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Camel milk (CM), known for its immune-regulatory, anti-inflammatory, antiapoptotic, and antidiabetic properties, is a natural healthy food. It is easily digestible due to the high levels of β-casein and diverse secreted antibodies, exhibiting superior antibacterial and antiviral activities compared with bovine milk. β-casein is less allergic and more digestible because it is more susceptible to digestive hydrolysis in the gut; therefore, higher levels of β-casein make CM advantageous for human health. Furthermore, antibodies help the digestive system by destroying the antigens, which are then overwhelmed and digested by macrophages. The connection between the gut microbiota and human health has gained substantial research attention, as it offers potential benefits and supports disease treatment. The gut microbiota has a vital role in regulating the host's health because it helps in several biological functions, such as protection against pathogens, immune function regulation, energy harvesting from digested foods, and reinforcement of digestive tract biochemical barriers. These functions could be affected by the changes in the gut microbiota profile, and gut microbiota differences are associated with several diseases, such as inflammatory bowel disease, colon cancer, irritable bowel disorder, mental illness, allergy, and obesity. This review focuses on the digestibility of CM components, particularly protein and fat, and their influence on gut microbiota modulation. Notably, the hypoallergenic properties and small fat globules of CM contribute to its enhanced digestibility. Considering the rapid digestion of its proteins under conditions simulating infant gastrointestinal digestion, CM exhibits promise as a potential alternative for infant formula preparation due to the high β-/α s -casein ratio and protective proteins, in addition to the absence of β-lactoglobulin., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The amalgamation of nanostructures with modern electrochemical and optical techniques gave rise to interesting devices, so-called biosensors. A biosensor is an analytical tool that incorporates various biomolecules with an appropriate physicochemical transducer. Over the past few years, metal oxide nanomaterials (MONMs) have significantly stimulated biosensing research due to their desired functionalities, versatile chemical stability, and low cost along with their unique optical, catalytic, electrical, and adsorption properties that provide an attractive platform for linking the biomolecules, for example, antibodies, nucleic acids, enzymes, and receptor proteins as sensing elements with the transducer for the detection of signals or signal amplifications. The signals to be measured are in direct proportionate to the concentration of the bioanalyte. Because of their simplicity, cost-effectiveness, portability, quick analysis, higher sensitivity, and selectivity against a broad range of biosamples, MONMs-based electrochemical and optical biosensing platforms are exhaustively explored as powerful early-diagnosis tools for point of care applications. Herein, we made a bibliometric analysis of past twenty years (2004-2023) on the application of MONMs as electrochemical and optical biosensing units using Web of Science database and the results of which clearly reveal the increasing number of publications since 2004. Geographical area distribution analysis of these publications shows that China tops the list followed by the United States of America and India. In this review, we first describe the electrochemical and optical properties of MONMs that are crucial for the creation of extremely stable, specific, and sensitive sensors with desirable characteristics. Then, the biomedical applications of MONMs-based bare and hybrid electrochemical and optical biosensing frameworks are highlighted in the light of recent literature. Finally, current limitations and future challenges in the field of biosensing technology are addressed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Single-cell multiplexed phenotypic analysis expands the biomarkers for diagnosis, heralding a new era of precision medicine. Cell secretions are the primary measures of immune function, but single-cell screening remains challenging. Here, a novel cell membrane-based assay was developed using cholesterol-linked antibodies (CLAbs), integrating immunosorbent assays and droplet microfluidics to develop a flexible high-throughput single-cell secretion assay for multiplexed phenotyping. CLAb-grafted single cells were encapsulated in water-in-oil droplets to capture their own secretions. Subsequently, the cells were extracted from droplets for fluorescence labeling and screening. Multiple secretions and surface proteins were simultaneously measured from single cells by flow cytometry. To validate the approach, THP-1 cells, THP-1-derived M1 macrophages, and dendritic cells were assayed, indicating the differentiation efficiency of THP-1 cells under different chemical stimulations. Moreover, peripheral blood mononuclear cells from healthy donors under various stimuli showed varied active immune cell populations (6.62-47.14%). The peripheral blood mononuclear cells (PBMCs) of nasopharyngeal carcinoma patients were analyzed to identify a higher percentage of actively cytokine-secreted single cells in the basal state (2.82 ± 1.48%), compared with that in the health donors (0.70 ± 0.29%).

Abstract: Platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies and anti-PF4 antibodies cause heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT), respectively. Diagnostic and treatment considerations differ somewhat between HIT and VITT. We identified patients with thrombocytopenia and thrombosis without proximate heparin exposure or adenovirus-based vaccination who tested strongly positive by PF4/polyanion enzyme-immunoassays and negative/weakly positive by heparin-induced platelet activation (HIPA) test but strongly positive by PF4-induced platelet activation (PIPA) test (ie, VITT-like profile). We tested these patients by a standard chemiluminescence assay that detects anti-PF4/heparin antibodies found in HIT (HemosIL AcuStar HIT-IgG(PF4-H)) as well as a novel chemiluminescence assay for anti-PF4 antibodies found in VITT. Representative control sera included an exploratory anti-PF4 antibody-positive but HIPA-negative/weak cohort obtained before 2020 (n = 188). We identified 9 patients with a clinical-pathological profile of a VITT-like disorder in the absence of proximate heparin or vaccination, with a high frequency of stroke (arterial, n = 3; cerebral venous sinus thrombosis, n = 4), thrombocytopenia (median platelet count nadir, 49 × 109/L), and hypercoagulability (greatly elevated D-dimer levels). VITT-like serological features included strong reactivity by PIPA (aggregation <10 minutes in 9/9 sera) and positive testing in the novel anti-PF4 chemiluminescence assay (3/9 also tested positive in the anti-PF4/heparin chemiluminescence assay). Our exploratory cohort identified 13 additional patient sera obtained before 2020 with VITT-like anti-PF4 antibodies. Platelet-activating VITT-like anti-PF4 antibodies should be considered in patients with thrombocytopenia, thrombosis, and very high D-dimer levels, even without a proximate exposure to heparin or adenovirus vector vaccines., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

