Nonspecific Antibody Research Articles

Successful treatment of pulmonary hypertension and right heart failure by antibody-based functional blocking of the extra-domain A of fibronectin in a preclinical animal model

Abstract Background Pulmonary Hypertension (PH) as heterogeneous disease group is pathogenetically accompanied by pulmonary vascular and right ventricular tissue remodelling showing similarities between the clinical groups 1 (Pulmonary Arterial Hypertension), 2 (PH associated with left heart disease) and 3 (PH associated with lung disease). Since the remodelling process entails the abundant re-expression of extra-domain A containing fibronectin (ED-A+ Fn), which is virtually absent in healthy adult organs, a potential functional role in PH development and progression has been suggested and recently proven by our group. The human recombinant antibody F8 (IgG) specifically recognises ED-A+ Fn and is capable to block its function at least in vitro. Thus, F8 might serve as a novel therapeutic agent for the treatment or even prevention of PH. Purpose Against that background, the current study was aimed to test the therapeutic efficacy of F8 after systemic administration in a well-established preclinical model of PH in comparison to a non-specific control antibody and a dual endothelin receptor antagonist as standard of care. Methods Induction of PH was realized by subcutaneous injection of Monocrotaline (MCT) in mice. In total, 41 animals were included in the study approach and were divided into the following experimental groups: sham-treated controls (controls; n=7), PH mice without a specific treatment (PH_untreated; n=12), PH mice treated with the dual endothelin receptor antagonist Macitentan (PH_MAC; n=6), PH mice treated with the F8 antibody (PH_F8; n=8) and PH mice treated with an antibody of irrelevant antigen specificity in the murine system (PH_KSF; n=8). Treatment effects were evaluated on the haemodynamic, echocardiographic and microscopic level. Results In comparison to controls, untreated PH mice showed significantly higher right ventricular systolic pressure values (RVPsys, p<0.05) as well as impaired echocardiographic parameters of right ventricular morphology and function, i.e., enlarged basal right ventricular (RVbasal, p=0.016) diameters and reduced tricuspid annular plane systolic excursion values (TAPSE, p=0.008). In the PH_F8 group, haemodynamic and echocardiographic parameters were significantly improved compared to the PH_untreated or the PH_KSF group (p<0.05 for the majority of parameters). Histological evaluation revealed significant lung tissue damage after in the PH_untreated group compared to controls (p=0.008), which were significantly attenuated in the PH_F8 (p=0.04) but not the PH_KSF group (p=n.s.). Conclusion The therapeutic efficacy of the human recombinant F8 antibody specific to ED-A+ Fn has been proven for the first time in vivo in a well-established preclinical model of PH. F8 acts as a disease-modifier by attenuating PH associated lung tissue remodelling resulting in remarkable improvements of pulmonary hemodynamics and echocardiographic parameters of right ventricular load.

B-087 The Antifouling Properties of BSA on Magnetic Beads are Affected by BSA Grade, Bead Type, and pH

Abstract Background BSA (bovine serum albumin) is a protein that is widely used for its antifouling properties in a variety of immunoassays. One such immunoassay is a bead-based ELISA (enzyme-linked immunosorbent assay), which can provide quantitative detection of many diverse analytes. BSA increases the sensitivity of a bead-based ELISA by coating the beads and blocking nonspecific interactions, thereby lowering background noise. However, the BSA grade chosen for the assay is frequently based on experience, as there is a lack of understanding of how BSA grades interact with different bead surfaces under various assay conditions. The objective of this study is to examine if different BSA grades work better with particular bead chemistries and how pH influences those interactions. Specifically, the aim is to quantify the amount of BSA on ELISA beads and assess the blocking of nonspecific antibody signal with different bead types, BSA grades, and pH. Methods The mass of BSA binding to beads was measured by heating BSA-coated beads in 1x SDS loading dye, then running on a denaturing PAGE gel. Total protein was measured by densitometry against a standard curve using ImageJ. The amount of blocking was measured using a modified bead-based ELISA, comparing the amount of antibody-HRP binding to the beads in the presence and absence of BSA. The amount of antibody-HRP was measured using a SpectraMax Plus 384 plate reader against a standard curve. All data were analyzed using GraphPad Prism and presented as the average of at least three independent replicates with error bars representing standard deviation. Conditions tested include different Dynabeads (hydrophilic vs. hydrophobic - Dynabeads M-270 Carboxylic Acid and M-280 Tosylactivated), BSA grade (fatty acid (FA) vs. fatty acid-free (FAF) - Proliant SKUs 68100 and 68700), and pH (7 vs. 5.2). Results The choice of pH is more influential than the BSA grade on bead surface coating and blocking, regardless of bead type. Lowering the pH from 7 to 5.2 increases the mass of BSA on the beads ∼1.7-fold for hydrophobic beads and ∼10-fold for hydrophilic beads. However, the mass of BSA on the beads is reduced after the pH returns to 7, indicating the increased BSA binding at pH 5.2 is reversible. We also observed BSA decreases nonspecific antibody-bead interactions by up to 90%, depending on assay conditions. Though the amount of BSA coating the beads between FA and FAF grade BSA is not significant, FAF grade BSA blocks the antibody-bead interactions ∼10% more than FA grade BSA, suggesting FAF grade BSA has better antifouling properties. Conclusion Herein, we show the presence of BSA reduces the noise within bead-based ELISAs by binding to the bead and blocking nonspecific antibody-bead interactions, regardless of BSA grade, bead type, and pH. FAF grade BSA demonstrates slightly better blocking than FA grade BSA, but both BSA grades decreased nonspecific antibody-bead interactions. Further, BSA shows antifouling properties at the typical assay pH of 7, but lowering the pH to 5.2 can significantly increase BSA bead coating.

