Competitive ELISA Assay Research Articles

Humoral Immunity Profiling to Pandemic and Bat‐Derived Coronavirus Variants: A Geographical Comparison

AbstractDynamic pathogen exposure may impact the immunological response to SARS‐CoV‐2 (SCV2). One potential explanation for the lack of severe SCV2‐related morbidity and mortality in Southeast Asia is prior exposure to related betacoronaviruses. Recent discoveries of SCV2‐related betacoronaviruses from horseshoe bats (Rhinolophus sinicus) in Thailand, Laos, and Cambodia suggest the potential for bat‐to‐human spillover exposures in the region. In this work, serum antibodies to protein constructs from SCV2 and a representative bat coronavirus isolated in Cambodia (RshSTT182) are measured in pre‐pandemic Cambodian human sera using ELISA assays. Of 293 Cambodian samples tested (N = 131 with acute malaria, n = 162 with acute undifferentiated febrile illness), 32 (10.9%) are seropositive for SCV2 based on established Spike and receptor‐binding domain (RBD) cutoffs. Within SCV2 seropositive samples, 16 (50%) have higher antibody levels to antigens from the representative virus RshSTT182 versus SCV2 antigens; competitive binding ELISA assays demonstrate inhibition of reactivity to SCV2 Spike after pre‐incubation with RshSTT182 Spike. Surrogate virus neutralization tests demonstrate that 8/30 (26.7%) SCV2 ELISA positive pre‐pandemic Cambodian samples have neutralizing activity against SCV2, while 14/30 (46.7%) have activity against other SCV2‐related betacoronaviruses. These data suggest that exposure to related betacoronaviruses may elicit cross‐reactive immunity to SCV2 prior to the global pandemic.

Adjuvanted Modified Bacterial Antigens for Single-Dose Vaccines

Alum is the most used vaccine adjuvant, due to its safety, low cost and adjuvanticity to various antigens. However, the mechanism of action of alum is complex and not yet fully understood, and the immune responses elicited can be weak and antigen-dependent. While several antigens rapidly desorb from alum upon exposure to serum, phosphorylated proteins remain tightly bound through a ligand-exchange reaction with surface hydroxyls on alum. Here, bacterial proteins and glycoconjugates have been modified with phosphoserines, aiming at enhancing the binding to alum and prolonging their bioavailability. Tetanus toxoid protein and Salmonella Typhi fragmented Vi-CRM conjugate were used. Both antigens rapidly and completely desorbed from alum after incubation with serum, verified via a competitive ELISA assay, and set up to rapidly evaluate in vitro antigen desorption from alum. After antigen modification with phosphoserines, desorption from alum was slowed down, and modified antigens demonstrated more prolonged retention at the injection sites through in vivo optical imaging in mice. Both modified antigens elicited stronger immune responses in mice, after a single injection only, compared to unmodified antigens. A stronger binding to alum could result in potent single-dose vaccine candidates and opens the possibility to design novel carrier proteins for glycoconjugates and improved versions of bacterial recombinant proteins.

B-043 Q-nab™ Igg kit: verification and validation of a novel cell-free quantitative neutralizing antibody assay

Abstract Background As the world exits the COVID-19 pandemic era, it is very important to assess population-level and individual seroprotectivity against multiple circulating and emerging SARS-CoV-2 strains, especially in vulnerable populations. Current serological measurements for neutralizing antibodies (NAbs) are either done using complex and time-intensive pseudovirus- or micro-neutralization assays or as a high throughput competitive ELISA assay. These assays, by design, cannot be multiplexed to measure NAb titers against multiple strains and suffer from a limited dynamic range. The Q-NAb IgG Tests, which rely on blocking the non-neutralizing antibodies (NNAbs) using a novel fusion protein, were developed and validated to quantitatively measure NAb titer against the SARS-CoV-2 strains used to develop the first two mRNA vaccines viz. ancestral and BA.4/5 strains. Methods The Q-NAb assay measures the NAb titer in a sample by first sequestering the NNAbs using the fusion protein (made by covalently coupling SARS-CoV-2 BA.4/5 RBD and h-ACE2 protein) thereby enriching the NAbs in the sample. Variant-specific NAbs are then detected using an indirect ELISA where the wells are coated with the variant RBD and yield a signal that is directly proportional to the concentration of NAb. Clinical samples from subjects that have either had a SARS-CoV-2 infection (N=27), taken the initial vaccine series (N=123) and/or the bivalent booster (N=151) and pre-pandemic samples (N=29) were commercially sourced. Q-NAb NAb titers were measured using the ancestral and BA.4/5 kits and were compared against corresponding MN50 values using both parametric and non-parametric models. Single-site precision, low-end sensitivity, dilutional linearity, and interference screening of Q-NAb IgG Kits were evaluated in accordance with appropriate CLSI guidelines. Traceability and total uncertainty of the Q-NAb IgG Kits (Ancestral and BA.4/5) to WHO International Standard 21/340 and 21/338 respectively was established using a calibration hierarchy adapted from ISO 17511:2020. Results Both the ancestral and BA.4/5 Q-NAb IgG Test Kits correlated with MN50 titers with a Spearman correlation coefficient of 0.90 and 0.89 respectively. The total uncertainty in traceability of calibration to WHO International standards 21/340 and 21/338 was found to be less than 10% for both tests. Single site precision across the assay range for BA.4/5 kit was found to be 6-10% with a limit of quantitation of 312 IU/mL. Assays exhibited dilutional linearity across their respective dynamic range and had no significant interference from common endogenous and exogenous substances. Conclusions We have successfully validated the novel Q-NAb IgG neutralization assay platform as individual assays for measuring neutralizing titers against ancestral and BA.4/5 SARS-CoV-2 strains. The assays have expanded dynamic range, excellent analytical performance and correlation to accepted gold-standard microneutralization assays. Both assays are calibrated and traceable to WHO international standards, thereby enabling the compilation and comparison of data from multiple clinical cohorts run across multiple laboratories. The design of the Q-NAb IgG Test kit makes it amenable to multiplexing (for example, on an MSD platform) to assess neutralization titers against multiple variants at the same time. Such a multiplexed platform can be used to assess vaccine efficacy and seroprotection against multiple variants including emerging variants.