A sensitive immunochromatographic assay (ICA) using time-resolved fluorescence microspheres (TRFMs) coupled with an indirect-labeling mode was developed for simultaneously determining 22 kinds of β-lactams in milk samples. The TRFMs labeled anti-receptor monoclonal antibodies (mAbs) conjugated to penicillin-binding proteins (PBPs) as ternary TRFMs-mAb-PBPs (TMP) nanoscaffolds provide excellent solubility, brightness, and stability. Thanks to the fact that they not only fully expose the binding sites of PBPs, thereby enhancing the biological affinity of PBPs towards the target, but also generated superb fluorescence signals, the versatile TMP manifested unique possibilities as efficient probes for ICA with remarkable enhancement in sensitivity in β-lactams screening. The results showed that the standard curves of the 22 varying β-lactams displayed linearity in their respective concentration ranges (R 2 > 0.98), with the cutoff values of 1-100 ng/mL. The constructed TMP-ICA was successfully applied to the analysis of real milk, with consistent results compared with liquid chromatography-tandem mass spectrometry (LC-MS), providing an effective method for sensing β-lactams in food matrices., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Methicillin-resistant Staphylococcus aureus (MRSA) is a high priority pathogen due to its life-threating infections to human health. Development of prophylactic or therapeutic anti-MRSA vaccine is a potential approach to treat S. aureus infections and overcome the resistance crisis. β-1,4-GlcNAc glycosylated wall teichoic acids (WTAs) derived from S. aureus are a new type of antigen that is closely associated with β-lactam resistance. In this study, structure-defined β-1,4-GlcNAc-modified WTAs varied in chain length and numbers of GlcNAc modification were synthesized by an ionic liquid-supported oligosaccharide synthesis (ILSOS) strategy in high efficiency and chromatography-free approach. Then the obtained WTAs were conjugated with tetanus toxin (TT) as vaccine candidates and were further evaluated in a mouse model to determine the structure-immunogenicity relationship. In vivo immunological studies revealed that the WTAs-TT conjugates provoked robust T cell-dependent responses and elicited high levels of specific anti-WTAs IgG antibodies production associated with the WTAs structure including chain length as well as the β-1,4-GlcNAc modification pattern. Heptamer WTAs conjugate T6, carrying three copy of β-1,4-GlcNAc modified RboP, was identified to elicit the highest titers of specific antibody production. The T6 antisera exhibited the highest recognition and binding affinity and the most potent OP-killing activities to MSSA and MRSA cells. This study demonstrated that β-1,4-GlcNAc glycosylated WTAs are promising antigens for further development against MRSA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Beta-casein is a primary milk protein that constitutes approximately 30% of the casein in bovine milk, with the two most common types in cattle being A1 and A2. The A2 protein differs from the A1 version due to a mutation in the codon at position 67, resulting in a histidine to proline substitution. However, the bioactive peptide, beta-casomorphine-7 (BCM7), which originates from partial proteolysis of the A1 variant, has been linked to several gastrointestinal disorders in humans. Production of A1 beta casein-free products is increasing demand in the milk market, worldwide. This study generated and characterized a polyclonal IgY antibody that specifically recognizes the A1 beta-casein protein present in cow's milk. A commercially available IgY anti-A1 antibody was used as a positive control, and the sensitivity and specificity of both the commercial and produced anti-A1 antibodies were evaluated. The results showed 100% sensitivity and specificity of 100% of the commercial IgY anti-A1. The in-house produced anti-A1 antibody demonstrated a sensitivity of 95.2% and a specificity of 100%, indicating its potential as a reliable and cost effective tool for detecting A1 beta-casein protein in milk samples., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Some authors of this manuscript are partners of the company Scienco Biotech, which may or may not be interested in the development of a commercial product related to the test developed in this work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Porphyromonas gingivalis (P. gingivalis, P. g) is the main pathogen of periodontal disease, which is treated with egg yolk immunoglobulin Y (IgY) against P. gingivalis. In order to quickly obtain IgY, 30 hens were immunized with inactivated P. gingivalis. The purification of IgY was carried out by the oleic acid (OA) method and the classical method (AS), respectively. The IgY antibody characteristics and antibacterial effects in HPDLF cells were detected by SDS-PAGE, indirect ELISA, Western blot and viability/toxicity assays. SDS-PAGE and Western blot analysis showed that IgY molecules which were rapidly purified by OA method were complete and specific to P. gingivalis. In addition, the results of crystal violet staining and bacterial staining indicated that IgY could agglutinate with P. gingivalis, inhibiting bacterial invasion of host cells. This study is the first to rapidly and efficiently purify IgY by OA method, and the purified IgY is expected to be used in the detection and treatment of P. gingivalis., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

An increasing demand for the development of immunoglobin Y (IgY) illustrates the necessity of the component analysis in the process of conduction and quality control. This study investigated the proteomic changes in crude IgY extracts and purified IgY products obtained by sequential polyethylene glycol precipitation (PEG) of egg yolks followed by human mycoplasma protein-based affinity chromatography compared with intact egg yolks. After confirming the extraction efficiency and purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, liquid chromatography tandem-mass spectrometry (LC-MS/MS) was performed with samples including fresh yolk, IgY extracted product and purified product. A total of 348 proteins were identified, with 36 proteins deleted and 209 newly detected proteins in the purified product compared to the intact egg yolk. The significantly decreased proteins mainly included phosvitin, albumin, and apolipoprotein B whereas the significantly increased proteins were mainly IgY-related proteins. GO analysis showed that the purified IgY product had ATPase activity and purine ribonucleoside triphosphate binding activity, and was mainly involved in purine and nucleic acid metabolism. This study will inevitably fasten the commercial application of IgY antibodies and is of greater significance for promotion, development and approval for new antibody derived drug products., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Azathioprine (AZA) interferes with the activation of T and B lymphocytes, which are the main cells involved in the pathogenesis of Graves' disease (GD). The aim of this study was to investigate the effectiveness of AZA as an adjuvant therapy to antithyroid drugs (ATDs) for moderate and severe GD. In addition, we conducted an incremental cost-effectiveness analysis of AZA to determine its cost-effectiveness., Methods: We conducted a randomized, open-label, and parallel-group clinical trial. We randomized untreated hyperthyroid patients with severe GD into three groups. All patients received 45-mg carbimazole (CM) as the starting dose and propranolol 40-120 mg daily. The first group (AZA1) received an additional 1 mg/kg/day AZA, the second group (AZA2) received an additional 2 mg/kg/day AZA, and the third group (control group) received only CM and propranolol. We measured thyroid-stimulating hormone (TSH) and TSH-receptor antibody (TRAb) levels at baseline and every 3 months, while free triiodothyronine (FT3) and free thyroxine (FT4) levels were measured at the time of diagnosis, 1 month after initiation of therapy, and every 3 months thereafter until 2 years after remission. Thyroid volume (TV) was assessed by ultrasound at baseline and 1 year after remission., Results: A total of 270 patients were included in this trial. By the end of follow-up, there was higher remission rate in the AZA1 and AZA2 groups compared with controls (87.5% and 87.5% vs . 33.4%, p = 0.002). Throughout the course of follow-up, FT3, FT4, TSH, and TRAb were significantly different between the AZA groups and the control group, but there was no significant difference regarding TV. The decline in the concentrations of FT4, FT3, and TRAb was significantly faster in the AZA2 group than in the AZA1 group. The relapse rate during the 12-month follow-up was insignificantly higher in the control group than in either the AZA1 or AZA2 group (10, 4.4, and 4.4%, p = 0.05, respectively). The median relapse time was 18 months for the control group and 24 months for the AZA1 and AZA2 groups. The incremental cost-effectiveness ratio for the AZA group compared with the conventional group was 27,220.4 Egyptian pounds per remission reduction for patients using AZA as an adjuvant for ATDs., Conclusion: AZA could be a novel, affordable, cost-effective, and safe drug offering hope for patients with GD to achieve early and long-lasting medical remission., Trial Registry: The trial is registered at the Pan African Clinical Trial Registry (Registration number: PACTR201912487382180)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Allam, El-Zawawy, Kader Okda, Ali Alshaikh and Ghazy.)

