Antibody Binding Research Articles

Mechanisms of autoimmune encephalitis.

To provide an overview of the pathogenic mechanisms involved in autoimmune encephalitides mediated by antibodies against neuronal surface antigens, with a focus on NMDAR and LGI1 encephalitis. In antibody-mediated encephalitides, binding of IgG antibodies to neuronal surface antigens results in different pathogenic effects depending on the type of antibody, IgG subclass and epitope specificity. NMDAR IgG1 antibodies cause crosslinking and internalization of the target, synaptic and brain circuitry alterations, as well as alterations of NMDAR expressing oligodendrocytes, suggesting a link with white matter lesions observed in MRI studies. LGI1 IgG4 antibodies, instead, induce neuronal dysfunction by disrupting the interaction with cognate proteins and altering AMPAR-mediated signaling. In-vitro findings have been corroborated by memory and behavioral changes in animal models obtained by passive transfer of patients' antibodies or active immunization. These models have been fundamental to identify targets for innovative therapeutic strategies, aimed at counteracting or preventing antibody effects, such as the use of soluble ephrin-B2, NMDAR modulators (e.g., pregnenolone, SGE-301) or chimeric autoantibody receptor T cells (CAART) in models of NMDAR encephalitis. A deep understanding of the pathogenic mechanisms underlying antibody-mediated encephalitides is crucial for the development of new therapeutic approaches targeting brain autoimmunity.

Rapid antibody conjugation strategy via instant charge inversion of AuNBPs toward ultrasensitive SERS-LFIA detection of AFP

Alpha-fetoprotein (AFP) is widely utilized as the primary serum marker for diagnosing Hepatocellular carcinoma. Integrating surface-enhanced Raman scattering (SERS) with lateral flow immunoassays (LFIA) displays significant promise for the rapid quantification of AFP. Gold nanobipyramids (AuNBPs) is a kind of SERS probe with high activity showing strong application potential in SERS-LFIA. However, the drastic electrostatic interaction between negative charged antibodies and positive charged AuNBPs will result in uncontrollable aggregation, which poses a challenge for antibody binding and limits its application in SERS-LFIA. Hence, we propose a rapid antibody conjugation strategy with the aid of instant charge inversion of AuNBPs, which enables efficient and expedited antibody coupling onto AuNBPs. The thiol-free fish sperm (FSDNA) was employed to adsorb on the cetyltrimethylammonium bromide (CTAB) coated AuNBPs for immediate charge inversion. The adsorption of FSDNA will cause partial stripping of CTAB from the AuNBPs surface to provide additional active sites for antibodies binding. In this study, approximately average 37 AFP-antibodies were verified bonding on one charge inverted AuNBPs by direct incubation for 30 min. The antibodies modified AuNBPs were further incubated with 4-nitrothiophenol for preparing SERS immune probes, which display excellent colloid stability and specificity. The probes were used to construct SERS-LFIA strips for AFP detection, enabling highly sensitive detection of AFP with the limit of detection (LOD) as low as 0.72 pg·mL−1. The developed SERS-LFIA strip demonstrates excellent stability and accuracy in detecting serum samples, with a recovery rate ranging from 84 % to 114 %. This rapid antibody coupling strategy is applicable to a wide range of positively charged nanoparticles, effectively addressing the challenging issue of utilizing such nanomaterials in LFIA.

DNA vaccine prime and replicating vaccinia vaccine boost induce robust humoral and cellular immune responses against MERS-CoV in mice

As of December 2022, 2603 laboratory-identified Middle East respiratory syndrome coronavirus (MERS-CoV) infections and 935 associated deaths, with a mortality rate of 36%, had been reported to the World Health Organization (WHO). However, there are still no vaccines for MERS-CoV, which makes the prevention and control of MERS-CoV difficult. In this study, we generated two DNA vaccine candidates by integrating MERS-CoV Spike (S) gene into a replicating Vaccinia Tian Tan (VTT) vector. Compared to homologous immunization with either vaccine, mice immunized with DNA vaccine prime and VTT vaccine boost exhibited much stronger and durable humoral and cellular immune responses. The immunized mice produced robust binding antibodies and broad neutralizing antibodies against the EMC2012, England1 and KNIH strains of MERS-CoV. Prime-Boost immunization also induced strong MERS-S specific T cells responses, with high memory and poly-functional (CD107a-IFN-γ-TNF-α) effector CD8+ T cells. In conclusion, the research demonstrated that DNA-Prime/VTT-Boost strategy could elicit robust and balanced humoral and cellular immune responses against MERS-CoV-S. This study not only provides a promising set of MERS-CoV vaccine candidates, but also proposes a heterologous sequential immunization strategy worthy of further development.