New benzophenone analogs from Nigrospora sphaerica and their inhibitory activity against PD-1/PD-L1 interactions

Four newly identified benzophenone analogs [nigrophenone A–D (1–4)] and a pyrrolidinone analog [nigropyrrolidinone (5)], alongside thirteen known congeners (6–18), were isolated from Nigrospora sphaerica. Transcriptome analysis revealed that 6 might have the potential to modulate T-cell immunity. Quantitative measurements of the binding affinities between eighteen natural molecules and the immunological checkpoint receptors PD-1 and PD-L1 were performed using Surface Plasmon Resonance (SPR). The results of SPR analysis showed that 1–18 have KD values ranging from 1.8 to 99.5 μM for PD-1 and from 10.6 to 99.5 μM for PD-L1. Competitive inhibition studies, employing SPR and ELISA assays, have indicated that compounds 6, 10, 15, and 18 are capable of inhibiting the PD-1/PD-L1 interaction. Additionally, compound 6 exhibited notable in vitro anticancer potency through the augmentation of activating signals and the upregulation of PD-1 expression on CD8+ T cells, concurrently elevating the secretion of IFN-γ and IL-2, thereby inhibiting the proliferation of LLC and MC38 cells and promoting MC38 apoptosis. Moreover, compound 6 modulates the PI3K/Akt pathway, which is a key downstream effector of the PD-1/PD-L1 axis. These compounds are considered promising candidates for more in-depth exploration because they could significantly inhibit PD-1/PD-L1 interactions in tumor immunotherapy.

Associations between APOE genotypes, urine 8-isoprostane and blood trace elements in middle-aged mothers (CROME study)

BackgroundThere is almost no data on the combined associations between apolipoprotein E gene (APOE) genotypes, trace elements (TEs), and lipid peroxidation in vivo. The aim of our study was to evaluate the association between APOE genotypes and TE levels in blood (B-TEs) and erythrocytes (E-TEs), and 8-isoprostane in urine (U-8-isoprostane) in women with low exposure to potentially toxic TEs and with adequate supply of essential TEs. MethodsB-TEs, E-TEs and U-8-isoprostane were determined in 172 healthy women of childbearing age (30.1–51.4 years) using ICP-MS and ELISA competitive assay, respectively. All women were divided into three APOE genotype groups according to the presence of the ɛ4 allele, ɛ2 allele or ɛ3 homozygotic allele. The associations between B-TEs, E-TE, U-8-isoprostane, and the APOE genotype groups were estimated by multiple variable linear regression models with relevant explanatory variables (e.g., age, BMI, and seafood). ResultsAll TE and U-8-isoprostane levels were inside the reference ranges for the healthy population. In the multiple variable linear regression models, our results showed that urine 8-isoprostane levels increased by up to 43.3% in the APOE4 group compared to the APOE3 group and a negligible negative modifying effect for essential TEs. However, the APOE genotype groups were associated also with some TEs. A clear positive association was found between the APOE2 and APOE4 groups (vs. APOE3) with B-molybdenum. ConclusionsOur study suggests that the APOE4 genotype played an important role in 8-isoprostane variability in a population with an adequate supply of essential and with low exposure to potentially toxic TEs. Adequate copper, zinc and selenium status seemed to be protective against, while the levels of nonessential TEs were probably too low to play a decisive role in 8-isoprostane formation. The observed impact of the APOE2 and APOE4 groups on increased B-molybdenum opens a new research topic.

