 44.77 nm and a surface potential of -38.5 mV. The purified anti-spike mAb SARS-CoV-2 yielded two protein bands, representing the mAb heavy chain at 55 kDa and its light chain at 25 kDa. The immobilization of anti-spike mAb onto the surface of AuNPs revealed that 25 g/ml of mAb at phosphate buffer pH 9 was required to stabilize the AuNPs. The functional test of this conjugate was performed using dipstick LFIA, and the result shows that the AuNPs-mAb conjugates could successfully detect commercial spike antigen of SARS-CoV-2 at 10 ng level. In this study, laser-ablated AuNPs were functionalized with anti-spike mAb SARS-CoV-2 and successfully used as a signal reporter in half-stick LFIA for detecting antigen spike SARS-CoV-2.

Establishing Costs for Commercial Chimeric Antigen Receptor T-Cell (Tisagenlecleucel; Kymriah) in Children and Young Adult B-Cell Acute Lymphoblastic Leukemia; A Merged Analysis from the Prwcc and PHIS

Background: Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable efficacy in children and young adult (CAYA) B-cell acute lymphoblastic leukemia (B-ALL). Nonetheless, this therapy is highly personalized, technically complex, and costly to manufacture. The USA FDA approved the first CAR T cell therapy (Kymriah) in Aug 2017, for relapsed/refractory CAYA B-ALL. The list price of Kymriah is $475,000, not accounting for costs related to delivery of care, inpatient hospitalization, toxicity management or follow-up. A half-decade of clinical experience has amassed since commercialization, yet financial analyses remain sparse. Here, we conduct the first study to understand costs for commercial CAR in CAYA B-ALL. Methods: Ten centers participating in both the Pediatric Real-world CAR Consortium (PRWCC) and Pediatric Health Information System (PHIS; Children's Hospital Association, Lenexa, KS) were eligible for study (08/2017-03/2020). Clinical data from the PRWCC database was merged with PHIS using age, diagnosis, center and lymphodepletion dates. Utilizing center and year specific charge-to-cost ratio, PHIS provided data for inpatient admission associated costs, but not outpatient care. For patients that were infused outpatient and subsequently admitted, only inpatient cost was available and analyzed for the first 60 days post-infusion. For patients that were infused inpatient, cost was calculated from the first day of admission for lymphodepletion or Kymriah infusion to 60 days post-infusion. The 60-day follow up was chosen as most of the clinical activity and toxicities occur within the 60 days post-infusion. Each patient was assigned a cost of $475,000 for Kymriah (list price). For the subset of patients who underwent allogeneic hematopoietic cell transplantation (alloHCT) following Kymriah, the median inpatient cost was assessed for 1-year post-alloHCT. Cost data was analyzed as medians and ranges. Univariable and multivariable logistic regression models were used to identify factors associated with the higher median cost associated with inpatient admissions. Results: 109 Kymriah-recipients were included. Patient characteristics are listed in Table 1. The incidence of grade 1-2 and ≥3 CRS was 42% and 23%, respectively, and grade 1-2 and ≥3 ICANS was 17% and 6%, respectively. Relapse/refractory disease at day 30 and 60 post-Kymriah infusion was 11% and 16%, respectively. AlloHCT was performed in 17 (16%) patients. Median time to alloHCT from Kymriah infusion was 148 days (36-354). The median number of admissions was 1 (0-5) and median length of all hospitalization was 18 days (0-69), 99% patients required inpatient admission. Thirty-six patients (33%) required intensive care unit (ICU) admission with a median ICU stay of 5 days (1-24). The median total cost (Kymriah + inpatient cost) per patient was $615,845 ($475,000-1,578,321). Breakdown of median total cost (excluding Kymriah) was: room/board $33,676 ($0-454,256), pharmacy $65,854 ($0-515,132), laboratory/pathology cost $10,496 ($0-201,900) and other cost $8,204 ($0-573,163). One-year median cost associated with inpatient admissions for alloHCT was $556,712 ($105,670-1,919,275), combined median cost associated with Kymriah + alloHCT was $1,263,316 ($696,768-2,404,447). Factors associated with higher inpatient cost were public insurance ($96,287, p=0.002); inpatient Kymriah infusion ($395,182, p<0.001); CRS grade≥3 ($342,176, p<0.001); ICANS grade ≥3 ($354,013, p=0.010); ICU stay ($346,737 p<0.001). Multivariable analysis demonstrated that only inpatient infusion of Kymriah significantly associated with higher cost (Table 2). Conclusion: In this cost analysis across CAYA receiving Kymriah, we describe the total inpatient treatment-associated cost, with inpatient infusion emerging as a main driver of cost. With increased clinical experience, outpatient infusions and early interventions to avoid high-grade toxicity and ICU-level care have become more common and potentially more cost-effective. Our data justifies efforts to deliver Kymriah in the outpatient setting, when safe and feasible, and to provide support systems to facilitate this. Additionally, Kymriah drug cost accounts for the bulk (73%) of the overall median cost, highlighting that efforts to lower drug costs may permit enhanced access globally.