DFO-Km: A Modular Chelator as a New Chemical Tool for the Construction of Zirconium-89-Based Radiopharmaceuticals.

Zirconium-89-labeled monoclonal antibodies and other large macromolecules such as nanoparticles hold great promise as positron emission tomography imaging agents. In general, zirconium-89 is an ideal radionuclide for long-circulating vectors such as antibodies or nanoparticles. It is also a promising radionuclide for theranostic radiopharmaceuticals due to its suitable match in half-life with actinium-225, thorium-227, lutetium-177, and others. As such, demand for new and optimized bifunctional chelators for zirconium-89 continues to grow. Herein, we present the modular chelator DFO-Km, which is octadentate and features lysine as a modular amino acid linker. The modular amino acid linker can be changed to other natural or unnatural amino acids to access different bioconjugation chemistries, while the chelating portion is unchanged thus retaining identical metal ion coordination properties to DFO-Km. The epsilon-amine in the DFO-Km linker (lysine) was used to complete synthesis of a bifunctional derivative bearing a p-SCN-Ph moiety. The chelator DFO-Km includes a redesigned hydroxamic acid, which provides more flexibility for metal ion coordination relative to the monomer used in the previously published DFO-Em. Moreover, a set of comprehensive DFT calculations were performed to model and evaluate 16 geometric isomers of Zr-(DFO-Km), which suggested the complex would form the optimum cic-cis-trans-trans octadentate Zr(IV) coordination geometry with no aqua or hydroxide ligands present. The bifunctional derivative p-SCN-Ph-DFO-Km was compared directly with the commercially available p-SCN-Ph-DFO, and both underwent efficient conjugation to a nonspecific human serum antibody (IgG) to yield two model immunoconjugates. The behavior of [89Zr]Zr-DFO-Km-IgG was studied in healthy mice for 2 weeks and compared to an equivalent cohort injected with [89Zr]Zr-DFO-IgG as a clinical "gold standard" control. PET-CT and biodistribution results revealed higher stability of [89Zr]Zr-(DFO-Km)-IgG in vivo over [89Zr]Zr-DFO-IgG, as demonstrated by the significant reduction of zirconium-89 in the whole skeleton as visualized and quantified by PET-CT at 1, 3, 7, and 14 days post-injection. Using CT-gated regions of interest over these PET-CT images, the whole skeleton was selected and uptake values were measured at 14 days post-injection of 3.6 ± 0.9 (DFO) vs 1.9 ± 0.1 (DFO-Km) %ID/g (n = 4, * p = 0.02), which represents a ∼48% reduction in bone uptake with DFO-Km relative to DFO. Biodistribution experiments performed on these same mice following the 14 day imaging time point revealed bone (both tibia) uptake values of 3.7 ± 1.3 (DFO) vs 2.0 ± 0.6 (DFO-Km) %ID/g (n = 6, * p < 0.05), with the tibia uptake values in close agreement with whole-skeleton ROI PET-CT data. These results indicate that DFO-Km is an improved chelator for [89Zr]Zr4+ applications relative to DFO. The bifunctional chelator p-SCN-Ph-DFO-Km shows potential as a new chemical tool for creating bioconjugates using targeting vectors such as antibodies, peptides, and nanoparticles.

Cerebrospinal fluid biomarkers in psychiatric autoimmune encephalitis: a retrospective cohort study.