N-formyl methionine (fMet)-containing proteins are produced in bacteria, eukaryotic organelles mitochondria and plastids, and even in cytosol. However, Nα-terminally formylated proteins have been poorly characterized because of the lack of appropriate tools to detect fMet independently of downstream proximal sequences. Using a fMet-Gly-Ser-Gly-Cys peptide as an antigen, we generated a pan-fMet-specific rabbit polyclonal antibody called anti-fMet. The raised anti-fMet recognized universally and sequence context-independently Nt-formylated proteins in bacterial, yeast, and human cells as determined by a peptide spot array, dot blotting, and immunoblotting. We anticipate that the anti-fMet antibody will be broadly used to enable an understanding of the poorly explored functions and mechanisms of Nt-formylated proteins in various organisms., Competing Interests: Conflict of interest All authors declare no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The value of the affinity constants (kd, ka, and KD) that are determined by label free interaction analysis methods are strongly affected by the ligand density at the sensor surface [1]. This paper outlines a new SPR-imaging method that applies a ligand density gradient enabling the analyte response to be extrapolated to R max  = 0 μRIU. The mass transport limited region is used to determine the analyte concentration. Cumbersome optimization procedures for tuning the ligand density is prevented and surface dependent effects as rebinding, strong biphasic behavior etcetera are minimized. The method can be fully automated for e.g. accurate determination of the quality of antibodies from commercial sources., (Copyright © 2023 The Author. Published by Elsevier Inc. All rights reserved.)

Heparin-induced thrombocytopenia (HIT) is suspected much more often than it is confirmed. Technically simple platelet factor 4 (PF4)-polyanion enzyme-linked immunosorbent assays (ELISAs) are sensitive but nonspecific. In contrast, accurate functional tests such as the serotonin release assay, heparin-induced platelet activation assay, and PF4-dependent P-selectin expression assay require fresh platelets and have complex assay end points, limiting their availability to specialized reference laboratories. To enable broad deployment of functional testing, we sought to extend platelet viability significantly by optimizing storage conditions and developed a simple functional assay end point by measuring the release of a platelet α-granule protein, thrombospondin-1 (TSP1), in an ELISA format. Platelet cryopreservation conditions were optimized by freezing platelets at controlled cooling rates that preserve activatability. Several-month-old cryopreserved platelets were treated with PF4 or heparin and were evaluated for their ability to be activated by HIT and vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies in the TSP1 release assay (TRA). HIT and spontaneous HIT patient samples induced significantly higher TSP1 release using both PF4-treated (PF4-TRA) and heparin-treated cryopreserved platelets relative to samples from patients suspected of HIT who lacked platelet-activating antibodies. This latter group included several patients that tested strongly positive in PF4-polyanion ELISA but were not platelet-activating. Four VITT patient samples tested in the TRA activated PF4-treated, but not heparin-treated, cryopreserved platelets, consistent with recent data suggesting the requirement for PF4-treated platelets for VITT antibody detection. These findings have the potential to transform the testing paradigm in HIT and VITT, making decentralized, technically simple functional testing available for rapid and accurate in-hospital diagnosis., (© 2022 by The American Society of Hematology.)

Zearalenone (ZEN), also known as an F-2 toxin, is a secondary metabolite in the toxic Fusarium species with estrogen properties. ZEN and its derivatives can cause developmental and reproductive disorders in humans and other mammals. In this study, colloidal Au spheres (AuSPs) and Au nanoflowers (AuNFs) were used as signal labels to detect ZEN in cereals, and the critical factors affecting the sensitivity of the immunochromatographic strip (ICS), namely the volume of antigen, antibody, and probe quantities were optimized and compared in detail. Since the large specific surface area of AuNFs reduces the steric hindrance of proteins, it is more conducive to improving the fixation rate of antibodies and proteins. Compared with the traditional colloidal AuSP immunochromatographic strip (AuSP-ICS), the volume of the antibody used in the AuNF immunochromatographic strip (AuNF-ICS) was 0.6 times that in the AuSPs-ICS. At the same antigen volume, a lower amount of probe can achieve the desired visual detection effect and higher sensitivity. For the AuNF-ICS, the limit of detection (LOD) was as low as 0.08 ng mL -1 . ZEN could be detected quickly and accurately from 0.08-10.2 ng mL -1 . And the AuNF-ICS had a high degree of specificity and sensitivity to ZEN. In summary, the AuNF-ICS serves as a valuable tool in large-scale on-site detection of ZEN.

The dysregulation of the hormone cortisol is related to several pathological states, and its monitoring could help prevent severe stress, fatigue, and mental diseases. While wearable antibody-based biosensors could allow real-time and simple monitoring of antigens, an accurate and low-cost antibody-based cortisol detection through electrochemical methods is considerably challenging due to its low concentration and the high ionic strength of real biofluids. Here, a label-free and fast sensor for cortisol detection is proposed based on antibody-coated organic electrochemical transistors. The developed devices show unprecedented high sensitivities of 50 μA/dec for cortisol sensing in high-ionic-strength solutions with effective cortisol detection demonstrated with real human sweat. The sensing mechanism is analyzed through impedance spectroscopy and confirmed with electrical models. Compared to existing methods requiring bulky and expensive laboratory equipment, these wearable devices enable point-of-care cortisol detection in 5 min with direct sweat collection for personalized well-being monitoring.

In the modern farming industry, the irrational or illegal use of veterinary drugs leads to residues in animal-derived food, which can seriously threaten human health. Efficient detection of low concentrations of drug residues in animal products in a short time is a key challenge for analytical methods. This study proposes to use an antibody chip biosensor for rapid and automated analysis of cephalosporins, aminoglycosides, and sulfonamide antibiotics in pork and milk. 3D polymer slides were applied for the preparation of antibody chips. Ovalbumin (OVA) or bovine serum albumin (BSA) conjugates of the haptens were immobilized as spots on disposable chips. Monoclonal antibodies (mAbs) against cefalexin, ceftiofur, gentamicin, neomycin, and sulfonamides allowed the simultaneous detection of the respective analytes. Antibody binding was detected by a second antibody labeled with Cy3-generating fluorescence, which was scanned a with chip scanner. The limits of detection (LOD) for all the analytes were far below the respective maximum residue limits (MRLs) and ranged from 0.51 to 4.3 µg/kg. The average recoveries of all the analytes in each sample were in the range of 81.6-113.6%. The intra- and inter-assay CV was less than 12.9% and showed good accuracy and precision for all the antibiotics at the MRL level. The sample pretreatment method is simple, and the results are confirmed to be accurate by LC-MS/MS; therefore, this method is valuable for the quality control of animal-derived food.

Antibody-based therapeutic agents and other biopharmaceuticals are now used in the treatment of many diseases. However, when these biopharmaceuticals are administrated to patients, an immune reaction may occur that can reduce the drug's efficacy and lead to adverse side-effects. The immunogenicity of biopharmaceuticals can be evaluated by detecting and measuring antibodies that have been produced against these drugs, or antidrug antibodies. Methods for antidrug antibody detection and analysis can be important during the selection of a therapeutic approach based on such drugs and is crucial when developing and testing new biopharmaceuticals. This review examines approaches that have been used for antidrug antibody detection, measurement, and characterization. Many of these approaches are based on immunoassays and antigen binding tests, including homogeneous mobility shift assays. Other techniques that have been used for the analysis of antidrug antibodies are capillary electrophoresis, reporter gene assays, surface plasmon resonance spectroscopy, and liquid chromatography-mass spectrometry. The general principles of each approach will be discussed, along with their recent applications with regards to antidrug antibody analysis., (© 2022 Wiley-VCH GmbH.)