Expression of Cell Surface Immunotherapy Targets across the Multiple Myeloma Disease Spectrum: Implications for Sequencing and Multi-Target Strategies

Introduction Targeting surface proteins with immunotherapy including monoclonal antibodies, bispecific antibodies, antibody drug conjugates and chimeric antigen receptor T-cell therapy (CAR-T) has improved outcomes for patients with multiple myeloma (MM). No strategy has proven curative to date and relapse invariably occurs. There is a paucity of literature describing how these cell surface targets change over the disease course or at relapse post-immunotherapy. We comprehensively profiled a large repository of patient samples across the spectrum of disease, including paired samples taken prior to and post commercial B-cell maturation antigen (BCMA) CAR-T failure (administered in patients with triple class exposed disease after ≥4 prior lines of therapy (PLOT), to better understand how targeted therapies could be optimally sequenced or combined for maximum therapeutic benefit. Methods Bulk RNA sequencing (RNAseq) from CD138+ selected cells obtained from patients across the disease course (smoldering (SMM), newly diagnosed (NDMM), early relapse (ERMM=1-3 PLOT) and late relapse (LRMM ≥4PLOT) was interrogated for expression of surface markers of known relevance in MM. Matched samples were also evaluated for expression of targets of interests with multiparameter flow cytometry. Antigen density assessment for each marker of interest was calculated utilizing the median fluorescence intensities (MFI) of CD38, CD138, CD20, TACI, CD70, CD44, GPRC5D and IGTB7 under saturating antibody conditions for antigen density measurements. Quantitative expression in molecules per cell (mol/c) was calculated by calibration with custom Quantibrite beads (BD Biosciences) for BCMA, Ly9, SLAMF7 and FCRL5 after controlling for the fluorophore to antibody ratio. Paired patient samples from baseline and at progression after BCMA directed CAR-T were also evaluated. As abundance of co-regulated proteins is often correlated, we also analyzed expression of key targets in the context of their known regulatory network (Renatino-Canevarolo et al, ASH 2022). Results In total, RNAseq data was available from N=731 CD138+ selected samples across disease states, (59 SMM, 187 NDMM, 303 ERMM, 182 LRMM) and flow cytometry was performed on N=192 samples (13 SMM, 65 NDMM, 66 ERMM, 48 LRMM) with matched RNAseq data. Examining the findings between paired samples demonstrated certain markers have better correlation between gene expression and surface protein expression than others, with highest concordance seen for LY9 (r=0.6, p<0.0001), BCMA (r=0.5, p<0.0001) and IGTB7 (r=0.5, p<0.0001). Weak correlation for CD38 (r=0.3, p=0.0004), SLAMF7 (r=0.3, p<0.0001), CD44 (r=0.3, p=0.0003), FCRL5 (r=0.3, p=0.0004), CD19 (r=0.2, p=0.01), CD138 (r=0.2, p=0.002) and TACI (r=0.2, p=0.008), and no significant correlation for CD70 (r=0.09, p=0.2) and GPRC5D (r=0.05, p=0.5) was observed. Bulk seq analysis also identified that several of the genes encoding targets of interest are regulated by a common geneset, e.g. BCMA/CD138/SLAMF7 and FCRL5/TACI. Certain markers were noted to have higher protein expression over the disease course, in particular BCMA (p=0.0036, Figure 1). In contrast, CD38 (p=0.004), CD19 (p<0.0001), TACI (p=0.008), SLAMF7 (p<0.0001), GPRC5D (p=0.02) had lower expression levels as the disease progressed, while LY9, CD138, FCRL5, ITGB7 were stable/similar across disease states. Median protein expression in mol/c in LRMM samples was 593 (range 179-4,678) for BCMA, 11,869 (range 251 - 50,482) for Ly9, 516 (range 84-1,998) for FCRL5 and 5,821 (range 1779 - 20,752) for SLAMF7. Looking at patients with paired plasma cells pre- and post- CAR-T, changes in protein expression in mol/c were variable, although the majority at baseline and relapse had surface expression levels below expected median values identified in the larger cohort with LRMM. Conclusion: This large dataset of paired patient samples across the MM disease spectrum provides a comprehensive description of the differences in expression of key immunotherapy targets with important implications for how agents might be sequenced, and what combinatorial strategies could be employed to optimize response. Expression levels of targets may be one of many factors involved in progression after administration of immunotherapy that may be of variable importance depending on the target, and timing and nature of therapy employed. KHS & FLL contributed equally