Pseudomonas aeruginosa exotoxin A as a novel allergen induced Non-TH2 inflammation in a murine model of steroid-insensitive asthma

BackgroundDespite the immediate in vivo occurrence of anaphylactic and allergic reactions following treatment with Pseudomonas aeruginosa exotoxin A (PEA)-based immunotoxins, the immunological role of PEA in asthma pathogenesis remains unclear. ObjectiveThis study investigated the allergenic potential of PEA and the specific type of asthma induced. MethodsRecombinant PEA (rPEA) lacking domain Ia (to eliminate non-specific cytotoxicity) was expressed, purified, and employed to detect serum PEA-specific IgE levels in asthmatic patients. Competitive ELISA assays were used to assess rPEA's IgE binding capacity and allergenicity. Additionally, rPEA-challenged C57BL/6 mice were subjected to inflammatory endotyping and therapeutic assays to characterize the allergic nature of PEA. ResultsPEA-specific IgE was identified in 17 (14.2 %) of 120 asthma patients. The rPEA-sensitized and challenged mice had increased PEA-specific immunoglobulins (such as IgE, IgG1 and IgG2a) and developed asthma-like phenotypes with airway hyperresponsiveness, severe airway inflammation, and airway remodeling. Lungs from these mice displayed significant increases in neutrophils and IL-17A+ cells. Innate lymphoid cells (ILCs) produced type 2 cytokines (IL-4, IL-5, and IL-13), whereas Th cells did not. Nonetheless, airway inflammation, rather than hyperresponsiveness, was elicited in non-sensitized mice upon challenge with rPEA. Importantly, rPEA-induced asthmatic mice were unresponsive to dexamethasone treatment. ConclusionPEA is a novel allergen that sensitizes asthmatic patients. Furthermore, mice developed steroid-resistant asthma, characterized by an atypical cytokine profile associated with non-TH2 inflammation, only after being sensitized and challenged with rPEA. These findings suggest a potentially significant role for PEA in asthma development, warranting consideration in clinical diagnosis and treatment strategies.

Interleukin-17A (IL-17A) is involved in antibody specificity to conformational epitopes

The specificity of antibodies (Ab) is essential for the precise recognition of foreign or dangerous molecules. We have shown that mice infected with non-pathogenic Lactate Dehydrogenase Elevating Virus (LDV) inoculated with human growth hormone (hGH) or Ovalbumin (OVA), exhibit modified specificity of anti-hGH or anti-OVA Ab associated with the secretion of IFN-γ, IL-13, and IL-17.Cytokines are directly or indirectly involved in the isotypes, specificity, and affinity of Ab. Accordingly, here we investigated the effect of IL-17 neutralization on Ab specificities to OVA and Diphtheria Toxoid (DTx) in a mouse model of viral infection. Thereby, we employed an anti-cytokine “auto-vaccination” with an OVA/IL-17A complex or a Monoclonal Ab (MAb) anti-IL-17A (MM17/F3).Competitive ELISA assays were used to estimate the quality of the humoral immune response and the amount of Abs to conformational versus linear antigenic determinants. Results indicated that the OVA/IL-17A complex increased Abs levels to conformational epitopes of OVA, while LDV prolonged antibodies for a longer period. Mice treated with MM17F3 MAb showed an increase in Abs to conformational epitopes of OVA. A similar effect, confirmed by a competitive Western-blot assay, was produced by LDV. Moreover, an increased level of IgM, IgG1, and IgG2a was found in infected animals. Similarly, MAb anti-IL-17A treatment increased the proportion of Ab to conformational epitopes of DTx in uninfected mice, while LDV decreased this parameter.In conclusion, our findings demonstrate a correlation between IL-17A neutralization and a change in Ab specificity to OVA or DTx, presenting a novel strategy for obtaining Abs with higher specificity.

Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2

Coronavirus disease 2019 (COVID-19) is an immune-related disorder caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The complete pathogenesis of the virus remains to be determined. Unraveling the molecular mechanisms governing SARS-CoV-2 interactions with host cells is crucial for the formulation of effective prophylactic measures and the advancement of COVID-19 therapeutics. We analyzed human lung single-cell RNA sequencing dataset to discern the association of butyrophilin subfamily 3 member A2 (BTN3A2) expression with COVID-19. The BTN3A2 gene edited cell lines and transgenic mice were infected by live SARS-CoV-2 in a biosafety level 3 (BSL-3) laboratory. Immunoprecipitation, flow cytometry, biolayer interferometry and competition ELISA assays were performed in BTN3A2 gene edited cells. We performed quantitative real-time PCR, histological and/or immunohistochemical analyses for tissue samples from mice with or without SARS-CoV-2 infection. The BTN3A2 mRNA level was correlated with COVID-19 severity. BTN3A2 expression was predominantly identified in epithelial cells, elevated in pathological epithelial cells from COVID-19 patients and co-occurred with ACE2 expression in the same lung cell subtypes. BTN3A2 targeted the early stage of the viral life cycle by inhibiting SARS-CoV-2 attachment through interactions with the receptor-binding domain (RBD) of the Spike protein and ACE2. BTN3A2 inhibited ACE2-mediated SARS-CoV-2 infection by reducing ACE2 invitro and invivo. These results reveal a key role of BTN3A2 in the fight against COVID-19. Identifying potential monoclonal antibodies which mimic BTN3A2 may facilitate disruption of SARS-CoV-2 infection, providing a therapeutic avenue for COVID-19. This study was supported by the National Natural Science Foundation of China (32070569, U1902215, and 32371017), the CAS "Light of West China" Program, and Yunnan Province (202305AH340006).

Quantitative proteomics reveals the complex regulatory networks of LTTR-type regulators in pleiotropic functions of Aeromonas hydrophila

LysR-type transcriptional regulators (LTTRs) are ubiquitously distributed and abundant transcriptional regulators in prokaryotes, playing pivotal roles in diverse physiological processes. Nonetheless, despite their prevalence, the intricate functionalities and physiological implications of this protein family remain incompletely elucidated. In this study, we employed a comprehensive approach to deepen our understanding of LTTRs by generating a collection of 20 LTTR gene-deletion strains in Aeromonas hydrophila, accounting for 42.6 % of the predicted total LTTR repertoire, and subjected them to meticulous assessment of their physiological phenotypes. Leveraging quantitative proteomics, we conducted a comparative analysis of protein expression variations between six representative mutants and the wild-type strain. Subsequent bioinformatics analysis unveiled the involvement of these LTTRs in modulating a wide array of biological processes, notably including two-component regulatory systems (TCSs) and intracellular central metabolism. Moreover, employing subsequent microbiological methodologies, we experimentally verified the direct involvement of at least six LTTRs in the regulation of galactose metabolism. Importantly, through ELISA and competitive ELISA assays, we demonstrated the competitive binding capabilities of these LTTRs with the promoter of the α-galactosidase gene AHA_1897 and identified that four LTTRs (XapR, YidZ, YeeY, and AHA_1805) do not engage in competitive binding with other LTTRs. Overall, our comprehensive findings not only provide fundamental insights into the regulatory mechanisms governing crucial physiological functions of bacteria through LTTR family proteins but also uncover an intricate and interactive regulatory network mediated by LTTRs.

Comparative characterization of two monoclonal antibodies targeting canine PD-1.

Monoclonal antibodies targeting immune checkpoints have revolutionized oncology. Yet, the effectiveness of these treatments varies significantly among patients, and they are associated with unexpected adverse events, including hyperprogression. The murine research model used in drug development fails to recapitulate both the functional human immune system and the population heterogeneity. Hence, a novel model is urgently needed to study the consequences of immune checkpoint blockade. Dogs appear to be uniquely suited for this role. Approximately 1 in 4 companion dogs dies from cancer, yet no antibodies are commercially available for use in veterinary oncology. Here we characterize two novel antibodies that bind canine PD-1 with sub-nanomolar affinity as measured by SPR. Both antibodies block the clinically crucial PD-1/PD-L1 interaction in a competitive ELISA assay. Additionally, the antibodies were tested with a broad range of assays including Western Blot, ELISA, flow cytometry, immunofluorescence and immunohistochemistry. The antibodies appear to bind two distinct epitopes as predicted by molecular modeling and peptide phage display. Our study provides new tools for canine oncology research and a potential veterinary therapeutic.

Abstract 1341: SM2235: A bispecific EGFR x CD16A innate immune cell engager for enhanced treatment of EGFR-expressing solid tumors, regardless of RAS mutation status

