Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states.

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

Anti-Drug Antibody Incidence Comparison of Therapeutic Proteins Administered Via Subcutaneous vs. Intravenous Route.

Subcutaneous (SC) administration of therapeutic proteins is perceived to pose higher risk of immunogenicity when compared with intravenous (IV) route of administration (RoA). However, systematic evaluations of clinical data to support this claim are lacking. This meta-analysis was conducted to compare the immunogenicity of the same therapeutic protein by IV and SC RoA. Anti-drug antibody (ADA) data and controlling variables for 7 therapeutic proteins administered by both IV and SC routes across 48 treatment groups were analyzed. RoA was the primary independent variable of interest while therapeutic protein, patient population, adjusted dose, and number of ADA samples were controlling variables. Analysis of variance was used to compare the ADA incidence between IV and SC RoA, while accounting for controlling variables and potential interactions. Subsequently, 10 additional therapeutic proteins with ADA data published for both IV and SC administration were added to the above 7 therapeutic proteins and were evaluated for ADA incidence. RoA had no statistically significant effect on ADA incidence for the initial dataset of 7 therapeutic proteins (p = 0.55). The only variable with a significant effect on ADA incidence was the therapeutic protein. None of the other controlling variables, including their interactions with RoA, was significant. When all data from the 17 therapeutic proteins were pooled, there was no statistically significant effect of RoA on ADA incidence (p = 0.81). In conclusion, there is no significant difference in ADA incidence between the IV and SC RoA, based on analysis of clinical ADA data from 17 therapeutic proteins.

Chromatin Immunoprecipitation of Retroviral Genomes with Antibodies Recognizing Modified Histones and Specific Viral Proteins.

Mammalian cells have developed and optimized defense mechanisms to prevent or hamper viral infection. The early transcriptional silencing of incoming viral DNAs is one such antiviral strategy and seems to be of fundamental importance, since most cell types silence unintegrated retroviral DNAs. In this chapter, a method for chromatin immunoprecipitation of unintegrated DNA is described. This technique allows investigators to examine histone and co-factor interactions with unintegrated viral DNAs as well as to analyze histone modifications in general or in a kinetic fashion at various time points during viral infection.

SERS biosensor with plastic antibodies for detection of a cancer biomarker protein.

Surface-enhanced Raman scattering (SERS) is a powerful method for detecting breast cancer-specific biomarkers due to its extraordinary enhancement effects obtained by localized surface plasmon resonance (LSPR) in metallic nanostructures at hotspots. In this research, gold nanostars (AuNSs) were used as SERS probes to detect a cancer biomarker at very low concentrations. To this end, we combined molecularly imprinted polymers (MIPs) as a detection layer with SERS for the detection of the biomarker CA 15-3 in point-of-care (PoC) analysis. This required two main steps: (i) the deposition of MIPs on a gold electrode, followed by a second step (ii) antibody binding with AuNSs containing a suitable Raman reporter to enhance Raman signaling (SERS). The MPan sensor was prepared by electropolymerization of the monomer aniline in the presence of CA 15-3. The template molecule was then extracted from the polymer using sodium dodecyl sulfate (SDS). In parallel, a control material was prepared in the absence of the protein (NPan). Surface modification for the control was performed using electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The performance of the sensor was evaluated using the SERS technique, in which the MPan sensor is first incubated with the protein and then exposed to the SERS probe. Under optimized conditions, the device showed a linear response to CA 15-3 concentrations from 0.016 to 248.51 U mL-1 in a PBS buffer at pH 7.4 in 1000-fold diluted serum. Overall, this approach demonstrates the potential of SERS as an optical reader and opens a new avenue for biosensing applications.

Brain white matter changes and their associations with non-motor dysfunction in orthostatic hypotension in α-synucleinopathy: A NODDI study.