Correlates of protection against symptomatic SARS-CoV-2 in vaccinated children

The paucity of information on longevity of vaccine-induced immune responses and uncertainty of the correlates of protection hinder the development of evidence-based COVID-19 vaccination policies for new birth cohorts. Here, to address these knowledge gaps, we conducted a cohort study of healthy 5–12-year-olds vaccinated with BNT162b2. We serially measured binding and neutralizing antibody titers (nAbs), spike-specific memory B cell (MBC) and spike-reactive T cell responses over 1 year. We found that children mounted antibody, MBC and T cell responses after two doses of BNT162b2, with higher antibody and T cell responses than adults 6 months after vaccination. A booster (third) dose only improved antibody titers without impacting MBC and T cell responses. Among children with hybrid immunity, nAbs and T cell responses were highest in those infected after two vaccine doses. Binding IgG titers, MBC and T cell responses were predictive, with T cells being the most important predictor of protection against symptomatic infection before hybrid immunity; nAbs only correlated with protection after hybrid immunity. The stable MBC and T cell responses over time suggest sustained protection against symptomatic SARS-CoV-2 infection, even when nAbs wane. Booster vaccinations do not confer additional immunological protection to healthy children.

PASC (Post Acute Sequelae of COVID-19) is associated with decreased neutralizing antibody titers in both biological sexes and increased ANG-2 and GM-CSF in females.

Post-acute sequelae of COVID-19 (PASC) or the continuation of COVID-19 (Coronavirus disease 2019) symptoms past 12weeks may affect as many as 30% of people recovering from a SARS-CoV-2 (severe acute respiratory coronavirus 2) infection. The mechanisms regulating the development of PASC are currently not known; however, hypotheses include virus reservoirs, pre-existing conditions, microblood clots, immune dysregulation, as well as poor antibody responses. Importantly, virus neutralizing antibodies are essential for COVID-19 recovery and protection from reinfection but there is currently limited information on these immune regulators and associated cytokines in PASC patients. Understanding the key drivers of general and specific symptoms associated with Long COVID and the presence of virus neutralizing antibodies in PASC will aid in the development of therapeutics, diagnostics, and vaccines which currently do not exist. We designed a cross-sectional study to investigate systemic antibody and cytokine responses during COVID-19 recovery and PASC. In total, 195 participants were recruited in one of four groups: (1) Those who never had COVID-19 (No COVID); (2) Those in acute COVID-19 recovery (Acute Recovery) (4-12weeks post infection); (3) Those who recovered from COVID-19 (Recovered) (+ 12weeks from infection); and (4) those who had PASC (PASC) (+ 12weeks from infection). Participants completed a questionnaire on health history, sex, gender, demographics, experiences with COVID-19 acute and COVID-19 recovery/continuing symptoms. Serum samples collected were evaluated for antibody binding to viral proteins, virus neutralizing antibody titers, and serum cytokine levels using Ella SimplePlex Immunoassay™ panels. We found participants with PASC reported more pre-existing conditions (e.g. such as hypertension, asthma, and obesity), and PASC symptoms (e.g. fatigue, brain fog, headaches, and shortness of breath) following COVID-19 than COVID-19 Recovered individuals. Importantly, we found PASC individuals to have significantly decreased levels of neutralizing antibodies toward both SARS-CoV-2 and the Omicron BA.1 variant. Sex analysis indicated that female PASC study participants had sustained antibody levels as well as levels of the inflammatory cytokines GM-CSF and ANG-2 over time following COVID-19. Our study reports people experiencing PASC had lower levels of virus neutralizing antibodies; however, the results are limited by the collection time post-COVID-19 and post-vaccination. Moreover, we found females experiencing PASC had sustained levels of GM-CSF and ANG-2. With lower levels of virus neutralizing antibodies, this data suggests that PASC individuals not only have had a suboptimal antibody response during acute SARS-CoV-2 infection but may also have increased susceptibility to subsequent infections which may exacerbate or prolong current PASC illnesses. We also provide evidence suggesting GM-CSF and ANG-2 to play a role in the sex-bias of PASC. Taken together, our findings maybe important for understanding immune molecular drivers of PASC and PASC subgroups.