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a highly prothrombotic disorder that like heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4 (PF4). However, unlike HIT-where heparin at low concentrations (0.1-0.5 U/mL) typically enhances antibody-induced platelet activation, platelet activation by VITT sera is usually inhibited by heparin. Further, conventional platelet activation assays for HIT, such as the serotonin-release assay (SRA) and heparin-induced platelet activation (HIPA) test, often yield negative or atypical results when testing VITT sera. Nevertheless, VITT (like HIT) is a "clinical-pathological" disorder whereby laboratory detectability of platelet-activating anti-PF4 antibodies is crucial for diagnosis. VITT antibodies follow 2 fundamental principles of HIT laboratory testing: (1) high probability of a positive PF4-dependent enzyme-immunoassay (EIA), and (2) high probability of a positive platelet activation assay. However, optimal detection of VITT in platelet activation assays requires the addition of PF4, for example, PF4-enhanced SRA (PF4-SRA) and PF4-enhanced HIPA (PIPA). A novel whole blood assay, called the PF4-induced flow cytometry-based platelet activation (PIFPA) assay, exhibits high sensitivity and specificity for VITT. HIT and VITT sera/plasmas differ in their reactivity in rapid HIT immunoassays (90-97% sensitivity for HIT, <25% sensitivity for VITT), consistent with distinct antigen sites on PF4 recognized by HIT and VITT antibodies., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: To evaluate the diagnostic accuracy of SSX and SSX::SS18 antibodies in decalcified surgical specimens and outcome of synovial sarcomas (SS) of bone., Methods and Results: Twenty-five cases were classified as bone SS (prevalence 0.32% among malignant primary bone sarcoma). Median age was 34 years (range = 9-79). Twenty-four of 25 patients presented with non-metastatic tumours, one with lung metastases. The majority of tumours involved the long bones of extremities with metaphyseal origin. Mean size of the tumour was 7.1 cm. Twenty cases (80%) were monophasic and five (20%) were biphasic. SS18::SSX fusion-specific antibody had 92% sensitivity and 99% specificity for primary bone SS, whereas SSX C-terminus antibody had 100% sensitivity and 94% specificity. Fluorescence in-situ hybridisation (FISH) analysis was feasible in nine (36%) cases and detected SS18 rearrangement in all nine cases. All patients underwent surgical removal of their primary tumour, with adequate margins in 18 (72%) patients. Chemotherapy with metothrexate, cisplatin, doxorubicin and ifosfamide was used in the seven patients. Two patients with inadequate surgical margins received radiotherapy. With a median follow-up of 80 months (range = 6-428), 5- and 10-year overall survival (OS) was 66.6% and 47.9%, respectively, and 5 and 10 years' disease-free survival (DFS) was 36.8% [95% confidence interval (CI) = 18.0-55.7%] and 32.2% (95% CI = 14.6-51.2%), respectively. A significant improvement in 10 years' DFS in patients undergoing chemotherapy compared with patients who did not was observed (P = 0.039)., Conclusions: Our series highlights the utility of SS18::SSX fusion-specific and SSX C-terminus antibodies to support the diagnosis of SS. Adjustment chemotherapy was associated with improved prognosis in this series., (© 2021 John Wiley & Sons Ltd.)

Introduction: This study aimed to investigate amniotic fluid (AF) proteins that were differentially expressed between patients with cervical insufficiency (CI) and asymptomatic short cervix (SCX, ≤ 25 mm), and whether these proteins could be predictive of spontaneous preterm birth (SPTB) in these patients., Method: This was a retrospective cohort study of 129 singleton pregnant women with CI (n = 80) or SCX (n = 49) at 17 to 26 weeks who underwent amniocentesis. An antibody microarray was used to perform comparative proteomic profiling of AF from matched CI (n = 20) and SCX (n = 20) pregnancies. In the total cohort, an ELISA validation study was performed for 15 candidate proteins of interest. Subgroup analyses of patients with CI and SCX were conducted to evaluate the association between the 15 proteins and SPTB at < 32 weeks of gestation., Results: Eighty-six proteins showed intergroup differences. ELISA validation confirmed significantly higher levels of AF EN-RAGE, IL-8, lipocalin-2, MMP-9, S100A8/A9, thrombospondin-2, and TNFR2 in patients with CI than in those with SCX. Multivariable analysis showed that increased AF levels of EN-RAGE, S100A8/A9, and uPA were independently associated with SPTB at < 32 weeks in patients with CI; whereas in patients with SCX, high AF levels of APRIL, EN-RAGE, LBP, and TNFR2 were independently associated with SPTB at < 32 weeks., Conclusions: Multiple AF proteins show altered expression in patients with CI compared with SCX controls. Moreover, several novel mediators involved in inflammation were identified as potential biomarkers for predicting SPTB after the diagnosis of CI and SCX. These results provide new insights into target-specific molecules for targeted therapies to prevent SPTB in patients with CI/SCX., Competing Interests: The authors have declared that no competing interests exist.

Background: Mass vaccination is the key element in controlling current COVID-19 pandemic. Studies comparing immunogenicity of different COVID-19 vaccines are largely lacking. We aimed at measuring anti-S antibody (Ab) levels in individuals fully vaccinated with BNT162b2, BBIBP-CorV and Gam-COVID-Vac, as well as in COVID-19 convalescents., Methods: In this cross-sectional study, serum was collected from 400 age- and sex-matched participants, 100 fully vaccinated with BNT162b2, 100 with BBIBP-CorV and 100 with Gam-COVID-Vac on the 28th day after the second vaccine dose, and 100 recovered from COVID-19 at least 28 days after symptom(s) resolution. Sera were analyzed using the LIAISON SARS-CoV-2 S1/S2 IgG assay (DiaSorin, Saluggia, Italy). Wilcoxon rank-sum or Kruskal-Wallis tests was used for comparison of Ab levels., Results: Highest mean value (210.11, SD = 100.42) was measured in the BNT162b2 group, followed by Gam-COVID-Vac (171.11, SD = 120.69) and BBIBP-CorV (68.50, SD = 72.78) AU/mL (p<0.001). Significant differences in antibody levels were found between BNT162b2 and BBIBP-CorV (p<0.001), BNT162b2 and Gam-COVID-Vac (p = 0.001), as well as BBIBP-CorV and Gam-COVID-Vac groups (p<0.001). Percentage of seropositive was 81% in the convalescent group, 83% in BBIBP-CorV vaccinated and 100% in BNT162b2 and Gam-COVID-Vac. When comparing measured antibody levels in vaccinated to those in COVID-19 recovered, significantly higher antibody levels were found for vaccinated with BNT162b2 (p<0.001), and with Gam-COVID-Vac (p<0.001), while for BBIBP-CorV there was no statistically significant difference (p = 0.641)., Conclusions: All three investigated vaccines, BNT162b2, BBIBP-CorV and Gam-COVID-Vac, provide robust immune response 28 days after the second dose of vaccine, in the majority of participants. All individuals vaccinated with BNT162b2 and Gam-COVID-Vac seroconverted, while in vaccinated with BBIBP-CorV and COVID-19 recovered seroconversion rates were lower. Although less potent compared to other two vaccines, immune response after BBIBP-CorV was similar to response measured in convalescents. Challenge still remains to examine dynamics and durability of immunoprotection., Competing Interests: The authors have declared that no competing interests exist.