BACKGROUND: The specific non-motor symptoms associated with α-synucleinopathies, including orthostatic hypotension (OH), cognitive impairment, and emotional abnormalities, have been a subject of ongoing controversy over the mechanisms underlying the development of a vicious cycle among them. The distinct structural alterations in white matter (WM) in patients with α-synucleinopathies experiencing OH, alongside their association with other non-motor symptoms, remain unexplored. This study employs axial diffusivity and density imaging (NODDI) to investigate WM damage specific to α-synucleinopathies with concurrent OH, delivering fresh evidence to supplement our understanding of the pathogenic mechanisms and pathological rationales behind the occurrence of a spectrum of non-motor functional impairments in α-synucleinopathies. METHODS: This study recruited 49 individuals diagnosed with α-synucleinopathies, stratified into an α-OH group (n = 24) and an α-NOH group (without OH, n = 25). Additionally, 17 healthy controls were included for supine and standing blood pressure data collection, as well as neuropsychological assessments. Magnetic resonance imaging (MRI) was utilized for the calculation of NODDI parameters, and tract-based spatial statistics (TBSS) were employed to explore differential clusters. The fibers covered by these clusters were defined as regions of interest (ROI) for the extraction of NODDI parameter values and the analysis of their correlation with neuropsychological scores. RESULTS: The TBSS analysis unveiled specific cerebral regions exhibiting disparities within the α-OH group as compared to both the α-NOH group and the healthy controls. These differences were evident in clusters that indicated a decrease in the acquisition of the neurite density index (NDI), a reduction in the orientation dispersion index (ODI), and an increase in the isotropic volume fraction (FISO) (p < 0.05). The extracted values from these ROIs demonstrated significant correlations with clinically assessed differences in supine and standing blood pressure, overall cognitive scores, and anxiety-depression ratings (p < 0.05). CONCLUSION: Patients with α-synucleinopathies experiencing OH exhibit distinctive patterns of microstructural damage in the WM as revealed by the NODDI model, and there is a correlation with the onset and progression of non-motor functional impairments.

Comparative performance of scFv-based anti-BCMA CAR formats for improved T cell therapy in multiple myeloma.

In multiple myeloma (MM), B cell maturation antigen (BCMA)-directed CAR T cells have emerged as a novel therapy with potential for long-term disease control. Anti-BCMA CAR T cells with a CD8-based transmembrane (TM) and CD137 (41BB) as intracellular costimulatory domain are in routine clinical use. As the CAR construct architecture can differentially impact performance and efficacy, the optimal construction of a BCMA-targeting CAR remains to be elucidated. Here, we hypothesized that varying the constituents of the CAR structure known to impact performance could shed light on how to improve established anti-BCMA CAR constructs. CD8TM.41BBIC-based anti-BCMA CAR vectors with either a long linker or a short linker between the light and heavy scFv chain, CD28TM.41BBIC-based and CD28TM.CD28IC-based anti-BCMA CAR vector systems were used in primary human T cells. MM cell lines were used as target cells. The short linker anti-BCMA CAR demonstrated higher cytokine production, whereas in vitro cytotoxicity, T cell differentiation upon activation and proliferation were superior for the CD28TM.CD28IC-based CAR. While CD28TM.CD28IC-based CAR T cells killed MM cells faster, the persistence of 41BBIC-based constructs was superior in vivo. While CD28 and 41BB costimulation come with different in vitro and in vivo advantages, this did not translate into a superior outcome for either tested model. In conclusion, this study showcases the need to study the influence of different CAR architectures based on an identical scFv individually. It indicates that current scFv-based anti-BCMA CAR with clinical utility may already be at their functional optimum regarding the known structural variations of the scFv linker.

Validation of GCN5L1/BLOC1S1/BLOS1 antibodies using knockout cells and tissue.

GCN5L1, also known as BLOC1S1 and BLOS1, is a small intracellular protein involved in many key biological processes. Over the last decade, GCN5L1 has been implicated in the regulation of protein lysine acetylation, energy metabolism, endo-lysosomal function, and cellular immune pathways. An increasing number of published papers have used commercially-available reagents to interrogate GCN5L1 function. However, in many cases these reagents have not been rigorously validated, leading to potentially misleading results. In this report we tested several commercially-available antibodies for GCN5L1, and found that two-thirds of those available did not unambiguously detect the protein by western blot in cultured mouse cells or ex vivo liver tissue. These data suggest that previously published studies which used these unverified antibodies to measure GCN5L1 protein abundance, in the absence of other independent methods of corroboration, should be interpreted with appropriate caution.