Longitudinal Analysis of Humoral and Cellular Immune Response up to 6 Months after SARS-CoV-2 BA.5/BF.7/XBB Breakthrough Infection and BA.5/BF.7-XBB Reinfection.

The rapid mutation of SARS-CoV-2 has led to multiple rounds of large-scale breakthrough infection and reinfection worldwide. However, the dynamic changes of humoral and cellular immunity responses to several subvariants after infection remain unclear. In our study, a 6-month longitudinal immune response evaluation was conducted on 118 sera and 50 PBMC samples from 49 healthy individuals who experienced BA.5/BF.7/XBB breakthrough infection or BA.5/BF.7-XBB reinfection. By studying antibody response, memory B cell, and IFN-γ secreting CD4+/CD8+ T cell response to several SARS-CoV-2 variants, we observed that each component of immune response exhibited distinct kinetics. Either BA.5/BF.7/XBB breakthrough infection or BA.5/BF.7-XBB reinfection induces relatively high level of binding and neutralizing antibody titers against Omicron subvariants at an early time point, which rapidly decreases over time. Most of the individuals at 6 months post-breakthrough infection completely lost their neutralizing activities against BQ.1.1, CH.1.1, BA.2.86, JN.1 and XBB subvariants. Individuals with BA.5/BF.7-XBB reinfection exhibit immune imprinting shifting and recall pre-existing BA.5/BF.7 neutralization antibodies. In the BA.5 breakthrough infection group, the frequency of BA.5 and XBB.1.16-RBD specific memory B cells, resting memory B cells, and intermediate memory B cells gradually increased over time. On the other hand, the frequency of IFN-γ secreting CD4+/CD8+ T cells induced by WT/BA.5/XBB.1.16 spike trimer remains stable over time. Overall, our research indicates that individuals with breakthrough infection have rapidly declining antibody levels but have a relatively stable cellular immunity that can provide some degree of protection from future exposure to new antigens.

Anti-Netrin-1 decorated nanoparticles combined with chemotherapy for the treatment of triple-negative breast cancer

Nanoparticle's success as drug delivery systems for cancer treatment has been achieved through passive targeting mechanisms. However, tumor heterogeneity and rapid drug clearance limit the treatment efficacy. Improved outcomes and selective drug release can be achieved by grafting ligands at the surface of nanocarriers that bind molecules overexpressed in the tumor microenvironment (TME). In this work, we developed a docetaxel-loaded nanoemulsions (NEs) binding an anti-netrin-1 monoclonal antibody (NP137) to selectively target the netrin-1 protein overexpressed in many different tumors. The goal is to refine a combined approach utilizing NP137 and docetaxel as an improved tumor-targeting chemotherapeutic agent for addressing triple-negative breast cancer (TNBC). Several factors have been considered for the optimization of the active targeted drug delivery system via the click-chemistry conjugation, as the impact of PEGylated surfactant that stabilize the NEs shell on conjugation efficiency, cytocompatibility with EMT6 cell line and colloidal stability over time of NEs. Results showed that a 660 Da PEG chain length contributed to NEs colloidal stability and had no impact on cell viability or on the antibody binding ability for its ligand after surface conjugation. Moreover, docetaxel was encapsulated into the oily core of NEs, with an encapsulation efficiency of 70 %. To validate our treatment strategy in vivo, the 4T1 murine breast cancer model was used. As a result, the comparison of active-targeted and non-targeted NEs revealed that only active-targeted NE could decrease the tumor growth rate.