Immunoproteomics has emerged as a versatile tool for analyzing the antibody repertoire in various disease contexts. Until recently, characterization of antibody molecules in biological fluids was limited to bulk serology, which identifies clinically relevant features of polyclonal antibody responses. The past decade, however, has seen the rise of mass-spectrometry-enabled proteomics methods that have allowed profiling of the antibody response at the molecular level, with the disease-specific serological repertoire elucidated in unprecedented detail. In this review, we present an up-to-date survey of insights into the disease-specific immunological repertoire by examining how quantitative proteomics-based approaches have shed light on the humoral immune response to infection and vaccination in pathogenic illnesses, the molecular basis of autoimmune disease, and the tumor-specific repertoire in cancer. We address limitations of this technology with a focus on emerging potential solutions and discuss the promise of high-resolution immunoproteomics in therapeutic discovery and novel vaccine design., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ionov and Lee.)

Background: Thyroid peroxidase antibodies (TPO-Abs) play an important role in autoimmune thyroid disease, but are also prevalent in healthy individuals. However, it is unclear what determinants may influence the occurrence of TPO-Abs in healthy individuals and how TPO-Abs may affect health outcomes in these individuals. We aimed to identify determinants of TPO-Abs in a large, prospective population-based cohort of middle-aged and elderly individuals and to subsequently assess the association between TPO-Abs and risk of overall and cause-specific mortality. Methods: We performed binomial and multinomial logistic regression analyses to obtain odds ratios (ORs) and 95% confidence intervals [95% CIs] for the association of potential determinants based on previous literature with TPO-Ab positivity (>35 kU/L), TPO-Ab detectability (>5 kU/L), and TPO-Ab categories. Cox proportional hazards regression analyses were performed to obtain hazard ratios (HRs) and CIs for the association between TPO-Abs and mortality risk. Results: In 9685 participants (57% women, median baseline age 63.3 years, median follow-up time 10.1 years), we identified female sex (OR = 2.47 [CI 2.13-2.86]) and current smoking (OR = 3.10 [CI 2.66-3.62]) as determinants of TPO-Ab positivity and TPO-Ab detectability, respectively. Higher age (OR = 0.98 [CI 0.97-0.98]) and all categories of alcohol consumption (ORs ranging from 0.71-0.78) were associated with lower odds of TPO-Ab detectability. TPO-Ab detectability was associated with a higher risk of overall (HR = 1.09 [CI 1.01-1.17]), cancer-related (HR = 1.18 [CI 1.01-1.38]), and cardiovascular mortality (HR = 1.21 [CI 1.01-1.45]). Interestingly, this was more prominent in men compared with women (HR for cardiovascular mortality 1.50 vs. 0.99, respectively). Conclusions: In community-dwelling middle-aged and elderly individuals, female sex and current smoking are the most important determinants associated with TPO-Ab levels in the detectable and positive range, whereas alcohol consumption is associated with lower odds of TPO-Abs. The clinical importance of detectable TPO-Ab levels is illustrated by the association with an increased mortality risk, mainly in men. Our results warrant further exploration of the clinical applicability of detectable TPO-Ab levels, potentially as a marker for low-grade inflammation. The Rotterdam Study has been entered into the Netherlands National Trial Register (NTR; www.trialregister.nl) and into the WHO International Clinical Trials Registry Platform (ICTRP; www.who.int/ictrp/network/primary/en/) under shared catalogue number NTR6831.

As a widely distributed arthropod and vector for various pathogens, Hyalomma asiaticum presents great risk and potential losses in animal husbandry. Effective measures, including the use of vaccines, are necessary for controlling ticks and tick-borne diseases. A concise understanding of the tick-host interaction associated molecules and pathways is required for vaccine development. In the present study, a protein containing a single-domain von Willebrand factor type C (HaSVC) was isolated from H. asiaticum and was subjected to functional identification. As a result, the full-length sequence of the HaSVC (506 bp) gene was obtained, which putatively encodes 100 amino acids with a predicted molecular mass of 11 kDa, excluding the 23-amino acid signal peptide. HaSVC contains 8 cysteines to form 4 disulfide bonds. The native HaSVC protein was detected in multiple tick organs. HaSVC neither attenuated the anti-coagulation process nor directly affected the blood feeding of adult ticks. However, the purified recombinant protein HaSVC (rHaSVC/GST) significantly increased the proliferation of mice spleen cells. This might suggest a regulatory function for HaSVC on inflammation, thus providing new information that may explain the "crosstalk" between ticks and hosts., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Antibody repertoire sequencing (Rep-seq) has been widely used to reveal repertoire dynamics and to interrogate antibodies of interest at single nucleotide-level resolution. However, polymerase chain reaction (PCR) amplification introduces extensive artifacts including chimeras and nucleotide errors, leading to false discovery of antibodies and incorrect assessment of somatic hypermutations (SHMs) which subsequently mislead downstream investigations. Here, a novel approach named DUMPArts, which improves the accuracy of antibody repertoires by labeling each sample with dual barcodes and each molecule with dual unique molecular identifiers (UMIs) via minimal PCR amplification to remove artifacts, is developed. Tested by ultra-deep Rep-seq data, DUMPArts removed inter-sample chimeras, which cause artifactual shared clones and constitute approximately 15% of reads in the library, as well as intra-sample chimeras with erroneous SHMs and constituting approximately 20% of the reads, and corrected base errors and amplification biases by consensus building. The removal of these artifacts will provide an accurate assessment of antibody repertoires and benefit related studies, especially mAb discovery and antibody-guided vaccine design., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Zeng, Zhu, Wang, Zhang, Yang, Tang, Li, Chen, Ma, Lan, Liu, Yang, Yu and Zhang.)

Background/aim: Children on dialysis are under increased risk of influenza and invasive pneumococcal disease. Although vaccination against these microorganisms are recommended in dialysis patients and despite the fact that these vaccines can reduce disease burden and rates of hospitalization due to infection, vaccination rates are below expected and desired. We aimed to evaluate influenza and pneumococcal vaccination and infection rates in European pediatric dialysis centers., Materials and Methods: In 16 centers from 11 countries, 357 pediatric dialysis patients were evaluated retrospectively during 1 year of observation period between 01.01.2014 and 01.01.2015., Results: In all centers, vaccination policy included immunization of dialysis patients with inactive influenza vaccine and pneumococcal conjugate vaccine (PCV). Fifty percent of the centers recommended pneumococcal polysaccharide vaccine following routine PCV series. A significantly higher pneumococcal vaccination rate (43.9%) was seen in peritoneal dialysis (PD) patients compared to those on hemodialysis (HD) (32.9%) (p = 0.035), while the rates for influenza were similar (42.4% and 46.1% respectively, p = 0.496). Among all dialysis patients, 2.2% (n = 8) developed pneumonia and 6.4% (n = 23) was infected by Influenza. Pneumococcic pneumonia rate was 5% for 140 patients who received antipneumococcal vaccine, while only one pneumonia episode was recorded out of 217 unvaccinated patients (p = 0.007). The influenza virus infection rates were similar for patients vaccinated and nonvaccinated (7 % and 6 %, respectively)., Conclusions: Although influenza and pneumococcal vaccines are highly recommended in pediatric dialysis patients, vaccination rates were lower than expected. Pneumococcal vaccination rates were higher in PD compared to the patients on HD. The rate of children with influenza infection was higher than pneumonia. The efficacy of influenza and pneumococcal vaccines was highlighted by the low infection rates. Higher pneumonia rates in patients vaccinated against pneumococcus compared to unvaccinated ones might be due to coexisting risk factors., (The authors declare that there are no competing conflicts of interest.)