A general approach for selection of epitope-directed binders to proteins.

Directing antibodies to a particular epitope among many possible on a target protein is a significant challenge. Here, we present a simple and general method for epitope-directed selection (EDS) using a differential phage selection strategy. This involves engineering the protein of interest (POI) with the epitope of interest (EOI) mutated using a systematic bioinformatics algorithm to guide the local design of an EOI decoy variant. Using several alternating rounds of negative selection with the EOI decoy variant followed by positive selection on the wild-type POI, we were able to identify highly specific and potent antibodies to five different EOI antigens that bind and functionally block known sites of proteolysis. Among these, we developed highly specific antibodies that target the proteolytic site on the CUB domain containing protein 1 (CDCP1) to prevent its proteolysis allowing us to study the cellular maturation of this event that triggers malignancy. We generated antibodies that recognize the junction between the pro- and catalytic domains for three different matrix metalloproteases (MMPs), MMP1, MMP3, and MMP9, that selectively block activation of each of these enzymes and impair cell migration. We targeted a proteolytic epitope on the cell surface receptor, EPH Receptor A2 (EphA2), that is known to transform it from a tumor suppressor to an oncoprotein. We believe that the EDS method greatly facilitates the generation of antibodies to specific EOIs on a wide range of proteins and enzymes for broad therapeutic and diagnostic applications.

Biosensing-based quality control monitoring of the higher-order structures of therapeutic antibody domains.

Advanced biopharmaceutical manufacturing requires novel process analytical technologies for the rapid and sensitive assessment of the higher-order structures of therapeutic proteins. However, conventional physicochemical analyses of denatured proteins have limitations in terms of sensitivity, throughput, analytical resolution, and real-time monitoring capacity. Although probe-based sensing can overcome these limitations, typical non-specific probes lack analytical resolution and provide little to no information regarding which parts of the protein structure have been collapsed. To meet these analytical demands, we generated biosensing probes derived from artificial proteins that could specifically recognize the higher-order structural changes in antibodies at the protein domain level. Biopanning of phage-displayed protein libraries generated artificial proteins that bound to a denatured antibody domain, but not its natively folded structure, with nanomolar affinity. The protein probes not only recognized the higher-order structural changes in intact IgGs but also distinguished between the denatured antibody domains. These domain-specific probes were used to generate response contour plots to visualize the antibody denaturation caused by various process parameters, such as pH, temperature, and holding time for acid elution and virus inactivation. These protein probes can be combined with established analytical techniques, such as surface plasmon resonance for real-time monitoring or plate-based assays for high-throughput analysis, to aid in the development of new analytical technologies for the process optimization and monitoring of antibody manufacturing.

Novel method for classification of prion diseases by detecting PrPres signal patterns from formalin-fixed paraffin-embedded samples.

Prion disease is an infectious and fatal neurodegenerative disease. Western blotting (WB)-based identification of proteinase K (PK)-resistant prion protein (PrPres) is considered a definitive diagnosis of prion diseases. In this study, we aimed to detect PrPres using formalin-fixed paraffin-embedded (FFPE) specimens from cases of sporadic Creutzfeldt-Jakob disease (sCJD), Gerstmann-Sträussler-Scheinker disease (GSS), glycosylphosphatidylinositol-anchorless prion disease (GPIALP), and V180I CJD. FFPE samples were prepared after formic acid treatment to inactivate infectivity. After deparaffinization, PK digestion was performed, and the protein was extracted. In sCJD, a pronounced PrPres signal was observed, with antibodies specific for type 1 and type 2 PrPres exhibited a strong or weak signals depending on the case. Histological examination of serial sections revealed that the histological changes were compatible with the biochemical characteristics. In GSS and GPIALP, prion protein core-specific antibodies presented as PrPres bands at 8-9 kDa and smear bands, respectively. However, an antibody specific for the C-terminus presented as smears in GSS, with no PrPres detected in GPIALP. It was difficult to detect PrPres in V180I CJD. Collectively, our findings demonstrate the possibility of detecting PrPres in FFPE and classifying the prion disease types. This approach facilitates histopathological and biochemical evaluation in the same sample and is safe owing to the inactivation of infectivity. Therefore, it may be valuable for the diagnosis and research of prion diseases.