Failure and out of Specification Manufacturing of Autologous CAR-T Cells Could be Associated with a High Concentration of Total Nucleated Cells, CD3 + Cells and Neutrophils in the Apheresis Product

Manufacturing capacities of commercial autologous chimeric antigen receptor (CAR)-T cells have been significantly increased during the last years, allowing treatment of a larger patient cohort. However, this was associated with the emergence of several failures and out of specifications (OOS), resulting in treatment delay. The aim of our study was to identify early indicators related to apheresis products that can predict CAR-T cells manufacturing failure or OOS. To this end, we analyzed manipulation processes of cellular starting materials (conservation, freezing, cryopreservation) and quality controls (QC) including count and/or concentration of total nucleated cells (TNC), CD3 +, CD4 +, CD8 + cells, platelets, monocytes and neutrophils. We analyzed 802 apheresis products that were collected for the manufacturing of axicabtagene ciloleucel (axi-cel, n=303), tisagenlecleucel (tisa-cel, n=403), brexucatagene autoleucel (brexu-cel, n=44) and idecabtagene vicleucel (ide-cel, n=52). This cohort was classified into 3 groups: failure (CAR-T cells not produced), OOS (CAR-T cells produced but non-compliant) and compliant. We identified 21 failures and 24 OOS, resulting in either no treatment or a significant delay when the OOS were injected (57.6±17.5d versus 45.9±16.3d in compliant group, p=0.0405). Among each CAR-T cells product, 13 (4.3%) axi-cel, 30 (7.4%) tisa-cel, 1 (2.3%) brexu-cel and 1 (1.9%) ide-cel were concerned. The main causes of failures were low viability (33.3%), non-compliant dose (28.6%), microbiological contamination (23.8%) and formulation concerns (14.3%). In OOS, low viability (41.7%), low dose (29.2%), microbiological contamination (4.2%), formulation (12.5%) and safety concerns (8.3%; i.e high vector copy number) were identified. 17 products (13 failures and 4 OOS) were successfully remanufactured without an increased delay comparing to compliant products (47.5±15.9 and 47.3±15.1 respectively versus 45.9±16.3 d). To identify risk factors of manufacturing failure or OOS due to non-compliant viability and/or dose, we analyzed processing and QC of collected cellular starting materials. OOS were associated with a high concentration of TNC (146.7±94.3 vs 61.6±36.1 x10 6/mL, p=0.009) and CD3 + cells (88.6±76.5 vs 26.0±21.9 x10 6/mL, p=0.0112) in cell collections, while failures were observed in apheresis with high neutrophils concentration (14.3±15.03 x10 3/µL vs 6.0±7.6 x10 3/µL, p=0.0195). Interestingly, neutrophils were significantly decreased in apheresis collected for CAR-T cells remanufacturing which succeed (p=0.0156). This preliminary study showed that high TNC, CD3 + cells and neutrophils concentration in the apheresis product might be predictive factors of failures and/or OOS of commercial autologous CAR-T cells manufacturing.