Psychiatric autoimmune encephalitis (pAE) is a growing field of interest in diagnosis and therapy in psychiatric hospitals and institutions. This study investigates the relevant extent to which there are potential biomarkers in cerebrospinal fluid (CSF) that can differentiate against a cohort with neurodegenerative disease. We included in this study a total of 27 patients with possible and definite psychiatric autoimmune encephalitis and compared with a cohort with CSF-based AD (n = 27) different biomarkers in CSF such as lactate, cell count, % lymphocytes, % monocytes, total protein content, albumin, immunoglobulins G (IgG), M (IgM) and A (IgA), CSF/serum albumin ratio, CSF/serum IgG ratio, CSF/serum IgA ratio, intrathecal IgG synthesis, blood-brain barrier disruption, specific antibody synthesis for measles, rubella, herpes simplex virus, varicella zoster virus, Ebstein-Barr virus and cytomegalovirus, total tau protein (t-tau), phosphorylated tau protein 181 (p-tau181), amyloid beta 42 (Aß42), amyloid beta 40 (Aß40) and the amyloid beta 42/ amyloid beta 40 (Aß42/40) ratio. The p-tau 181 was elevated above cut-off values in both possible pAE and AD. However, in definitive pAE, p-tau181 levels were not elevated. When elevated p-tau181 levels in possible AE were compared with those in AD, we found relevant differences, such as a relative increase in p-tau181 in AD patients. Elevated p-tau181 levels were detected in possible psychiatric AEs with IgLON5, glycine, recoverin, titin, and nonspecific neuropil antibodies in serum and IgLON5, titin, Yo, and nonspecific neuropil autoantibodies in CSF. In addition, we detected elevated levels of p-tau181 and IgLON5 autoantibodies in serum and CSF, and Yo autoantibodies in CSF in patients with definitive pAE. Interestingly, we observed a higher CSF/serum IgM ratio in possible and definitive pAE than in AD patients. Our results suggest that neuroaxonal brain damage may occur in specific psychiatric AEs associated with IgLON5, glycine, recoverin, and titin autoantibodies. Further research should focus on the CSF/serum IgM ratio as an early marker of autoantibody production in pAE compared to AD as a potential biomarker for differential diagnosis.

Substantiation for the relationship and «closed loop effect» between post-traumatic stress disorder and recurrent pregnancy loss

The objective: to identify and evaluate the changes in immune homeostasis in women with recurrent pregnancy loss (RPL) who had posttraumatic stress disorder (PTSD), and to establish the relationship between psycho-emotional status, the immune system and the role of chronic stress in the pathogenesis of autoimmune disorders as a cause of RPL.Materials and methods. An examination of 150 women of reproductive age was carried out, who were divided into three groups: I group (main group) – 100 patients with RPL and PTSD; II group (comparison one) – patients with RPL without PTSD (30 women) and the III group (control one) – 20 patients with PTSD who are planning pregnancy.Indicators of cellular immunity were studied and antiphospholipid syndrome was diagnosed. The electronic database of primary data was created using the Microsoft Excel 2016 spreadsheet program. Statistical data analysis was performed using the Statistica10.0 statistical analysis package.Results. Based on the results of the screening it was found that there are non-specific antibodies to anionic phospholipids, also known as antiphospholipid antibodies, in some patients of the main group. In the presence of antiphospholipid antibodies, the number of NK cells (CD3-CD16/56+) doubled, increasing the probability of the pregnancy interruption that had already occurred. There was also an increased number of NK cells (CD3-CD16/56+) in the blood of patients with PTSD compared to the control group – the patients with PTSD who are planning pregnancy.Conclusions. The results of the study confirmed the connection between PTSD and RPL and demonstrated significant immune homeostasis disorders, as well as the structural and morphological changes in the reproductive system.Women with RPL may have PTSD, which affects the pathogenesis of autoimmune disorders and RPL. These results lead to more deep understanding of RPL and will help in the development of new preventive and therapeutic measures.