Can antibodies be "vegan"? A guide through the maze of today's antibody generation methods.

There is no doubt that today's life sciences would look very different without the availability of millions of research antibody products. Nevertheless, the use of antibody reagents that are poorly characterized has led to the publication of false or misleading results. The use of laboratory animals to produce research antibodies has also been criticized. Surprisingly, both problems can be addressed with the same technology. This review charts today's maze of different antibody formats and the various methods for antibody production and their interconnections, ultimately concluding that sequence-defined recombinant antibodies offer a clear path to both improved quality of experimental data and reduced use of animals.

SARS-CoV-2 booster vaccine dose significantly extends humoral immune response half-life beyond the primary series.

SARS-CoV-2 lipid nanoparticle mRNA vaccines continue to be administered as the predominant prophylactic measure to reduce COVID-19 disease pathogenesis. Quantifying the kinetics of the secondary immune response from subsequent doses beyond the primary series and understanding how dose-dependent immune waning kinetics vary as a function of age, sex, and various comorbidities remains an important question. We study anti-spike IgG waning kinetics in 152 individuals who received an mRNA-based primary series (first two doses) and a subset of 137 individuals who then received an mRNA-based booster dose. We find the booster dose elicits a 71-84% increase in the median Anti-S half life over that of the primary series. We find the Anti-S half life for both primary series and booster doses decreases with age. However, we stress that although chronological age continues to be a good proxy for vaccine-induced humoral waning, immunosenescence is likely not the mechanism, rather, more likely the mechanism is related to the presence of noncommunicable diseases, which also accumulate with age, that affect immune regulation. We are able to independently reproduce recent observations that those with pre-existing asthma exhibit a stronger primary series humoral response to vaccination than compared to those that do not, and further, we find this result is sustained for the booster dose. Finally, via a single-variate Kruskal-Wallis test we find no difference between male and female humoral decay kinetics, however, a multivariate approach utilizing  Least Absolute Shrinkage and Selection Operator (LASSO) regression for feature selection reveals a statistically significant (p < 1 × 10 - 3 ), albeit small, bias in favour of longer-lasting humoral immunity amongst males.

Adaptive immune responses to two-dose COVID-19 vaccine series in healthy Canadian adults ≥ 50 years: a prospective, observational cohort study.

To evaluate immune responses to COVID-19 vaccines in adults aged 50 years and older, spike protein (S)-specific antibody concentration, avidity, and function (via angiotensin-converting enzyme 2 (ACE2) inhibition surrogate neutralization and antibody dependent cellular phagocytosis (ADCP)), as well as S-specific T cells were quantified via activation induced marker (AIM) assay in response to two-dose series. Eighty-four adults were vaccinated with either: mRNA/mRNA (mRNA-1273 and/or BNT162b2); ChAdOx1-S/mRNA; or ChAdOx1-S/ChAdOx1-S. Anti-S IgG concentrations, ADCP scores and ACE2 inhibiting antibody concentrations were highest at one-month post-second dose and declined by four-months post-second dose for all groups. mRNA/mRNA and ChAdOx1-S/mRNA schedules had significantly higher antibody responses than ChAdOx1-S/ChAdOx1-S. CD8+ T-cell responses one-month post-second dose were associated with increased ACE2 surrogate neutralization. Antibody avidity (total relative avidity index) did not change between one-month and four-months post-second dose and did not significantly differ between groups by four-months post-second dose. In determining COVID-19 correlates of protection, a measure that considers both antibody concentration and avidity should be considered.

Immune response stability to the SARS-CoV-2 mRNA vaccine booster is influenced by differential splicing of HLA genes.