Introduction: As several clinical trials have revealed that angiotensin-converting enzyme inhibitors and angiotensin II (Ang II) receptor blockers may be efficient in treating vascular dementia (VaD), the long-acting blockade of the renin-angiotensin system (RAS) would be useful considering the poor adherence of antihypertensive drugs. Accordingly, we continuously blocked RAS via vaccination and examined the effectiveness of the VaD model in rats., Methods: Male Wistar rats were exposed to two-vessel occlusions (2VO) after three injections of Ang II peptide vaccine. The effects of the vaccine were evaluated in the novel object recognition test, brain RAS components, and markers for oligodendrocytes., Results: In the vaccinated rats, anti-Ang II antibody titer level was increased in serum until Day 168, but not in cerebral parenchyma. Vaccinated rats showed better object recognition memory with inhibited demyelination in the corpus callosum and activation of astrocytes and microglia. Also, levels of BrdU/GSTπ-positive cells and the phosphorylation of cAMP response element binding protein was increased in vaccinated rats, indicating that the differentiation of oligodendrocyte progenitor cells to mature oligodendrocytes was accelerated. Vaccinated rats showed increased expression of fibroblast growth factor-2 (FGF2), which was observed in endothelial cells. Angiotensinogen mRNA was decreased at 7 days after 2VO but increased at 14 and 28 days., Conclusion: Ang II vaccine might have promoted oligodendrocyte differentiation and inhibited astrocytic and microglial activation by stimulating FGF2 signaling in the endothelial cells-oligodendrocyte/astrocyte/microglia coupling. These data indicate the feasibility of Ang II vaccine for preventing progression of vascular dementia., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Using a recombinant protein antigen for antibody testing shows a sum of antibody responses to multiple different immune epitopes existing in the protein antigen. In contrast, the antibody testing to an immunogenic peptide epitope reflects a singular antibody response to the individual peptide epitope. Therefore, using a panel of peptide epitopes provides an advantage for profiling multiple singular antibody responses with potential to estimate recent malaria exposure in human infections. However, transitioning from malaria immune epitope peptide-based ELISA to an all peptide bead-based multiplex Luminex assay presents some challenges including variation in the ability of different peptides to bind beads. The aim of this study was to develop a peptide coupling method while demonstrating the utility of these peptide epitopes from multiple stage antigens of Plasmodium falciparum for measuring antibodies. Successful coupling of peptide epitopes to beads followed three steps: 1) development of a peptide tag appended to the C-terminus of each peptide epitope consisting of beta-alanine-lysine (x 4)--cysteine, 2) bead modification with a high concentration of adipic acid dihydrazide, and 3) use of the peptide epitope as a blocker in place of the traditional choice, bovine serum albumin (BSA). This new method was used to couple 12 peptide epitopes from multiple stage specific antigens of P. falciparum, 1 Anopheles mosquito salivary gland peptide, and 1 Epstein-Barr virus peptide as an assay control. The new method was applied to testing of IgG in pooled samples from 30 individuals with previously repeated malaria exposure in western Kenya and IgM and IgG in samples from 37 U.S. travelers with recent exposure to malaria. The new peptide-bead coupling method and subsequent multiplex Luminex assay showed reliable detection of IgG to all 14 peptides in Kenyan samples. Among 37 samples from U.S. travelers recently diagnosed with malaria, IgM and IgG to the peptide epitopes were detected with high sensitivity and variation. Overall, the U.S. travelers had a much lower positivity rates of IgM than IgG to different peptide epitopes, ranging from a high of 62.2% positive for one epitope to a low of only 5.4% positive for another epitope. In contrast, the travelers had IgG positive rates from 97.3% to 91.9% to various peptide epitopes. Based on the different distribution in IgM and IgG positivity to overall number of peptide epitopes and to the number of pre-erythrocytic, erythrocytic, gametocytic, and salivary stage epitopes at the individual level, four distinct patterns of IgM and IgG responses among the 37 samples from US travelers were observed. Independent peptide-bead coupling and antibody level readout between two different instruments also showed comparable results. Overall, this new coupling method resolves the peptide-bead coupling challenge, is reproducible, and can be applied to any other immunogenic peptide epitopes. The resulting all peptide bead-based multiplex Luminex assay can be expanded to include other peptide epitopes of P. falciparum, different malaria species, or other diseases for surveillance, either in US travelers or endemic areas., (Published by Elsevier B.V.)

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (K D ) of a protein-antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled ("conformational") antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA-aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.

Immunotherapies are revolutionizing cancer care, producing durable responses and potentially cures in a subset of patients. However, response rates are low for most tumors, grade 3/4 toxicities are not uncommon, and our current understanding of tumor immunobiology is incomplete. While hundreds of immunomodulatory proteins in the tumor microenvironment shape the anti-tumor response, few of them can be reliably quantified. To address this need, we developed a multiplex panel of targeted proteomic assays targeting 52 peptides representing 46 proteins using peptide immunoaffinity enrichment coupled to multiple reaction monitoring-mass spectrometry. We validated the assays in tissue and plasma matrices, where performance figures of merit showed over 3 orders of dynamic range and median inter-day CVs of 5.2% (tissue) and 21% (plasma). A feasibility study in clinical biospecimens showed detection of 48/52 peptides in frozen tissue and 38/52 peptides in plasma. The assays are publicly available as a resource for the research community., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Whiteaker, Lundeen, Zhao, Schoenherr, Burian, Huang, Voytovich, Wang, Kennedy, Ivey, Lin, Murillo, Lorentzen, Thiagarajan, Colantonio, Caceres, Roberts, Knotts, Reading, Kaczmarczyk, Richardson, Garcia-Buntley, Bocik, Hewitt, Murray, Do, Brophy, Wilz, Yu, Ajjarapu, Boja, Hiltke, Rodriguez and Paulovich.)

Background: Six Sigma (6σ) is an efficient laboratory management method. We aimed to analyze the performance of immunology and protein analytes in terms of Six Sigma., Methods: Assays were evaluated for these 10 immunology and protein analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C-reactive protein (CRP), and Cystatin C (Cys C). The Sigma values were evaluated based on bias, four different allowable total error (TEa) and coefficient of variation (CV) at QC materials levels 1 and 2 in 2020. Sigma Method Decision Charts were established. Improvement measures of analytes with poor performance were recommended according to the quality goal index (QGI), and appropriate quality control rules were given according to the Sigma values., Results: While using the TEa NCCL , 90% analytes had a world-class performance with σ>6, Cys C showed marginal performance with σ<4. While using minimum, desirable, and optimal biological variation of TEa, only three (IgG, IgM, and CRP), one (CRP), and one (CRP) analytes reached 6σ level, respectively. Based on σ NCCL that is calculated from TEa NCCL , Sigma Method Decision Charts were constructed. For Cys C, five multi-rules (1 3s /2 2s /R 4s /4 1s /6 X , N = 6, R = 1, Batch length: 45) were adopted for QC management. The remaining analytes required only one QC rule (1 3s , N = 2, R = 1, Batch length: 1000). Cys C need to improve precision (QGI = 0.12)., Conclusions: The laboratories should choose appropriate TEa goals and make judicious use of Sigma metrics as a quality improvement tool., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