Trial of the Pluslife SARS-CoV-2 Nucleic Acid Rapid Test Kit: Prospective Cohort Study

Background In response to the SARS-CoV-2 epidemic, a convenient, rapid, and sensitive diagnostic method for detecting COVID-19 is crucial for patient control and timely treatment. Objective This study aimed to validate the detection of SARS-CoV-2 with the Pluslife SARS-CoV-2 rapid test kit developed based on a novel thermostatic amplification technique called RNase hybridization-assisted amplification. Methods From November 25 to December 8, 2022, patients with suspected or confirmed COVID-19, close contacts, and health care workers at high risk of exposure were recruited from 3 hospitals and 1 university. Respiratory specimens were collected for testing with the Pluslife SARS-CoV-2 rapid test kit and compared with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and a commercial antigen assay kit. Samples from 1447 cases were obtained from 3 “ready-to-test” scenarios in which samples were collected on site and tested immediately, and samples from 503 cases were obtained from a “freeze-thaw test” scenario in which samples were collected, frozen, and thawed for testing. Results Pluslife SARS-CoV-2 rapid testing of samples from the “ready-to-test” scenario was found to be accurate (overall sensitivity and specificity of 98.3% and 99.3%, respectively) and diagnostically useful (positive and negative likelihood ratios of 145.45 and 0.02, respectively). Pluslife SARS-CoV-2 rapid testing of samples from the “freeze-thaw test” scenario was also found to be accurate (overall sensitivity and specificity of 71.2% and 98.6%, respectively) and diagnostically useful (positive and negative likelihood ratios of 51.01 and 0.67, respectively). Our findings demonstrated that the time efficiency and accuracy of the results in a “ready-to-test” scenario were better. The time required from sample preparation to the seeing the result of the Pluslife SARS-CoV-2 rapid test was 10 to 38 minutes, which was substantially shorter than that of RT-qPCR (at least 90 minutes). In addition, the diagnostic efficacy of the Pluslife SARS-CoV-2 rapid test was better than that of a commercial antigen assay kit. Conclusions The developed RNase hybridization-assisted amplification assay provided rapid, sensitive, and convenient detection of SARS-CoV-2 infection and may be useful for enhanced detection of COVID-19 in homes, high-risk industries, and hospitals.

Organ complications after CD19 CAR T-cell therapy for large B cell lymphoma: a retrospective study from the EBMT transplant complications and lymphoma working party.

We investigated ≥ grade 3 (CTC-AE) organ toxicities for commercial CD19 chimeric antigen receptor T cell (CAR-T cell) products in 492 patients (Axi-Cel; n = 315; Tisa-Cel; n = 177) with Large B-cell Lymphoma in the European Society for Blood and Marrow Transplantation (EBMT) CAR-T registry. The incidence of ≥ grade 3 organ toxicities during the first 100 days after CAR-T was low and the most frequent were: renal (3.0%), cardiac (2.3%), gastro-intestinal (2.3%) and hepatic (1.8%). The majority occurred within three weeks after CAR-T cell therapy. Overall survival was 83.1% [79.8-86.5; 95% CI] at 3 months and 53.5% [49-58.4; 95% CI] at one year after CAR-T. The most frequent cause of death was tumour progression (85.1%). Non-relapse mortality was 3.1% [2.3-4.1; 95% CI] at 3 months and 5.2% [4.1-6.5; 95% CI] at one year after CAR-T. The most frequent causes of non-relapse mortality were cell-therapy-related toxicities including organ toxicities (6.4% of total deaths) and infections (4.4% of total deaths). Our data demonstrates good safety in the European real-world setting.