Many molecular mechanisms that lead to the host antibody response to COVID-19 vaccines remain largely unknown. In this study, we used serum antibody detection combined with whole blood RNA-based transcriptome analysis to investigate variability in vaccine response in healthy recipients of a booster (third) dose schedule of the mRNA BNT162b2 vaccine against COVID-19. The cohort was divided into two groups: (1) low-stable individuals, with antibody concentration anti-SARS-CoV IgG S1 below 0.4 percentile at 180 days after boosting vaccination; and (2) high-stable individuals, with antibody values greater than 0.6 percentile of the range in the same period (median 9525 [185-80,000] AU/mL). Differential gene expression, expressed single nucleotide variants and insertions/deletions, differential splicing events, and allelic imbalance were explored to broaden our understanding of the immune response sustenance. Our analysis revealed a differential expression of genes with immunological functions in individuals with low antibody titers, compared to those with higher antibody titers, underscoring the fundamental importance of the innate immune response for boosting immunity. Our findings also provide new insights into the determinants of the immune response variability to the SARS-CoV-2 mRNA vaccine booster, highlighting the significance of differential splicing regulatory mechanisms, mainly concerning HLA alleles, in delineating vaccine immunogenicity.

Comparison of Brn3a and RBPMS Labeling to Assess Retinal Ganglion Cell Loss During Aging and in a Model of Optic Neuropathy.

Purpose: Retinal ganglion cell (RGC) loss provides the basis for diagnosis and stage determination of many optic neuropathies, and quantification of RGC survival is a critical outcome measure in models of optic neuropathy. This study examines the accuracy of manual RGC counting using two selective markers, Brn3a and RBPMS. Methods: Retinal flat mounts from 1- to 18-month-old C57BL/6 mice, and from mice after microbead (MB)-induced intraocular pressure (IOP) elevation, are immunostained with Brn3a and/or RBPMS antibodies. Four individuals masked to the experimental conditions manually counted labeled RGCs in three copies of five images, and inter- and intra-person reliability was evaluated by the intraclass correlation coefficient (ICC). Results: A larger population (approximately 10% higher) of RGCs are labeled with RBPMS than Brn3a antibody up to 6 months of age, but differences decrease to approximately 1% at older ages. Both RGC-labeled populations significantly decrease with age. MB-induced IOP elevation is associated with a significant decrease of both Brn3a- and RBPMS-positive RGCs. Notably, RGC labeling with Brn3a provides more consistent cell counts than RBPMS in interpersonal (ICC = 0.87 to 0.11, respectively) and intra-personal reliability (ICC = 0.97 to 0.66, respectively). Conclusions: Brn3a and RBPMS markers are independently capable of detecting significant decreases of RGC number with age and in response to IOP elevation despite RPBMS detecting a larger number of RGCs up to 6 months of age. Brn3a labeling is less prone to manual cell counting variability than RBPMS labeling. Overall, either marker can be used as a single marker to detect significant changes in RGC survival, each offering distinct advantages.

Immunological and molecular diagnostic techniques in fish health: present and future prospectus.

Fish health management is critical to aquaculture and fisheries as it directly affects sustainability and productivity. Fish disease diagnosis has taken a massive stride because of advances in immunological and molecular diagnostic tools which provide a sensitive, quick, and accurate means of identifying diseases. This review presents an overview of the main molecular and immunological diagnostic methods for determining the health of fish. The immunological techniques help to diagnose different fish diseases by detecting specific antigens and antibodies. The application of immunological techniques to vaccine development is also examined in this review. The genetic identification of pathogens is made possible by molecular diagnostic techniques that enable the precise identification of bacterial, viral, and parasitic organisms in addition to evaluating host reactions and genetic variation associated with resistance to disease. The combination of molecular and immunological methods has resulted in the creation of novel techniques for thorough evaluation of fish health. These developments improve treatment measures, pathogen identification and provide new information about the variables affecting fish health, such as genetic predispositions and environmental stresses. In the framework of sustainable fish farming and fisheries management, this paper focuses on the importance of these diagnostic techniques that play a crucial role in protecting fish populations and the aquatic habitats. This review also examines the present and potential future directions in immunological and molecular diagnostic techniques in fish health.

One-pot green synthesis of ZIF-8/IgG composite for the precise orientation and protection of antibody and its application in purification and detection of aflatoxins in peanut oil.