Abstract Background: Monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) are frontline therapies for numerous EGFR-dependent epithelial tumors. However, their efficacy faces challenges due to on-target skin toxicity and intrinsic or acquired resistance. Within the tumor microenvironment, innate immune cells, including macrophages and natural killer (NK) cells, play a significant role but are hindered by the lack of specific cancer cell targeting and a suppressive microenvironment. To enhance innate immune cell cytotoxicity, we have developed SM2235, a humanized tetravalent bispecific VHH antibody. SM2235 partially blocks EGFR and EGF interaction while conditionally activating innate immune cells through CD16A upon engagement with cancer cells. Methods: SM2235 was engineered by combining a partially blocking EGFR single-domain antibody with a high-affinity CD16A-activating single-domain antibody on an inactivated human IgG1 Fc backbone. The binding affinity and specificity were confirmed using bio-layer interferometry and ELISA with recombinant proteins, followed by flow cytometry with EGFR+ human cancer cell lines and CD16A+ primary NK cells. SM2235's efficacy in blocking EGFR-EGF interaction and downstream signaling was assessed through competitive ELISA and proliferation assays in both EGFR-dependent and independent cancer cell lines. Its ability to induce target-dependent NK cell and macrophage activation was evaluated through activation markers as well as cytotoxicity and phagocytosis assays. In vivo efficacy of SM2235 against EGFR+ human tumors was measured using xenograft assays in NSG mice, covering cetuximab-sensitive and resistant cancer cell lines. Preliminary pharmacokinetics and safety studies were conducted in cynomolgus monkeys. Results: SM2235 exhibits sub-nanomolar affinity for EGFR+ human cancer cell lines and CD16A+ primary human NK cells. It partially inhibits EGFR-dependent cancer cell proliferation. Compared to cetuximab, SM2235 significantly enhances target-dependent CD16A-mediated NK cell and macrophage activation and cytotoxicity against EGFR+ cancer cells in ADCC and ADCP assays in vitro. SM2235 effectively inhibits EGFR+ xenograft tumors in vivo, irrespective of KRAS mutation status. In cynomolgus monkeys, SM2235 demonstrates excellent tolerance with a 20-hour serum half-life of approximately 20 hours and no dose-dependent skin toxicity. Conclusions: Preclinical characterization of SM2235 shows promising in vivo efficacy against EGFR+ human cancers, regardless of KRAS mutation, with a favorable safety profile marked by minimal skin toxicity. Citation Format: Huiqin Geng, Wenjing Song, Xiaodan Liu, Shihao Lu, Hong Zhou, Simin Yang, Siyao Xie, Yi Wei, Xing Zhang, Sheila Zhou, Yanbin Liang. SM2235: A bispecific EGFR x CD16A innate immune cell engager for enhanced treatment of EGFR-expressing solid tumors, regardless of RAS mutation status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1341.

Gentulizumab, a novel anti-CD47 antibody with potent antitumor activity and demonstrates a favorable safety profile

BackgroundTargeting CD47/SIRPα axis has emerged as a promising strategy in cancer immunotherapy. Despite the encouraging clinical efficacy observed in hematologic malignancies through CD47-SIRPα blockade, there are safety concerns related to the binding of anti-CD47 antibodies to CD47 on the membrane of peripheral blood cells.MethodsIn order to enhance the selectivity and therapeutic efficacy of the antibody, we developed a humanized anti-CD47 monoclonal antibody called Gentulizumab (GenSci059). The binding capacity of GenSci059 to CD47 was evaluated using flow cytometry and surface plasmon resonance (SPR) methods, the inhibitory effect of GenSci059 on the CD47-SIRPα interaction was evaluated through competitive ELISA assays. The anti-tumor activity of GenSci059 was assessed using in vitro macrophage models and in vivo patient-derived xenograft (PDX) models. To evaluate the safety profile of GenSci059, binding assays were conducted using blood cells. Additionally, we investigated the underlying mechanisms contributing to the weaker binding of GenSci059 to erythrocytes. Finally, toxicity studies were performed in non-human primates to assess the potential risks associated with GenSci059.ResultsGenSci059 displayed strong binding to CD47 in both human and monkey, and effectively inhibited the CD47-SIRPα interaction. With doses ranging from 5 to 20 mg/kg, GenSci059 demonstrated potent inhibition of the growth of subcutaneous tumor with the inhibition rates ranged from 30.3% to complete regression. Combination of GenSci059 with 2.5 mg/kg Rituximab at a dose of 2.5 mg/kg showed enhanced tumor inhibition compared to monotherapy, exhibiting synergistic effects. GenSci059 exhibited minimal binding to hRBCs compared to Hu5F9-G4. The binding of GenSci059 to CD47 depended on the cyclization of N-terminal pyroglutamic acid and the spatial conformation of CD47, but was not affected by its glycosylation modifications. A maximum tolerated dose (MTD) of 450 mg/kg was observed for GenSci059, and no significant adverse effects were observed in repeated dosages up to 10 + 300 mg/kg, indicating a favorable safety profile.ConclusionGenSci059 selectively binds to CD47, effectively blocks the CD47/SIRPα axis signaling pathway and enhances the phagocytosis effects of macrophages toward tumor cells. This monoclonal antibody demonstrates potent antitumor activity and exhibits a favorable safety profile, positioning it as a promising and effective therapeutic option for cancer.