Histone crotonylation of peripheral blood mononuclear cells is a potential biomarker for diagnosis of colorectal cancer

BackgroundBlood-based tests have public appeal in screening cancers due to their minimally invasive nature, ability to integrate with other routine blood tests, and high compliance. This study aimed to investigate whether certain epigenetic modulation of peripheral blood mononuclear cells (PBMCs) could be a biomarker of colorectal cancer (CRC).ResultsWestern blotting of histones in the PBMCs from 40 colorectal cancer patients and 40 healthy controls was performed to identify the crotonylation sites of proteins. The correlation of crotonylation with tumor staging and diagnostic efficacy were analyzed. Crotonylation of H2BK12 (H2BK12cr) was identified significantly upregulated in the PBMCs of CRC patients compared to healthy controls, and were closely related to distant metastasis (P = 0.0478) and late TNM stage (P = 0.0201). Receiver operator characteristic curve (ROC) analysis demonstrated that the area under curve (AUC) of H2BK12cr was 0.8488, the sensitivity was 70%, and the specificity was 92.5%. The H2BK12cr parameter significantly increased the diagnostic effectiveness of CRC compared with the commercial carcinoembryonic antigen assays.ConclusionsThe H2BK12cr level in PBMCs of CRC patients has a potential to be a biomarker for distinguishing CRC patients from healthy controls with the advantages of easy operation and high diagnostic efficacy.

Highly sensitive digital detection of SARS-CoV-2 nucleocapsid protein through single-molecule counting.

Nucleocapsid protein (NP) is one of the structural proteins of SARS-CoV-2 which is stable, well-conserved, highly immunogenic, and abundantly expressed due to the host's adaptive immune response, making it a promising antigenic biomarker for the early and rapid identification and diagnosis of SARS-CoV-2. Traditional antigen analytical methods with NP as the detection marker often have insufficient sensitivity. To achieve rapid and highly sensitive detection of NP, we constructed a novel single-molecule (digital) fluorescence-linked immunosorbent assay (FLISA) based on streptavidin-modified transparent 96-well microplates. Streptavidin was immobilized on the microplate under optimized conditions with a 15mM carbonate buffer solution (pH 9.6) as the coating solution, biotinylated antibodies conjugated with streptavidin as capture probes, and carboxylated fluorescent microsphere-conjugated monoclonal antibodies (FMs-mAbs) as fluorescent probes. Individual sandwich immunolabeled complexes of the SARS-CoV-2 diagnostic marker NP were detected and counted though wide-field inverted fluorescence microscopy (1.1 × 1.4 mm2). FLISA had a linear detection range of 0.2pg/mL to 200ng/mL and a limit of detection (LOD) of 0.73fg/mL and 8fg/mL for NP in phosphate buffer saline and spiked nasal swab samples, respectively. The sensitivity was much higher than commercial antigen detection kits, providing wide detection prospects in future clinical diagnosis, environmental monitoring, and other fields.

Targeting of human fibroblast growth factor receptor 2 by a novel specific nanobody

Inhibition of FGFR2 signaling is promising in targeted therapy of FGFR2-related tumors. In this study, anti-FGFR2 nanobodies (Nbs) were isolated through screening of an immune camelid phage display library. Four rounds of biopanning were carried out with commercial human FGFR2 antigen and enrichment was assessed by ELISA and phage titration. The gene of Nb was sub-cloned into the expression vector, and the recombinant vector was transformed into Escherichia coli WK6 cells. The recombinant protein was purified using Ni–NTA affinity chromatography. The anti-FGFR2 Nb (C13) was characterized by SDS-PAGE, western blotting, competitive inhibition ELISA, flow cytometry, MTT, and migration assay. C13 Nb recognized FGFR2 with high specificity and no cross-reactivity was observed with other tested antigens. The affinity of C13 Nb was calculated to be 1.5 × 10−9 M. Results of cytotoxicity showed that C13 Nb (10 µg/ml) inhibited 85% of the proliferation of T-47D cells (p < 0.001). In addition, C13 inhibited the migration of 68% of T-47D toward the source of the growth factor (p < 0.01). The flow cytometry showed that C13 Nb bound to the surface of FGFR2+ cells, T-47D cell line (96%). Results indicate the potential of anti-FGFR2 Nb for targeted therapy of FGFR2-overexpressing tumors after complementary investigations.