Structural basis of Zika virus NS1 multimerization and human antibody recognition

Zika virus (ZIKV) belongs to the Flavivirus genus of the Flaviviridae family along with the four serotypes of dengue virus (DENV1–4). The recent global outbreaks of contemporary ZIKV strains demonstrated that infection can lead to neurological sequelae in adults and severe abnormalities in newborns that were previously unreported with ancestral strains. As such, there remains an unmet need for efficacious vaccines and antiviral agents against ZIKV. The non-structural protein 1 (NS1) is secreted from the infected cell and is thought to be associated with disease severity besides its proven usefulness for differential diagnoses. However, its physiologically relevant structure and pathogenesis mechanisms remain unclear. Here, we present high-resolution cryoEM structures of ZIKV recombinant secreted NS1 (rsNS1) and its complexes with three human monoclonal antibodies (AA12, EB9, GB5), as well as evidence for ZIKV infection-derived secreted NS1 (isNS1) binding to High Density Lipoprotein (HDL). We show that ZIKV rsNS1 forms tetramers and filamentous repeats of tetramers. We also observed that antibody binding did not disrupt the ZIKV NS1 tetramers as they bound to the wing and connector subdomain of the β-ladder. Our study reveals new insights into NS1 multimerization, highlights the need to distinguish the polymorphic nature of rsNS1 and isNS1, and expands the mechanistic basis of the protection conferred by antibodies targeting NS1.

Highly sensitive in-situ growth gold dendrite structure electrochemical sensor for early Alzheimer's disease screening

Beta-amyloid 42 (Aβ-42) peptide is an essential biomarker for Alzheimer's disease (AD) diagnosis. This study presents high sensitivity and stability sensors for Aβ-42 peptide detection, along with an integrated handheld analysis and display device. The sensors, with a wide response range and high accuracy, capable of measuring Aβ-42 peptide concentration at the femtogram level, could have enabled early screening for AD. Employing an in-situ grown three-dimensional sensor, the specific surface area of the sensor was increased by 200 % which, thereby, provided more antibody binding sites to enhance detection accuracy. This electrochemical sensor demonstrated a broad linear response range from 4.5 × 10−8 pg/mL to 4.5 × 102 pg/mL and could accurately determine the concentration of spiked analytes (including artificial saliva, human plasma, and human blood). Furthermore, the sensor exhibited satisfactory stability, with only a 5 % sensitivity loss after 35 days of storage at 4 °C. Additionally, this handheld Aβ-42 peptide testing device incorporates a display for result delivery. Such innovative technology could fill the gap in the market for portable testing instruments and could provide an efficient and reliable solution for early AD screening.

Peripherally-derived LGI1-reactive monoclonal antibodies cause epileptic seizures in vivo.

One striking clinical hallmark in patients with autoantibodies to leucine-rich glioma inactivated 1 (LGI1) is the very frequent focal seizure semiologies, including faciobrachial dystonic seizures (FBDS), in addition to the amnesia. Polyclonal serum IgGs have successfully modelled the cognitive changes in vivo but not seizures. Hence, it remains unclear whether LGI1-autoantibodies are sufficient to cause seizures. We tested this with the molecularly precise monoclonal antibodies directed against LGI1 (LGI1-mAbs), derived from patient circulating B cells. These were directed towards both major domains of LGI1, LRR and EPTP and infused intracerebroventricularly over 7 days into juvenile male Wistar rats using osmotic pumps. Continuous wireless EEG was recorded from a depth electrode placed in hippocampal CA3 plus behavioural tests for memory and hyperexcitability were performed. Following infusion completion (Day 9), post-mortem brain slices were studied for antibody binding and effects on Kv1.1. The LGI1-mAbs bound most strongly in the hippocampal CA3 region and induced a significant reduction in Kv1.1 cluster number in this subfield. By comparison to control-Ab injected rats video-EEG analysis over 9 days revealed convulsive and non-convulsive seizure activity in rats infused with LGI1-mAbs, with a significant number of ictal events. Memory was not impaired in the novel object recognition test. Peripherally-derived human LGI1-mAbs infused into rodent CSF provide strong evidence of direct in vivo epileptogenesis with molecular correlations. These findings fulfill criteria for LGI1-antibodies in seizure causation.

Safety and Immunogenicity of Accelerated Heterologous Two-dose Ebola Vaccine Regimens in Adults With and Without HIV in Africa.