Estrogen receptor beta expression in triple negative breast cancers is not associated with recurrence or survival

BackgroundTriple negative BCa (TNBC) is defined by a lack of expression of estrogen (ERα), progesterone (PgR) receptors and human epidermal growth factor receptor 2 (HER2) as assessed by protein expression and/or gene amplification. It makes up ~ 15% of all BCa and often has a poor prognosis. TNBC is not treated with endocrine therapies as ERα and PR negative tumors in general do not show benefit. However, a small fraction of the true TNBC tumors do show tamoxifen sensitivity, with those expressing the most common isoform of ERβ1 having the most benefit. Recently, the antibodies commonly used to assess ERβ1 in TNBC have been found to lack specificity, which calls into question available data regarding the proportion of TNBC that express ERβ1 and any relationship to clinical outcome.MethodsTo confirm the true frequency of ERβ1 in TNBC we performed robust ERβ1 immunohistochemistry using the specific antibody CWK-F12 ERβ1 on 156 primary TNBC cancers from patients with a median of 78 months (range 0.2–155 months) follow up.ResultsWe found that high expression of ERβ1 was not associated with increased recurrence or survival when assessed as percentage of ERβ1 positive tumor cells or as Allred > 5. In contrast, the non-specific PPG5-10 antibody did show an association with recurrence and survival.ConclusionsOur data indicate that ERβ1 expression in TNBC tumours does not associate with prognosis.

Optimized multiplex immunofluorescence for the characterization of tumor immune microenvironment in neoplastic paraffin-preserved tissues.

The study of neoplastic cells enabled the discovery of important tumor-related biomarkers which resulted in new forms of early diagnosis, therapeutic options, and prognostic markers. Thus, immunofluorescence (IF), a high throughput imaging technology, represents a valuable method that enables the virtual characterization and localization of diverse cell types and targets, preserving tissue architecture and spatial surroundings. IF staining and analysis of formalin-fixed paraffin-embedded (FFPE) tissues are considered a challenge due to several difficulties, such as tissue autofluorescence, non-specific antibody binding, and image acquisition and quality. This study aimed to develop a multiplex-fluorescence staining technique with high-contrast and high-quality multiple-color images to enrich the investigation of important biomarkers. We present a robust optimized multiple-immunofluorescence procedure that reduced sample autofluorescence, enabled the use of simultaneous antibodies on the same sample, and showed super-resolution imaging through precise antigen localization. We illustrated the utility of this powerful method in FFPE neoplastic appendix, lymph node and bone marrow biopsies, and a 3D-coculture system, in which cells are enabled to grow and interact with their surroundings in all 3D dimensions. Our optimized multiple-immunofluorescence method represents a powerful tool for better understanding the complexity of tumor cells, characterizing cell populations and spatial localization, revealing predictive and prognostic biomarkers, and identifying immunologic phenotypes in a single and limited sample. Thisvaluable IF protocol successfully enables tumor microenvironment profiling that could contribute to the study of cellular crosstalk and the niche, and to the identification of predictive biomarkers for neoplasms.

Serum Level of Antibodies Against Novel Acinetobacter Baumannii OmpA-selected Peptides in ICU Staff: Promise for the Future of Vaccine Development.

Extensively drug-resistant Acinetobacter baumannii is considered one of the most dangerous threats to global health, requiring novel therapeutic interventions. The outer membrane protein A (OmpA) is an immunogenic agent that triggers immune responses. The current study evaluated serum antibody levels against previously determined immunogenic OmpA peptides from A baumannii in ICU staff. Serum samples were collected from 62 ICU staff members (representing the exposed group), healthy controls (representing the nonexposed group), and patients with systemic lupus erythematosus (SLE) (as controls for nonspecific antibody reactions). After excluding the cross-reactive antibodies via Escherichia coli lysate pretreatment, all the samples were assessed in the vicinity of A baumannii lysate by enzyme-linked immunosorbent assay (ELISA). All the positive samples were assessed for interaction with previously designed and selected peptides using ELISA. The protective potential of positive serum antibodies was surveyed in vitro using an opsonophagocytic study. The most antibody positive samples against one of the dominant peptides were determined in the ICU personnel (75%). SLE serum samples did not react with candidate peptides. The strongest positive reaction was observed in serum treatment with one of the OmpA peptides (No. 5) with significant differences compared to other designed peptides. Our findings showed that ICU samples have substantially higher antibody levels than the nonexposed group; Positive samples show strong results in the opsonophagocytosiis assay. This study demonstrates A baumannii colonization at human mucosal surfaces, especially in exposed healthy workers. Novel OmpA-derived peptides could be used to identify immunogenic vaccine candidates. Therefore, more studies are needed before this peptide and antibody levels are used in diagnosis, prevention, or treatment.