In a prospective observational study, seroconversion to a specific pathogen can serve as a marker of an incident infection, whether or not that infection is symptomatic or clinically diagnosed. While self-reported symptoms can be affected by reporting bias, seroconversion is likely to be free of this bias as it is based on objective measurements of antibody response. Non-invasive salivary antibody tests can be used instead of serum tests to detect seroconversions in prospective studies. In the present study, individuals and families were recruited at a Lake Michigan beach in Wisconsin in August 2011. Data on recreational water exposure and baseline saliva samples (S1) were collected at recruitment. Follow-up data on gastrointestinal symptoms were collected via a telephone interview approximately 10 days post-recruitment. Follow-up saliva samples were self-collected approximately 2 weeks (S2) and 30-40 days post-recruitment (S3) and mailed to the study laboratory. Samples were analyzed for immunoglobulin (Ig) G responses to recombinant antigens of three noroviruses and Cryptosporidium, as well as protein purification tags as internal controls, using an in-house multiplex suspension immunoassay on the Luminex platform. Responses were defined as ratios of antibody reactivities with a target protein and its purification tag. Seroconversions were defined as at least four-fold and three-fold increases in responses in S2 and S3 samples compared to S1, respectively. In addition, an S2 response had to be above the upper 90% one-sided prediction limit of a corresponding spline function of age. Among 872 study participants, there were seven (0.8%) individuals with seroconversions, including six individuals with seroconversions to noroviruses and two to Cryptosporidium (one individual seroconverted to both pathogens). Among 176 (20%) individuals who reported swallowing lake water, there were six (3.4%) seroconversions compared to one (0.14%) seroconversion among the remaining 696 individuals: the crude and age-standardized risk differences per 1000 beachgoers were 32.7 (95% confidence limits 5.7; 59.6) and 94.8 (4.6; 276), respectively. The age-adjusted odds ratio of seroconversion in those who swallowed water vs. all others was 49.5 (4.5; 549), p = 0.001. Individuals with a norovirus seroconversion were more likely to experience vomiting symptoms within 4 days of the index beach visit than non-converters with an odds ratio of 34 (3.4, 350), p = 0.003. This study contributed further evidence that recreational water exposure is associated with symptomatic and asymptomatic waterborne infections, and that salivary antibody assays can be used in epidemiological surveys of norovirus and Cryptosporidium infections., (© 2021. The Author(s).)

Background: Some studies have indicated that interferon (IFN) may be valuable in COVID-19. We aimed to evaluate the impact of short-term IFN on incident thyroid dysfunction and autoimmunity among COVID-19 survivors., Methods: We included consecutive adults without known thyroid disorder admitted to Queen Mary Hospital for COVID-19 from July 2020 to January 2021 who had thyroid function tests (TFTs) and anti-thyroid antibodies measured both on admission and at three months., Results: 226 patients were included (median age 55.0 years; 49.6% men): 135 were IFN-treated. There tended to be more abnormal TFTs upon reassessment in IFN-treated patients (8.1% vs 2.2%, p=0.080). 179 patients (65.4% IFN-treated) had a complete reassessment of anti-thyroid antibodies. There were significant increases in titres of both anti-thyroid peroxidase antibodies (anti-TPO: baseline 29.21 units [IQR: 14.97 - 67.14] vs reassessment 34.30 units [IQR: 18.82 - 94.65], p<0.001) and anti-thyroglobulin antibodies (anti-Tg: baseline 8.23 units [IQR: 5.40 - 18.44] vs reassessment 9.14 units [IQR: 6.83 - 17.17], p=0.001) in the IFN-treated group but not IFN-naïve group. IFN treatment (standardised beta 0.245, p=0.001) was independently associated with changes in anti-TPO titre. Of the 143 patients negative for anti-TPO at baseline, 8 became anti-TPO positive upon reassessment (seven IFN-treated; one IFN-naïve). Incident anti-TPO positivity was more likely to be associated with abnormal TFTs upon reassessment (phi 0.188, p=0.025)., Conclusion: IFN for COVID-19 was associated with modest increases in anti-thyroid antibody titres, and a trend of more incident anti-TPO positivity and abnormal TFTs during convalescence. Our findings suggest that clinicians monitor the thyroid function and anti-thyroid antibodies among IFN-treated COVID-19 survivors, and call for further follow-up studies regarding the clinical significance of these changes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lui, Hung, Lee, Lee, Tam, Pang, Ho, Cheung, Fong, Law, To, Lam, Chow, Woo, Lam and Tan.)

FluorAcryl 3298 (FA) is a UV-curable fluoroacrylate polymer commonly employed as a chemically resistant, hydrophobic, and oleophobic coating. Here, FA was used in a cleanroom-based microstructuring process to fabricate hydrophilic-in-hydrophobic (HiH) micropatterned surfaces containing femtoliter-sized well arrays. A short protocol involving direct UV photopatterning, an etching step, and final recovery of the hydrophobic properties of the polymer produced patterned substrates with micrometer resolution. Specifically, HiH microwell arrays were obtained with a well diameter of 10 μm and various well depths ranging from 300 nm to 1 μm with high reproducibility. The 300 nm deep microdroplet array (MDA) substrates were used for digital immunoassays, which presented a limit of detection in the attomolar range. This demonstrated the chemical functionality of the hydrophilic and hydrophobic surfaces. Furthermore, the 1 μm deep wells could efficiently capture particles such as bacteria, whereas the 300 nm deep substrates or other types of flat HiH molecular monolayers could not. Capturing a mixture of bacteria expressing red- and green-fluorescent proteins, respectively, served as a model for screening and selection of specific phenotypes using FA-MDAs. Here, green-fluorescent bacteria were specifically selected by overlaying a solution of gelatin methacryloyl (GelMA) mixed with a photoinitiator and using a high-magnification objective, together with custom pinholes, in a common fluorescence microscope to cross-link the hydrogel around the bacteria of interest. In conclusion, due to the straightforward processing, versatility, and low-price, FA is an advantageous alternative to more commonly used fluorinated materials, such as CYTOP or Teflon-AF, for the fabrication of HiH microwell arrays and other biphilic microstructures.

The ability to perform multiplexed detection of various biomarkers within complex biological fluids in a robust, rapid, sensitive, and cost-effective manner could transform clinical diagnostics and enable personalized healthcare. Electrochemical (EC) sensor technology has been explored as a way to address this challenge because it does not require optical instrumentation and it is readily compatible with both integrated circuit and microfluidic technologies; yet this approach has had little impact as a viable commercial bioanalytical tool to date. The most critical limitation hindering their clinical application is the fact that EC sensors undergo rapid biofouling when exposed to complex biological samples (e.g., blood, plasma, saliva, urine), leading to the loss of sensitivity and selectivity. Thus, to break through this barrier, we must solve this biofouling problem.In response to this challenge, our group has developed a rapid, robust, and low-cost nanocomposite-based antifouling coating for multiplexed EC sensors that enables unprecedented performance in terms of biomarker signal detection compared to reported literature. The bioinspired antifouling coating that we developed is a nanoporous composite that contains various conductive nanomaterials, including gold nanowires (AuNWs), carbon nanotubes (CNTs), or reduced graphene oxide nanoflakes (rGOx). Each study has progressively evolved this technology to provide increasing performance while simplifying process flow, reducing time, and decreasing cost. For example, after successfully developing a semipermeable nanocomposite coating containing AuNWs cross-linked to bovine serum albumin (BSA) using glutaraldehyde, we replaced the nanomaterials with reduced graphene oxide, reducing the cost by 100-fold while maintaining similar signal transduction and antifouling properties. We, subsequently, developed a localized heat-induced coating method that significantly improved the efficiency of the drop-casting coating process and occurs within the unprecedented time of <1 min (at least 3 orders of magnitude faster than state-of-the-art). Moreover, the resulting coated electrodes can be stored at room temperature for at least 5 months and still maintain full sensitivity and specificity. Importantly, this improved coating showed excellent antifouling activity against various biological fluids, including plasma, serum, whole blood, urine, and saliva.To enable affinity-based sensing of multiple biomarkers simultaneously, we have developed multiplexed EC sensors coated with the improved nanocomposite coating and then employed a sandwich enzyme-linked immunosorbent assay (ELISA) format for signal detection in which the substrate for the enzyme bound to the secondary antibody precipitates locally at the molecular binding site above the electrode surface. Using this improved EC sensor platform, we demonstrated ultrasensitive detection of a wide range of biomarkers from biological fluids, including clinical biomarkers, in both single and multiplex formats ( N = 4) with assay times of 37 and 15 min when integrated with a microfluidic system. These biosensors developed demonstrate the vast potential of solving the biofouling problem, and how it can enable potential clinically important diagnostic applications. This Account reviews our antifouling surface chemistry and the multiplexed EC sensor-based biodetection method we developed and places it in context of the various innovative contributions that have been made by other researchers in this field. We are optimistic that future iterations of these systems will change the way diagnostic testing is done, and where it can be carried out, in the future.