Clinical Evaluation of Commercial HARDSON COVID-19 Antigen Rapid Test Kit for Routine COVID-19 Diagnosis.

This study aimed to evaluate the sensitivity, specificity, and accuracy of the commercial HARDSON COVID-19 Antigen Rapid Test Kit for diagnosing COVID-19 among the Iranian population by compared with the results of commercial RT-PCR. Two nasopharyngeal swabs were collected from each patient. One swab was tested with HARDSON COVID-19 Antigen Rapid Test Kit, and the second swab was placed in 3 mL of a virus-transmitted inactivated media for RT-PCR testing. Then, the results of both tests were compared to investigate the diagnostic accuracy of the rapid antigen test. A total of 275 suspected COVID-19 patients' samples were collected to investigate the diagnostic accuracy of HARDSON COVID-19 Antigen Rapid Test Kit. In this study, 162 positive and 113 negative samples were evaluated. As a result, the sensitivity, specificity, and accuracy of HARDSON COVID-19 Antigen Rapid Test Kit were 90%, 100%, and 94%, respectively. The diagnostic kit analyzed in this study indicated excellent specificity and a relatively good overall sensitivity for the diagnosis of COVID-19 when compared with the RT-PCR detection kit. Based on the result of this study, COVID-19 Antigen Rapid Test Kit indicated a good sensitivity (96%) in low cycle threshold (Ct) value, and it would be recommended to be integrated into routine diagnostic laboratories and used as an at-home rapid antigen test.

Dual-clamped one-pot SERS-based biosensors for rapid and sensitive detection of SARS-CoV-2 using portable Raman spectrometer

Rapid and sensitive diagnostics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of utmost importance to control the widespread coronavirus disease 2019 (COVID-19) upsurge. This study demonstrated a novel one-pot surface-enhanced Raman scattering (SERS) based immunoassay to detect SARS-CoV-2, without any washing process using a portable Raman spectrometer. The SERS-immune assay was designed using a regular digital versatile disk (DVD) substrate integrated with Raman reporter labeled silver nanoparticles for double clamping effects. The disks were molded to form nanopillar arrays and coated with silver film to enhance the sensitivity of immunoassay. The SERS platform demonstrated a limit of detection (LoD) up to 50 pg mL−1 for SARS-CoV-2 spike protein and virus-like-particle (VLP) protein in phosphate buffer saline within a turnaround time of 20 mins. Moreover, VLP protein spiked in untreated saliva achieved an LoD of 400 pg mL−1, providing a cycle threshold (Ct) value range of 30–32, closer to reverse transcription-polymerase chain reaction (RT-PCR) results (35−40) and higher than the commercial rapid antigen tests, ranging from 25 to 28. Therefore, the developed one-pot SERS based biosensor exhibited highly sensitive and rapid detection of SARS-CoV-2, which could be a potential point-of-care platform for early and cost-effective diagnosis of the COVID-19 virus.

STUDYING THE DIAGNOSTIC VALUE OF RECOMBINANT CAMPYLO-BACTER JEJUNI ANTIGENS

This article describes the results of a study on the diagnostic value of Campylobacter jejuni recombinant antigens, Campylobacter Omp18 protein and major outer membrane protein (MOMP). In the present study, these proteins were used as antigens in enzyme im-munoassays to detect specific antibodies in the serum of cattle. Commercial native protein antigens were used to compare the effectiveness with similar studies. In total, 95 blood se rum samples from cattle from various farms in the northern region of the Republic of Ka-zakhstan were used. The greatest number of positive results was observed when using a commercial native antigen (52.6%); 47.4% of sera reacted positively with respect to the Omp18 antigen, and 38.9% with respect to the MOMP antigen. Statistical analysis revealed a high correlation between the native and Omp18 antigens and, to a lesser extent, with MOMP32. Because native antigens contain many different cell-wall proteins, they are less specific. Therefore, for serological diagnosis of cattle using enzyme immunoassays, it is ad-visable to use the C. jejuni outer membrane recombinant antigen MOMP32, as it has valua-ble diagnostic properties.