Thresholding CITE-Seq-based Antibody Expression Data to Improve Cell Type Identification in scRNA-Seq

Abstract scRNA-seq is widely used to assess immune cell heterogeneity by measuring individual cell transcriptomes. Surface markers have been used for decades to type immune cells by flow and more recently mass cytometry. Expression of surface markers is poorly correlated with the mRNA levels of their encoding transcripts. CITE-Seq provides a systematic approach to analyze surface phenotypes along with cell transcriptomes by using oligonucleotide-tagged antibodies. Like flow cytometry, CITE-seq-based assessment of surface markers is complicated by non-specific binding. Here, we introduce and validate a systematic method of thresholding the levels of protein marker expression to address the issue of non-specific antibody binding. To remove the non-specific signal, a threshold value separating noise from expression for each surface marker is obtained by comparing the antibody signal in a cell type known to not express this surface marker. If no negative cell type is available, we use deconvolution of overlapping normal distributions to find the best separator in density plots (Ridgeline plots) by deconvolution (normalmixEM tool) of overlapping normal distributions. In total, 34 of 49 antibodies tested were reliably thresholded, thus effectively removing the noise from the signal. In a validation experiment testing 188 antibodies at 4 different concentrations, 124 were successfully thresholded, including all 34 mentioned above. Supported by the National Institutes of Health (NIH) P01 HL136275

Microwave resonator–based sensor system for specific antibody detection

An antibody-detecting sensor is described that is based on a microwave electrodynamic resonator. A polystyrene film with immobilized bacteria deposited on a lithium niobate plate was placed at one end of the resonator and was used as the sensing element. The second end was electrically shorted. The frequency and depth of the reflection coefficient S11 for three resonances in the range 6.5–8.5 GHz were used as an analytical signal to examine antibody interactions with bacteria and determine the time required for cell immobilization. The sensor distinguished between situations in which bacteria interacted with specific antibodies and those in which no such interaction occurred (control). Although the cell–antibody interaction changed the frequency and depth of the second and third resonance peaks, the parameters of the first resonance peak did not change. The interaction of cells with nonspecific antibodies did not change the parameters of any of the peaks. These results are promising for use in the design of methods to detect specific antibodies, which can supplement the existing methods of antibody analysis.

Abstract 6008: Antibody validation for clinical pharmacodynamic assay reveals cross-reactivity of commercial DNMT3B clones

Abstract Cooperativity between the maintenance DNA methyltransferase, DNMT1, and de novo methyltransferases, DNMT3A and 3B, is essential for proper maintenance and integrity of the methylome, and deregulation of these enzymes and associated transcriptional silencing of multiple genes has been shown in many types of cancer. Two DNA methyltransferase inhibitors are currently in clinical use with several others undergoing clinical evaluation, and the pharmacodynamic response of DNMT enzymes is a crucial aspect of understanding therapeutic effects in preclinical studies and clinical trials. Vigorous fit-for-purpose validation of research-grade reagents and the application of effective quality control testing procedures to assess lot-to-lot variability (and reject unacceptable lots) are essential to assure robust and reproducible pharmacodynamic assay performance. During the antibody validation process for the DNMT3B enzyme target, two commercial monoclonal antibody clones were found to exhibit significant cross-reactivity to another cellular protein and inappropriate subcellular localization via immunohistochemistry (IHC), including diffuse cytoplasmic staining and significant staining in a DNMT3B knock out (KO) cell line. Two other commercial monoclonal antibody clones showed the expected nuclear expression in the HCT116 parental cell line and lack of staining in the DNMT KO cells. Further evaluation via western blot revealed that the cross-reacting antibodies detected a band of the approximate expected molecular weight (MW) of DNMT3B but of a protein that was expressed equivalently in the parental and DNMT3B KO cell line pair, whereas the two other antibody clones only detected a significant band of correct MW in the parental but not the KO cell line. These results suggest that some commercial antibody clones are binding to a protein other than DNMT3B and that they are unsuitable for use with IHC and western blot to evaluate subcellular localization and expression levels of DNMT3B protein. We presume that these non-specific antibody clones were chosen for commercial development based on target screening via Western blot that did not include evaluation of a validated DNMT3B knock out cell line, as is not uncommon for research-grade antibody clones. This data highlights the critical importance of thorough fit-for-purpose validation and quality control for crucial reagents used for pharmacodynamic biomarker studies. Funded by NCI Contract No HHSN261200800001E Citation Format: Gabriel J. Benton, Manisha Mohandoss, James H. Doroshow, Ralph E. Parchment, Katherine V. Ferry-Galow. Antibody validation for clinical pharmacodynamic assay reveals cross-reactivity of commercial DNMT3B clones. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6008.

Loss of anti-AT1R reactivity in ELISA post-adsorption - False reactivity or interference in the assay?