Shorter prophylactic vaccine schedules may offer more rapid protection against Ebola in resource-limited settings. This randomized, observer-blind, placebo-controlled, phase 2 trial conducted in five sub-Saharan African countries included people without HIV (PWOH, n = 249) and people living with HIV (PLWH, n = 250). Adult participants received one of two accelerated Ebola vaccine regimens (MVA-BN-Filo, Ad26.ZEBOV administered 14 days apart [n = 79] or Ad26.ZEBOV, MVA-BN-Filo administered 28 days apart [n = 322]) or saline/placebo (n = 98). The primary endpoints were safety (adverse events [AEs]) and immunogenicity (Ebola virus [EBOV] glycoprotein-specific binding antibody responses). Binding antibody responders were defined as participants with a > 2.5-fold increase from baseline or the lower limit of quantification if negative at baseline. The mean age was 33.4 years, 52% of participants were female, and among PLWH, the median (interquartile range) CD4+ cell count was 560.0 (418.0-752.0) cells/μL. AEs were generally mild/moderate with no vaccine-related serious AEs or remarkable safety profile differences by HIV status. At 21 days post-dose 2, EBOV glycoprotein-specific binding antibody response rates in vaccine recipients were 99% for the 14-day regimen (geometric mean concentrations [GMCs]: 5168 enzyme-linked immunosorbent assay units (EU)/mL in PWOH; 2509 EU/mL in PLWH), and 98% for the 28-day regimen (GMCs: 6037 EU/mL in PWOH; 2939 EU/mL in PLWH). At 12 months post-dose 2, GMCs in PWOH and PLWH were 635 and 514 EU/mL, respectively, for the 14-day regimen and 331 and 360 EU/mL, respectively, for the 28-day regimen. Accelerated 14- and 28-day Ebola vaccine regimens were safe and immunogenic in PWOH and PLWH in Africa. NCT02598388.

Determination of optimum levels of binding antibody units (BAU) of new quantitative chemiluminescent immuno-assay (CLIA) in COVID-19 vaccinated volunteer blood donors

BackgroundFor assessment of COVID-19 vaccine efficacy, neutralization activity of anti-SARS-CoV-2 antibody is measured. This study was undertaken to determine optimum levels of binding antibody units (BAU/ml) in new quantitative chemiluminescent assay (CLIA) that corresponded to neutralizing potential (30% inhibition) of sVNT assay. Methods: Ninety-one blood samples were analyzed by CLIA and sVNT assays. Test samples (n = 75) were collected from blood donors post-2nd vaccination dose, while control samples (n = 16) were archived pre-COVID donor samples. Correlation between CLIA and sVNT was calculated and receiver operating characteristic (ROC) curve was drawn and analyzed. Results: Results indicated excellent correlation between 57.5 BAU/ml on CLIA and 30%inhibition on sVNT assay. ROC curve analysis revealed that the area under the curve (AUC) was 0.971. Discussion: The present study determined that 57.5 BAU/ml on CLIA corresponded to 30% inhibition on sVNT assay. Periodic quantitative analysis

Development of Detection Antibody Targeting the Linear Epitope in SARS-CoV-2 Nucleocapsid Protein with Ultra-High Sensitivity.

The COVID-19 pandemic caused by SARS-CoV-2 highlighted the importance of reliable detection methods for disease control and surveillance. Optimizing detection antibodies by rational screening antigens would improve the sensitivity and specificity of antibody-based detection methods such as colloidal gold immunochromatography. In this study, we screened three peptide antigens with conserved sequences in the N protein of SARS-CoV-2 using bioinformatical and structural biological analyses. Antibodies that specifically recognize these peptides were prepared. The epitope of the peptide that had the highest binding affinity with its antibody was located on the surface of the N protein, which was favorable for antibody binding. Using the optimal antibody that can recognize this epitope, we developed colloidal gold immunochromatography, which can detect the N protein at 10 pg/mL. Importantly, this antibody could effectively recognize both the natural peptide antigen and mutated peptide antigen in the N protein, showing the feasibility of being applied in the large-scale population testing of SARS-CoV-2. Our study provides a platform with reference significance for the rational screening of detection antibodies with high sensitivity, specificity, and reliability for SARS-CoV-2 and other pathogens.

M6ACali: machine learning-powered calibration for accurate m6A detection in MeRIP-Seq.