Development of swine immune reagents for swine immunity and biomedical research

Abstract The USDA-NIFA Swine Immune Toolkit Initiative has been involved in generating priority immune reagents for swine immunity and biomedical research and pipelining them for marketing. With the help of commercial partners we expressed each target immune molecule as a soluble protein using yeast expression systems and then produced panels of monoclonal antibodies (mAbs) against each target. Most recently we generated panels of mAbs reactive with porcine IL-5, IL-6, IL-13, IL-21, IL-28B, CXCL10, and BAFF and screened their reactivity in multiple immune assays. We determined mAb reactivity with orthologous proteins for most panels of mAbs. A sensitive sandwich ELISA is now available for IL-13 and CXCL10; other targets are being screened for best mAb pairs. Reactivity tests of labeled α-CXCL10, α-IL-6 and α-IL-13 mAbs for intracellular staining of porcine immune cells using flow cytometry assays are underway using different cell stimulation conditions. Immunohistochemistry analyses for binding of α-CXCL10 and other mAbs on formalin fixed pig lymph nodes and spleen tissues are in progress. Further, analysis of a large panel of commercial α-human CD antigen mAbs for cross-reactivity with porcine cells is ongoing. For each target, our goal is to provide the veterinary community with new commercial reagents and standardized assay techniques for their research efforts. Finally, efforts are proceeding to develop SLA tetramers for antigen presentation studies. Tools and reagents generated by this project will undoubtedly advance our understanding of swine immune responses to disease and vaccine and use for biomedical research efforts. Supported by USDA-NIFA AFRI grant # 2019-67015-29815 and USDA ARS project 8042-32000-117.

Heartworm (Dirofilaria immitis) in carnivores kept in zoos in Texas, USA: risk perception, practices, and antigen detection

BackgroundDirofilaria immitis is the causative agent of heartworm disease in wild and domestic canids, felids, and mustelids. Recent studies demonstrate that additional families in the order Carnivora are also susceptible to infection. Therefore, the objectives of this study were to (1) better understand current practices surrounding heartworm prevention and diagnostics in zoological facilities located in the state of Texas, USA, and (2) assess archival serum samples of carnivores kept in these facilities for the presence D. immitis antigen and/or antibody.MethodsA questionnaire was completed by veterinarians or veterinary technicians representing 10 zoological facilities across Texas. This questionnaire was designed at the taxonomic family level, encompassing the 12 terrestrial carnivore families Ailuridae, Canidae, Eupleridae, Felidae, Herpestidae, Hyaenidae, Mephitidae, Mustelidae, Prionodontidae, Procyonidae, Ursidae, and Viverridae. The second objective was achieved with the use of archival serum samples made available by six zoo facilities.ResultsRisk perception varied across facilities for every family, including among species belonging to Canidae. All facilities used monthly heartworm prevention in canids and felids, with more variation existing in the other families. The use of diagnostic testing and type and route of administration of preventive varied by facility, with oral ivermectin the most commonly used preventive. A total of 217 archival serum samples, belonging to 211 individual animals encompassing 11 families and 39 species, were tested with a commercial heartworm antigen ELISA test, pre- and post-immune-complex dissociation. A subset of samples was also assessed for the presence of feline anti-heartworm antibodies using a commercial ELISA test. Two animals, both of which were Asian small-clawed otters from the same facility, had antigen detected (0.95%).ConclusionsThis study demonstrates that while the zoo veterinary community is aware of the risk and health impact of heartworm disease in canids and felids, there is still a great deal of uncertainty regarding the risks and ideal strategies for prevention in other carnivore families. The low proportion of antigen detection may serve as a baseline for future prevalence studies across the southern United States, where there is an emerging concern of macrocyclic lactone resistance in heartworm.Graphical