Autoantibodies to Angiotensin II type 1 receptor (AT1R) are associated with detrimental outcomes in organ transplants. However, reports showed that adsorption with latex beads reduced positive anti-AT1R antibodies, suggesting possible false reactivity. To investigate this conundrum, we studied 11 samples positive for AT1R antibodies with an ELISA kit before and after adsorption. Adsorption significantly reduced the measurable level of AT1R antibodies (28.3±9.8 vs. 6.3±3.0 U/ml, p<0.001). AT1R antibodies were lower when post-adsorption serum was added back at 1:1 ratio to the neat serum compared to the diluent control (8.6±4.2 vs. 18.1±10.3 U/ml, p=0.02). Sham adsorption with the buffer from Adsorb Out™ kit without beads also suppressed the detection of anti-AT1R antibodies (32.7±9.1 vs. 8.1±3.9 U/ml, p<0.001). Thus, rather than actively removing nonspecific antibodies by the beads, the adsorption process introduces soluble factors that interfere with the detection of anti-AT1R antibodies with the ELISA kit.

Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins.

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute alterations in protein activation states. It facilitates the assessment of RBC treatments, characterization of differences in certain disease states, and description of cellular coherencies. The detection of acutely altered protein activation (e.g., through mechanotransduction) requires adequate sample preparation to preserve otherwise temporary protein modifications. The basic principle includes immobilizing the target binding sites of the desired RBC proteins to enable the initial binding of specific primary antibodies. The sample is further processed to guarantee optimal conditions for the binding of the secondary antibody to the corresponding primary antibody. The selection of non-fluorescent secondary antibodies requires additional treatment, including biotin-avidin coupling and the application of 3,3-diaminobenzidine-tetrahydrochloride (DAB) to develop the staining, which needs to be controlled in real-time under a microscope in order to stop the oxidation, and thus staining intensity, on time. For staining intensity detection, images are taken using a standard light microscope. In a modification of this protocol, a fluorescein-conjugated secondary antibody can be applied instead, which has the advantage that no further development step is necessary. This procedure, however, requires a fluorescence objective attached to a microscope for staining detection. Given the semi-quantitative nature of these methods, it is imperative to provide several control stains to account for non-specific antibody reactions and background signals. Here, we present both staining protocols and the corresponding analytical processes to compare and discuss the respective results and advantages of the different staining techniques.

Development of machine learning models for prediction of antibody non-specificity.

For the development of therapeutic antibodies, it is acknowledged that optimal target binding affinity should be accompanied by high specificity. In recent years, there has been an increased focus on lowering non-specific binding during early-stage drug development. In particular, non-specificity has been recognised as a root-cause for failure in numerous antibody-based drug programs due to unexpected pharmacokinetics and elevated toxicity. Hence, there is a need for predictive tools for flagging and deselecting non-specific antibodies at an early stage to mitigate failure at clinical late-stage development. From a computational design perspective, prediction has remained a challenge as non-specificity is currently a poorly understood biophysical phenomena, typically involving weak interactions to various, unrelated targets and surfaces. In this work, we describe the development of sequence-based machine learning (ML) models for prediction of antibody non-specificity. A dataset of >1000 antibody sequences paired with their respective non-specificity flag, defined by ELISA using a panel of common antigens, were retrieved from public sources (Jain et al., 2017; Boughter et al., 2020). The sequences were embedded using state-of-the-art protein language models for representation of their physiochemical properties, whereafter the vectorized embeddings were served as features for training of binary classifiers for non-specificity flags. A total of 12 different antibody domain-specific models were generated. Highest predictability of non-specificity was obtained for ML algorithms trained on the heavy-chain variable domain (VH), and the complementarity determining regions (CDRs), and resulted in a mean accuracy of 73% across 3-, 5-, and 10-fold cross-validations. The classification probability of non-specificity followed an uptrend with the experimental data (non-specificity flags), thereby mimicking regression behaviour. Altogether, our work demonstrates feasibility for prediction of non-specific binding from the antibody sequence, and that the non-specificity primarily stems from the VH domain, with main contributions from the heavy-chain CDRs.