Composition and diversity of culturable cyanobacteria in sediment samples from the upper layers of two tropical reservoirs

Benthic cyanobacteria research in high mountain reservoirs remains limited, mainly due to their complexity and knowledge gaps that persist in relation to their ecology in tropical regions. This study aimed to explore the composition, diversity, and toxic potential of cyanobacterial in the upper sediment of two Colombian reservoirs. Our investigation involved multiple methodologies, such as germination experiments, that allowed us to assess the presence and viability of cyanobacteria in upper sediment samples, while the competitive ELISA assay allowed for the quantification of toxins within the cultures. The molecular analysis of Operational Taxonomic Units (OTUs) from subsamples of sediment cultures focused on evaluating cyanobacterial diversity and richness among prokaryotic phyla, and the Phylogenetic analysis of culturable cyanobacteria. Chlorophyll-a measurements confirmed the presence of viable populations in sediment cultures, while microscopic identification demonstrated the growth capacity of cyanobacteria from the orders Nostocales, Chroococcales, Oscillatoriales, and Synechococcales under controlled laboratory conditions. Despite low microcystin levels in culture, the prior detection of mcy genes in direct sediment samples suggests a possible toxic potential of cyanobacterial inhabiting the upper sediments. Community analysis, based on the OTUs abundance, revealed a notably diverse microbial community in both reservoir sediments, with a higher relative abundance of cyanobacteria compared to other prokaryotic phyla. These findings support the hypothesis that surface sediments play a fundamental role as a repository for cyanobacteria that may pose inherent risks to ecosystem health. In conclusion, this research underscores the necessity of further studies to achieve a holistic comprehension of benthic cyanobacteria dynamics in high-mountain tropical reservoirs.

A new pepper allergen Zan b 1.01 of 2S albumins: Identification, cloning, characterization, and cross-reactivity.

Zanthoxylum bungeanum (Sichuan pepper; in Chinese) is used as a spice worldwide and is a potentially life-threatening allergenic food source, as first reported by our team in 2005. However, its allergen components are unknown. We aim to identify and characterize its major allergen and determine its cross-reactivities with citrus seeds, pistachios, and cashew seeds. Ionic exchange and molecular exclusion chromatography were used to isolate the protein components from Sichuan pepper seed. A protein fraction was characterized by SDS-PAGE, analytical ultracentrifugation, mass spectrometry, and circular dichroism spectroscopy. The coding region of it was amplified from the genome. ELISA and competitive ELISA assays were used to investigate the allergenicity and cross-reactivity of allergens. This protein allergen was around 14 kDa. It was a 2S albumin similar to an α-Amylase inhibitor (AAI) domain-containing protein of Citrus sinensis. Circular dichroism spectroscopy showed its thermal stability was high. A 303 bps DNA sequence of the AAI domain was cloned from the genome of the Sichuan pepper. Competitive ELISA assays showed positive cross-reactivities between this allergen and citrus seeds, pistachios, and cashew seeds. A major allergen of around 14 kDa from Sichuan pepper seed was confirmed, which belongs to the 2S albumin of plant seed storage proteins. Based on the nomenclature of the IUIS Subcommittee for Allergen Nomenclature, this allergen is designated as Zan b 1.01. The cross-reactivities were demonstrated between Zan b 1.01 and citrus seeds, pistachios, and cashew seeds.

Characterization of a Panel of Monoclonal Antibodies Targeting the F-Protein of the Respiratory Syncytial Virus (RSV) for the Typing of Contemporary Circulating Strains.

Respiratory syncytial virus (RSV) is the most common cause of upper and lower respiratory tract infections in infants and young children. Virus-specific monoclonal antibodies (mAbs) can be used for diagnosis, prophylaxis, and research of RSV pathogenesis. A panel of 16 anti-RSV mAbs was obtained from mice immunized by RSV strain Long. Half of them had virus-neutralizing activity. According to Western blot all of these mAbs effectively bound native oligomeric (homodimeric and homotrimeric) forms of the RSV fusion (F) protein. Only five of the mAbs interacted with the monomeric form, and only one of these possessed neutralizing activity. None of these mAbs, nor the commercial humanized neutralizing mAb palivizumab, reacted with the denaturated F protein. Thus, interaction of all these mAbs with F protein had clear conformational dependence. Competitive ELISA and neutralization assays allowed the identification of nine antigenic target sites for the interaction of mAb with the F protein. Five partially overlapping sites may represent a complex spatial structure of one antigenic determinant, including one neutralizing and four non-neutralizing epitopes. Four sites (three neutralizing and one non-neutralizing) were found to be distinct. As a result of virus cultivation RSV-A, strain Long, in the presence of a large amount of one of the neutralizing mAbs, an escape mutant with a substitution, N240S, in the F protein, was obtained. Thus, it was shown for the first time that position 240 is critical for the protective effect of an anti-RSV antibody. To assess the ability of these mAbs to interact with modern RSV strains circulating in St. Petersburg (Russia) between 2014 and 2022, 73 RSV-A and 22 RSV-B isolates were analyzed. Six mAbs were directed to conserved epitopes of the F protein as they interacted most efficiently with both RSV subtypes in a fixed cell-ELISA and could be used for diagnostic assays detecting RSV.