We present m6ACali, a novel machine-learning framework aimed at enhancing the accuracy of N6-methyladenosine (m6A) epitranscriptome profiling by reducing the impact of non-specific antibody enrichment in MeRIP-Seq. The calibration model serves as a genomic feature-based classifier that refines the identification of m6A sites, distinguishing those genuinely present from those that can be detected in in-vitro transcribed (IVT) control experiments. We find that m6ACali effectively identifies non-specific binding peaks reported by exomePeak2 and MACS2 in novel MeRIP-Seq datasets without the need for paired IVT controls. The model interpretation revealed that off-target antibody binding sites commonly occur at short exons and short mRNAs, originating from high read coverage regions that share the motif sequence with true m6A sites. We also reveal that the ML strategy can efficiently adjust differentially methylated peaks and other antibody-dependent, base-resolution m6A detection techniques. As a result, m6ACali offers a promising method for the universal enhancement of m6A profiles generated by MeRIP-Seq experiments, elevating the benchmark for omics-level m6A data integration.

Incidence of SARS-CoV-2 seropositivity in pediatric healthcare workers prior to widespread vaccination: A 5-month longitudinal cohort study

ObjectivesDetermine SARS-CoV-2 IgG antibody incidence over time in unvaccinated pediatric healthcare workers (pHCWs). DesignA prospective longitudinal cohort of unvaccinated pHCWs measuring the incidence of new infection after initial prevalence was established at 4.1% with seropositive predominance in emergency department (ED)-based pHCWs. Serum samples were collected at follow-up visits to detect new SARS-CoV-2 seropositivity. Univariate analysis was performed to estimate different incidence rates between participant demographics, job, employment location, and community risk factors. Anxiety levels about COVID-19 were collected. SARS-CoV-2 antibody decay postinfection and neutralization antibodies were evaluated. Log-linear Poisson regression models were used to estimate incidence. ResultsOf 642 initially enrolled, 390 pHCWs presented for at least one follow-up serology test after baseline analysis. The incidence of SARS-CoV-2 seropositivity was 8.2%. The seropositive cohort, like the negative one, consisted mainly of females in non-ED settings and nonphysician roles. There were no statistically significant differences in incidence across variables. Seropositive participants dropped antibody titers by 50% at 3 months. Neutralization antibodies correlated to SARS-CoV-2 binding antibodies (r = 0.43, P < 0.0001). ConclusionThe incidence of seropositivity was 8.2%. Although seropositivity was higher among ED staff during the early stages of the pandemic, this difference declined over time, likely due to the universal adoption of personal protective equipment.

Binding of Natural Antibodies Generated after COVID-19 and Vaccination with Individual Peptides Corresponding to the SARS-CoV-2 S-Protein.

The rapid development of vaccines is a crucial objective in modern biotechnology and molecular pharmacology. In this context, conducting research to expedite the selection of a potent immunogen is imperative. The candidate vaccine should induce the production of antibodies that can recognize the immunogenic epitopes of the target protein, resembling the ones found in recovered patients. One major challenge in vaccine development is the absence of straightforward and reliable techniques to determine the extent to which the spectrum of antibodies produced after vaccination corresponds to antibodies found after recovery. This paper describes a newly developed method to detect antibodies specific to immunogenic epitopes of the target protein in blood plasma and to compare them with antibody spectra generated post vaccination. Comparing the antibody pool generated in the human body after recovering from an infectious disease with the pool formed through vaccination can become a universal method for screening candidate vaccines. This method will enable the identification of candidate vaccines that can induce the production of antibodies similar to those generated in response to a natural infection. Implementing this approach will facilitate the rapid development of new vaccines, even when faced with a pandemic.

Higher correlation between neutralizing antibodies and surrogate neutralizing or binding antibodies in COVID-19 patients than vaccine recipients.