Intraocular neutralizing antibodies against aflibercept in patients with age related macular degeneration

PurposeTo detect immunoglobulins in aqueous humour of AMD patients after repeated administration of intravitreal aflibercept.Patients and methodsTwenty-one patients (age: 77.85 ± 9.21 years) previously treated with intravitreal aflibercept due to wet type age-related macular degeneration (AMD group) and 18 age-matched control subjects (age: 69.75 ± 12.67 years) were included in this study. Patients in the AMD group received a mean of 5 intravitreal injections (min: 1 max: 17) prior to the cataract surgery. Samples of aqueous humour (50 μl) were obtained by anterior chamber paracentesis as the first step of routine cataract surgery. The IgG content of the samples was analysed by an in-house developed ELISA system.ResultsA significant increase in nonspecific IgG levels in the AMD group was detected compared to the control group (13.37 ± 6.65 vs. 9.44 ± 6.55 μg/ml; p = 0.03). In 11 patients, intraocular anti-aflibercept immunoglobulins could be detected (0.05 ± 0.01 μg/ml) which was significantly higher than the limit of detection for anti-aflibercept (0.04 μg/ml; p = 0.001). No correlation was found between the number of injections or the type of CNV and the aqueous level of anti-aflibercept (r = 0.02; p = 0.95).ConclusionAccording to our results, penetration of non-specific systemic antibodies through the impaired blood-retinal barrier is higher in patients with neovascular AMD than in subjects with an intact structural barrier. Evaluation of neutralizing antibodies to anti-VEGF agents in the aqueous humour can lead us to understanding tachyphylaxis and changes in intraocular immune mechanisms due to AMD.

Removing Negative Impacts from Inevitable Nonreproducible and Nonspecific Antibody-Probe Interactions in Viral Serology.

Serological assays are indispensable tools in public health. Presently deployed serological assays, however, largely overlook research progress made in the last two decades that jeopardizes the conceptual foundation of these assays, i.e., antibody (Ab) specificity. Challenges to traditional understanding of Ab specificity include Ab polyspecificity and most recently nonreproducible Ab-probe interactions (NRIs). Here, using SARS-CoV-2 and four common livestock viruses as a test bed, we developed a new serological platform that integrates recent understanding about Ab specificity. We first demonstrate that the response rate (RR) from a large-sized serum pool (∼100) is not affected by NRIs or by nonspecific Ab-probe interactions (NSIs), so RR can be incorporated into the diagnostic probe selection process. We subsequently used multiple probes (configured as a "protein peptide hybrid microarray", PPHM) to generate a digital microarray index (DMI) and finally demonstrated that DMI-based analysis yields an extremely robust probabilistic trend that enables accurate diagnosis of viral infection that overcomes multiple negative impacts exerted by NSI/NRI. Thus, our study with SARS-CoV-2 confirms that the PPHM-RR-DMI platform enables very rapid development of serological assays that outperform traditional assays (for both sensitivity and specificity) and supports that the platform is extendable to other viruses.

Quantitative and quantitative analysis of spleen and bone marrow CD8&lt;sup&gt;+&lt;/sup&gt; and CD4&lt;sup&gt;+&lt;/sup&gt; T cell populations in lactating mice

Purpose: to investigate the phenotype of adaptive immunity cells in the spleen and bone marrow of lactating mice.Materials and methods. The studies were carried out on lactating mice. For the experiment, the spleen and bone marrow were taken from animals. A suspension of individual splenocytes was prepared by grinding the spleen. Bone marrow was obtained by rinsing the medullary cavity with a syringe with 1–2 ml of a balanced salt solution. The phenotype of adaptive immunity cells was determined using a set of fluorochrome-conjugated antibodies: CD4-PerCP-Cy5.5, CD8-PE/Cy7, CD62L-APC/Cy7, CD44-BV510 (Biolegend, USA), in the presence of True Stain reagent containing antibodies to CD16/CD32 (Biolegend, USA) to block nonspecific antibody binding. Data collection was carried out on a CytoFlex flow cytometer (Beckman Coulter, USA). The results were analyzed using the Kaluza Analysis 2.1 program (Beckman Coulter, USA).Results. As a result of the study, it was found that there are 2.2 times more CD8+ T-cells of effector (TEM) and central memory (TCM) in the red bone marrow, while all subpopulations of CD4+ T-memory cells (TCM; TEM; TNV) predominate in the spleen . The content of the subpopulation of CD8+ T-cells of naive memory (TNV) in the bone marrow and spleen was almost the same and amounted to 52.57±1.58 % and 57.40±2.63 %, respectively. A significantly low content (p&lt;0.001) of populations of CD8+ T-cells of effector memory (TEM; CD44+CD62L-) in the spleen was found to be 4.9±1.39 % compared with T-cells (TEM; CD44+CD62L-) of red bone marrow brain 11.04±2.58 %.Conclusion. The population of CD4+ T-cells of effector memory (TEM) accumulates in large numbers in the spleen in order to further respond with a cellular or humoral mechanism in response to the penetration of an antigen into the body. The bone marrow quantitatively and qualitatively surpasses the spleen in the accumulation and placement of effector (TEM) and central memory (TCM) CD8+ T cells, which are responsible for immunological memory and adaptive immune response.