Monoclonal Antibodies Targeting LIGHT Block Ltbr Signalling and Eliminate Acute Myeloid Leukemia Stem Cells

Introduction Acute myeloid leukemia (AML) is a molecular heterogeneous disease that originates from the malignant transformation of a hematogenic stem- or progenitor cells resulting in a leukemia stem cell (LSC). LSCs are a main cause of resistance to therapy and relapse. Despite recent advances in targeted therapy and immunotherapy, prognosis for elderly patients not fit do undergo intensive therapy remains poor. We recently documented that targeting CD70, the ligand of the tumor necrosis factor receptor (TNFR) CD27, by the antibody-dependent cell-mediated cytotoxicity (ADCC)-enhanced monoclonal antibody (mAb) cusatuzumab eliminates LSC. We now studied the role of the related TNFR lymphotoxin b receptor (LTbR) with its ligand LIGHT in the pathogenesis of AML in mice and humans and developed a LIGHT targeting mAb to specifically eliminate LSCs. Experimental design We investigated the impact of LIGHT/LTbR signalling in AML development by using the retrovirally-induced MLL-AF9 syngeneic AML mouse model. Light-, Ltbr -deficient and control MLL-AF9 transduced LSCs were injected intravenously into non-irradiated BL/6 recipient mice. AML development was evaluated by analysing MLL-AF9-GFP-positive leukemic cells in blood, spleen, and bone marrow (BM). BM LSC were phenotypically characterized by FACS and functionally by performing colony forming unit (CFU) assays in vitro and by secondary transplantations of LSCs including limited dilution assays (LDA) in vivo. Correlative survival analysis for LIGHT and LTbR expression was performed on a publicly available gene dataset (GSE6891). LIGHT and LTBR protein expression was analysed on primary AML CD34 + leukemic stem and progenitor cells (LSPCs) and control human CD34 + hematopoietic stem cell and progenitor cells (HSCPs). LIGHT/LTbR signalling was blocked by either siRNA treatment or by treatment with novel LIGHT-targeting mAb. The effect of blocking LIGHT/LTbR-signalling was analysed in vitro by CFU assays, RNA-sequencing and xenotransplant experiments in vivo. LIGHT-targeting mAbs were generated by immunization of llamas and phage display selections followed by cloning of FABs into a human backbone (hIgG1). Clones were selected based on their blocking potency of receptor/ligand interaction using competition ELISA and FACS-based cell binding affinity assays. ADCC-enhanced anti-LIGHT mAbs were generated via POTELLIGENT® technology. Anti-LIGHT mAbs that specifically block the interaction of LIGHT/LTBR with deficiency in effector functions (Fc-death) were produced by amino acid substitutions in the CH2 domain of the parental clone. Results LTbR was expressed by HSCs and AML LSCs. In contrast, LIGHT was only expressed on murine and human LSCs but not normal HSCs. The absence of LIGHT or its receptor LTbR on LSCs resulted in significantly prolonged survival and decreased numbers of LSC in BM of AML mice. Light -/- and Ltbr -/- LSCs had a reduced CFU capacity compared to control LSCs. Similarly, inhibition of LIGHT-signalling on LSCs via LTbR-Ig significantly reduced colony formation. Moreover, secondary transplantation of Light -/-LSCs resulted in prolonged survival compared to Light-proficient LSCs. An LDA analysis revealed a 11-fold reduction of the LSC frequency in BM in the absence of LIGHT or LTBR. Similarly, inhibiting LIGHT signalling by siLIGHT RNA in human AML LSPCs resulted in reduced CFU capacity compared to control siRNA treatment. In addition, a GEO analysis revealed that low LIGHT or LTbR mRNA levels correlated with improved overall survival in AML patients. After immunizations of llamas and phage-display selection three humanized anti-LIGHT mAbs with high blocking and high binding efficiency were selected (mAb10F11 and 12C2 specific for human LIGHT and the cross-reactive murine/human 9F6 mAB). Incubation of LSCs with the blocking mAbs significantly reduced CFU capacity. HSPCs from healthy donors remained unaffected by the treatment. A gene expression analysis revealed that blocking LIGHT/LTbR-signalling in LSCs reduced stemness and promoted differentiation. In addition, ADCC-enhanced mAbs activated NK cells to eliminate LIGHT-expressing LSCs. Our findings reveal that LIGHT/LTbR-signalling is crucial for the pathogenesis of AML and especially for the maintenance and expansion of LSCs. We developed and validated novel LIGHT targeting mAbs in vitro and in vivo for further development in clinical trials.