This study determined the seropositive rates and levels of antibodies to severe acute respiratory syndrome coronavirus-2 in 50 patients and 108 vaccinees using microneutralization test (MNT), surrogate virus neutralization test (sVNT), chemiluminescent microparticle immunoassay (CMIA), and electrochemiluminescence immunoassay (ECLIA). MNT, as the reference method, employed living clade S and Delta viruses to measure neutralizing (NT) antibodies, while sVNT employed wild type strain and Delta receptor-binding domains (RBD) as the test antigens to measure sVNT antibodies. CMIA and ECLIA employed only one version of RBD to measure the binding antibodies. Our study performed S gene sequencing of the test virus to exclude undesired mutants that might lead to changes in antibody levels in MNT assay. We showed that spike protein amino acid sequences of our Delta virus contained 13 amino acid changes, with 3 related to the reduced neutralization. The MNT assay showed a significant reduction in seropositive rates and antibody levels in the patients' sera when the Delta variant replaced clade S as the test virus. In contrast, the seropositive rates determined by sVNT assay using wild type strain RBD and Delta RBD were non-significantly different, suggesting that sVNT assay could not identify the difference between the antigenicity of wild type RBD and Delta RBD. Furthermore, the correlation between the levels of NT and sVNT antibodies was moderate with the patients' sera but modest with the post-vaccination sera. The seropositive rates in the patients, as determined by CMIA or ECLIA, were not different from the MNT assay using clade S, but not Delta, as the test virus. In all analyses, the correlations between the antibody levels measured by MNT and the other 3 assays were modest to moderate, with the r-values of 0.3500-0.7882.

The physiological interactome of TCR-like antibody therapeutics in human tissues.

Selective binding of TCR-like antibodies that target a single tumour-specific peptide antigen presented by human leukocyte antigens (HLA) is the absolute prerequisite for their therapeutic suitability and patient safety. To date, selectivity assessment has been limited to peptide library screening and predictive modeling. We developed an experimental platform to de novo identify interactomes of TCR-like antibodies directly in human tissues using mass spectrometry. As proof of concept, we confirm the target epitope of a MAGE-A4-specific TCR-like antibody. We further determine cross-reactive peptidesequences for ESK1, a TCR-like antibody with known off-target activity, in human liver tissue. We confirm off-target-induced T cell activation and ESK1-mediated liver spheroid killing. Off-target sequences feature an amino acid motif that allows a structural groove-coordination mimicking that of the target peptide, therefore allowing the interaction with the engager molecule. We conclude that our strategy offers an accurate, scalable route for evaluating the non-clinical safety profile of TCR-like antibody therapeutics prior to first-in-human clinical application.

A flow cytometry-based assay to measure neutralizing antibodies against SARS-CoV-2 virus.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has highlighted the need for serological assays that can accurately evaluate the neutralizing efficiency of antibodies produced during infection or induced by vaccines. However, conventional assays often require the manipulation of live viruses on a level-three biosafety (BSL3) facility, which presents practical and safety challenges. Here, we present a novel, alternative assay that measures neutralizing antibodies (NAbs) against SARS-CoV-2 in plasma using flow cytometry. This assay is based on antibody binding to the S protein and has demonstrated precision in both intra- and inter-assay measurements at a dilution of 1:50. The cut-off was determined using Receiver Operating Characteristic (ROC) analysis and the value of 36.01% has shown high sensitivity and specificity in distinguishing between pre-pandemic sera, COVID-19 patients, and vaccinated individuals. The efficiency significantly correlates with the gold standard test, PRNT. Our new assay offers a safe and efficient alternative to conventional assays for evaluating NAbs against SARS-CoV-2.

Walking a thin line between fixation and epitope binding - characterization of antigen retrieval methods suitable for eosinophil and HSV-2 staining in formalin-fixed female reproductive tissue.

Antibody-based fluorescence analysis of female reproductive tissues in research of sexually transmitted diseases allows for an in-depth understanding of protein localization, interactions, and pathogenesis. However, in many cases, cryosectioning is not compatible with biosafety regulations; at all times, exposure of lab personnel and the public to potentially harmful pathogens from biological infectious material must be avoided; thus, formaldehyde fixation is essential. Due to formaldehyde's cross-linking properties, protein detection with antibodies can be impeded. To allow effective epitope binding during immunofluorescence of formalin-fixed paraffin-embedded vaginal tissue, we investigated two antigen retrieval methods. We tested these methods regarding their suitability for automated image analysis, facilitating reproducible quantitative microscopic data acquisition in sexually transmitted disease research. Heat-based retrieval at 80°C in citrate buffer proved to increase antibody binding to eosinophil protein and HSV-2 visibly and tissue morphology best, and was the most efficient for sample processing and quantitative analysis.