Immunohematological Discrepancy – Due to Nuisance Antibody!

Timely provision of blood plays an important role in the management algorithm of patients. Interference by minor blood group (BG) antibodies has become a common entity among multi-transfused patients; other nonsignificant antibodies are too encountered rarely. We report one such interfering nonspecific antibody in a 55-year-old female patient posted for splenectomy at our institute. BG discrepancy (type one) with a pan-positive (4+) antibody screening panel was found in her testing results. Crossmatches were compatible at antihuman globulin phase and at room temperature, and autocontrol was negative. Detailed meticulous immunohematological workup helped us in concluding that the offending antibody was not directed against the ingredients of column matrix of gel cards or the enhancement media (low ionic strength solution); rather it was directed against the chemical constituents in the suspension medium of commercial cell panel. Bearing the presence of nonspecific “nuisance” antibodies in mind becomes extremely important so that they can be detected and managed quickly.

Molecular states during acute COVID-19 reveal distinct etiologies of long-term sequelae

Post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are debilitating, clinically heterogeneous and of unknown molecular etiology. A transcriptome-wide investigation was performed in 165 acutely infected hospitalized individuals who were followed clinically into the post-acute period. Distinct gene expression signatures of post-acute sequelae were already present in whole blood during acute infection, with innate and adaptive immune cells implicated in different symptoms. Two clusters of sequelae exhibited divergent plasma-cell-associated gene expression patterns. In one cluster, sequelae associated with higher expression of immunoglobulin-related genes in an anti-spike antibody titer-dependent manner. In the other, sequelae associated independently of these titers with lower expression of immunoglobulin-related genes, indicating lower non-specific antibody production in individuals with these sequelae. This relationship between lower total immunoglobulins and sequelae was validated in an external cohort. Altogether, multiple etiologies of post-acute sequelae were already detectable during SARS-CoV-2 infection, directly linking these sequelae with the acute host response to the virus and providing early insights into their development.

Autoimmune Encephalitis Misdiagnosis in Adults; A Multicenter Observational Study of Outpatient Subspecialty Clinics

Objective To determine the diseases misdiagnosed as AE and potential reasons for misdiagnosis. Background Misdiagnosis of autoimmune encephalitis (AE) may harm patients. Design/Methods Patients with AE misdiagnosis were identified (1/1/2014-12/31/2020) from outpatient AE subspecialty clinics including: Mayo Clinic (n = 44); Oxford (n = 18); UT Southwestern (n = 18); UCSF (n = 17); Washington University (n = 6); University of Utah (n = 4). Inclusion criteria were adults (=18 years) with: 1) A prior diagnosis of AE; and 2) An alternative diagnosis made at a participating center. We collected data on clinical features, investigations, fulfillment of possible AE criteria, alternative diagnoses, and potential contributors to misdiagnosis. Results We identified 107 patients misdiagnosed with AE. Thirty (28%) fulfilled diagnostic criteria for “possible AE”. Median onset age was 48 years (inter-quartile range, 35.5-60.5) and 65 (61%) were female. Correct diagnoses included: functional neurologic disorder, 27 (25%); neurodegenerative disease, 22 (21%); primary psychiatric disease, 19 (18%); cognitive deficits from comorbidities, 11 (10%); cerebral neoplasm, 10 (9%); and other, 18 (17%). Onset was insidious (&gt;3 months) in 51 (48%). MRI brain was suggestive of encephalitis in 19/104 (18%) and CSF pleocytosis occurred in 16/84 (19%). Thyroid-peroxidase antibodies were elevated in 24/62 (39%). Positive neural autoantibodies were more frequent in serum (48/105[46%]) than CSF (7/91[8%]; p&lt;0.001) and serum antibodies included: GAD65, 14; voltage-gated-potassium-channel-complex [LGI1, CASPR2 negative], 10; NMDA-receptor by cell-based assay only, 10 (6 negative in CSF); and other, 18. Immunotherapy adverse effects were observed in 17/84 (20%). Potential contributors to misdiagnosis included: over-interpretation of a non-specific positive serum antibody, 53 (50%); misinterpretation of functional, psychiatric, or non-specific cognitive dysfunction as encephalopathy, 41 (38%). Conclusions Red flags suggesting alternative diagnoses to AE include lack of fulfillment of “possible autoimmune encephalitis” criteria, positive non-specific serum antibody, and insidious onset. Avoiding AE misdiagnosis will prevent morbidity from unnecessary immunotherapies and delayed treatment of the correct diagnosis.