Immunotechniques for the Group Determination of Macrolide Antibiotics Traces in the Environment Using a Volume-Mediated Sensitivity Enhancement Strategy.

Macrolide antibiotics, which are effective antimicrobial agents, are intensively used in human and veterinary medicine, as well as in agriculture. Consequently, they are found all over the world as environmental pollutants, causing harm to sensitive ecological communities and provoking a selection of resistant forms. A novel azithromycin derivative, which was used as hapten conjugate, ensured the group immunorecognition of six major macrolide representatives (105-41%), namely erythromycin, erythromycin ethylsuccinate, clarithromycin, roxithromycin, azithromycin, and dirithromycin in a competitive immunoassay based on anti-clarithromycin antibodies. The heterologous hapten-based ELISA format resulted in a 5-fold increase in sensitivity, with an IC50 value of 0.04 ng/mL for erythromycin. In this study, we proposed an underexploited strategy in an immunoassay field to significantly improve the detectability of analytes in environmental samples. Unlike most approaches, it does not require special enhancers/amplifiers or additional concentration/extraction procedures; instead, it involves analyzing a larger volume of test samples. A gradual volume increase in the samples (from 0.025 to 10 mL) analyzed using a direct competitive ELISA, immunobeads, and immunofiltration assay formats based on the same reagents resulted in a significant improvement (more than 50-fold) in assay sensitivity and detection limit up to 5 and 1 pg/mL, respectively. The suitability of the test for detecting the macrolide contamination of natural water was confirmed by the recovery of macrolides from spiked blank samples (71.7-141.3%). During 2022-2023, a series of natural water samples from Lake Onega and its influents near Petrozavodsk were analyzed, using both the developed immunoassay and HPLC-MS/MS. The results revealed no contamination of macrolide antibiotic.

A Comprehensive Technology Platform for the Rapid Discovery of Peptide Inhibitors against SARS-CoV-2 Pseudovirus Infection.

We developed and validated a technology platform for designing and testing peptides inhibiting the infectivity of SARS-CoV-2 spike protein-based pseudoviruses. This platform integrates target evaluation, in silico inhibitor design, peptide synthesis, and efficacy screening. We generated a cyclic peptide library derived from the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor. The cell-free validation process by ELISA competition assays and Surface Plasmon Resonance (SPR) studies revealed that the cyclic peptide c9_05, but not its linear form, binds well to ACE2. Moreover, it effectively inhibited the transduction in HEK293, stably expressing the human ACE2 receptor of pseudovirus particles displaying the SARS-CoV-2 spike in the Wuhan or UK variants. However, the inhibitory efficacy of c9_05 was negligible against the Omicron variant, and it failed to impede the entry of pseudoviruses carrying the B.1.351 (South African) spike. These variants contain three or more mutations known to increase affinity to ACE2. This suggests further refinement is needed for potential SARS-CoV-2 inhibition. Our study hints at a promising approach to develop inhibitors targeting viral infectivity receptors, including SARS-CoV-2's. This platform also promises swift identification and evaluation of inhibitors for other emergent viruses.

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.

Identification of a common epitope in knottins and phospholipases D present in Loxosceles sp venom by a monoclonal antibody

In the Americas and specially in Brazil, the Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta are the three most medically relevant brown spider species, and whose bites can lead to the condition known as loxoscelism. Here, we report the development of a tool capable of identifying a common epitope amongst Loxosceles sp. venom's toxins. A murine monoclonal antibody (LmAb12) and its recombinant fragments (scFv12P and diabody12P) have been produced and characterized. This antibody and its recombinant constructs were able to recognize proteins of Loxosceles spider venoms with specificity. The scFv12P variant was also able to detect low concentrations of Loxosceles venom in a competitive ELISA assay, displaying potential as a venom identification tool. The primary antigenic target of LmAb12 is a knottin, a venom neurotoxin, that has a shared identity of 100 % between the L. intermedia and L. gaucho species and high similarity to L. laeta. Furthermore, we observed LmAb12 was able to partially inhibit in vitro hemolysis, a cellular event typically induced by the Loxosceles sp. venoms. Such behavior might be due to LmAb12 cross-reactivity between the antigenic target of LmAb12 and the venom's dermonecrotic toxins, the PLDs, or even the existence of synergism between these two toxins.