This study presents a novel approach toward the one-pot green synthesis of ZIF-8/IgG composite, focusing on its precise orientation and protection of the anti-aflatoxins antibody. The antibody orientation is achieved through the specific binding of IgG to the Fc region of the antibody, while the antibody protection is accomplished by the structural change restriction of ZIF-8 framework to the antibody. Consequently, the antibody exhibits enhanced target capability and significantly improved tolerance to organic solvents. The ZIF-8/IgG/anti-AFT was employed for the purification and detection of AFTs by coupling with UPLC. Under optimized conditions, the recoveries of spiked AFTs in peanut oils are between 86.1% and 106.4%, with relative standard deviations (RSDs) ranging from 0.8% to 8.8%. The linearity range is 0.5-20.0 ng for AFB1 and AFG1, 0.125-5.0 ng for AFB2 and AFG2, the limit of detection is 0.1 ng for AFB1 and AFG1, 0.03 ng for AFB2 and AFG2.

Hotspot sheep scab management: A community-based regional approach in England.

BACKGROUND: Ovine psoroptic mange (sheep scab), caused by an infestation of the mite Psoroptes ovis, leads to clinical disease, economic loss and severely compromised animal welfare. Here, a community-based approach to the management of scab in three high-risk areas of England is described. METHODS: For each of the 254 farms included in the study, an initial survey of their clinical sheep scab history was followed up by a blood test (ELISA) to detect the presence of antibodies to P. ovis. This facilitated the coordination of treatment across groups of farms in each region. Blood testing was then repeated at the end of the treatment programme. RESULTS: On the first blood test in 2021/2022, 25.6% (±5.5%) of the flocks were positive for sheep scab. On the second test in 2022/2023, 9% (±3.94%) of the flocks tested were positive, showing a highly statistically significant reduction in prevalence overall, but with strong regional variation. LIMITATIONS: generating an understanding of the flock-level nature of the blood test and confidence in its detection of scab where clinical signs were not apparent provided ongoing challenges. CONCLUSIONS: The programme demonstrated that a focused community-based approach can be used to significantly reduce the prevalence of sheep scab in high-risk areas of England. The use of the blood test on all farms allowed the identification of subclinical sheep scab. The programme provides an effective model for sheep scab management on a national scale.

For antibody sequence generative modeling, mixture models may be all you need.

MOTIVATION: Antibody therapeutic candidates must exhibit not only tight binding to their target but also good developability properties, especially low risk of immunogenicity. RESULTS: In this work, we fit a simple generative model, SAM, to sixty million human heavy and seventy million human light chains. We show that the probability of a sequence calculated by the model distinguishes human sequences from other species with the same or better accuracy on a variety of benchmark datasets containing >400 million sequences than any other model in the literature, outperforming large language models (LLMs) by large margins. SAM can humanize sequences, generate new sequences, and score sequences for humanness. It is both fast and fully interpretable. Our results highlight the importance of using simple models as baselines for protein engineering tasks. We additionally introduce a new tool for numbering antibody sequences which is orders of magnitude faster than existing tools in the literature. AVAILABILITY AND IMPLEMENTATION: All tools developed in this study are available at https://github.com/Wang-lab-UCSD/AntPack.

Specific features of a scaffolding antibody light chain.

The antigen-binding sites in conventional antibodies are formed by hypervariable complementarity-determining regions (CDRs) from both heavy chains (HCs) and light chains (LCs). A deviation from this paradigm is found in a subset of bovine antibodies that bind antigens via an ultra-long CDR. The HCs bearing ultra-long CDRs pair with a restricted set of highly conserved LCs that convey stability to the antibody. Despite the importance of these LCs, their specific features remained unknown. Here, we show that the conserved bovine LC found in antibodies with ultra-long CDRs exhibits a distinct combination of favorable physicochemical properties such as good secretion from mammalian cells, strong dimerization, high stability, and resistance to aggregation. These physicochemical traits of the LCs arise from a combination of the specific sequences in the germline CDRs and a lambda LC framework. In addition to understanding the molecular architecture of antibodies with ultra-long CDRs, our findings reveal fundamental insights into LC characteristics that can guide the design of antibodies with improved properties.