Background: The pathways that control protein transport across the blood-brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development., Methods: We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis., Results: BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis., Conclusions: Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies., (© 2021. The Author(s).)

Immune infertility occurs due to the presence of antisperm antibodies (ASA). This type of infertility has a relatively low prevalence (2.6-6.6% in infertile men), and its etiology, risk factors, targets, and consequences for male fertility are not completely understood. While it is largely accepted that abnormalities in the blood-testis barrier and/or blood-epididymal barrier are the main factors behind its etiology, and that sperm motility is the most frequently reported altered parameter, few are the well-defined risk factors and ASA targets only now started to be disclosed, with proteins involved in sperm-oocyte interaction rising as the most significant. The development of potential treatments is also limited, being the corticosteroids the more promising. Overall, there are still many knowledge gaps related to immune infertility. With this review, we aimed to gather all the information collected from studies developed in humans in the last decade. Despite the controversial results/inconsistencies, that are not only a result of the complexity of mechanisms/variables involved in ASA infertility but also from the technical approaches to assess ASA and the lack of a consensus regarding the thresholds to be used, this manuscript aims to bring a fresh update on the field. It has become clear that, to obtain more/reliable data, there is a need to assess ASA in all the routine seminal analyses, following WHO recommendations. In this way, it will be possible to obtain consistent and comparable information, that can add to current knowledge. Additionally, multicentric studies with large cohorts are also missing, and future research should take this into consideration.

Immuno-mass spectrometry imaging (iMSI) uses laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to determine the spatial expression of biomolecules in tissue sections following immunolabelling with antibodies conjugated to a metal reporter. As with all immunolabelling techniques, the binding efficiency of multiplexed staining can be affected by a number of factors including epitope blocking and other forms of steric hindrance. To date, the effects on the binding of metal-conjugated antibodies to their epitopes in a multiplexed analysis have yet to be quantitatively explored by iMSI. Here we describe a protocol to investigate the effects of multiplexing on reproducible binding using the muscle proteins, dystrophin, sarcospan, and myosin as a model, with antibodies conjugated with Maxpar® reagents before histological application to murine quadriceps sections using standard immunolabelling protocols and imaging with LA-ICP-MS. The antibodies were each individually applied to eight sections, and multiplexed to another eight sections. The average concentrations of the lanthanide analytes were determined, before statistical analyses found there was no significant difference between the individual and multiplexed application of the antibodies. These analyses provide a framework for ensuring reproducibility of antibody binding during multiplexed iMSI, which will allow quantitative exploration of protein-protein interactions and provide a greater understanding of fundamental biological processes during healthy and diseased states., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Sericin, as the main component of silkworm cocoon silk, surrounds and protects the silk fibroin. Sericin is a natural macromolecular protein complex encoded by the genes Ser1, Ser2, and Ser3. At present, there are no available antibodies against sericin that may be used to identify and locate it at the protein level, hindering the study of its secretion mechanism and materials application. Therefore, the development of effective antibodies against sericin is an urgent necessity. To address this problem, we prepared polyclonal antibodies against the Ser1, Ser2 and Ser3 proteins using synthesized peptides for the first time. The specificity of the antibodies was confirmed using dot blot, immunoblotting and mass spectrometry on the hybrid bands of the middle silk gland. The immunoblotting results of anti-sericin antibodies showed that sericin has different molecular weights in different regions of the middle silk gland and strains in the 5th instar. Through immunohistochemistry, anti-sericin antibodies revealed that sericin presented different distributions in the anterior part of the middle silk gland of 872 strain at the 7th day of 5th instar. In addition, the prepared antibodies not only detected intact sericin molecules, but also detected degraded sericin that was dissolved in five different solvents. In summary, this work prepared effective sericin antibodies for silk protein synthesis and secretion research and provides a possible molecular detection method for biological products containing silkworm sericin., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objectives: To report the neuropsychiatric features and frequency of NMDA receptor (NMDAR) and other neuronal immunoglobulin G antibodies in patients with first episode psychosis (FEP) and to assess the performance of reported warning signs and criteria for autoimmune psychosis (AP)., Methods: This was a prospective observational study of patients with FEP assessed for neuropsychiatric symptoms, serum and CSF neuronal antibodies (brain immunohistochemistry, cell-based assays, live neurons), and warning signs and criteria of AP. Previous autoimmune FEP series were reviewed., Results: One hundred five patients were included; their median age was 30 (range 14-75) years, and 44 (42%) were female. None had neuronal antibodies. Two of 105 (2%) had CSF pleocytosis, 4 of 100 (4%) had brain MRI abnormalities, and 3 of 73 (4%) EEG alterations. Thirty-four (32%) and 39 (37%) patients fulfilled 2 sets of warning signs of AP, and 21 (20%) fulfilled criteria of possible or probable AP, yet none developed AP. The cause of FEP was psychiatric in 101 (96%) and nonpsychiatric in 4 (4%). During this study, 3 patients with psychosis caused by anti-NMDAR encephalitis were transferred to our center; 2 did not meet criteria for possible AP. Of 1,159 reported patients with FEP, only 7 (1%) had CSF studies; 36 (3%) had serum NMDAR antibodies (without definite diagnosis of AP), and 4 had CSF NMDAR antibodies (3 classic anti-NMDAR encephalitis and 1 with isolated psychiatric features)., Conclusions: NMDAR antibodies were not found in patients with FEP unless they had anti-NMDAR encephalitis. Warning signs and criteria for AP have limited utility when neurologic symptoms are absent or paraclinical tests are normal. A diagnostic algorithm for autoimmune FEP is provided., (© 2021 American Academy of Neurology.)

Four programmed death ligand 1 (PD-L1) immunohistochemistry assays (28-8, 22C3, SP263, and SP142) have been approved for use by the US Food and Drug Administration (FDA). Analytical concordance between these assays has been evaluated in multiple studies. This systematic review included studies that investigated the analytical concordance of immunohistochemistry assays utilizing two or more PD-L1 antibodies from FDA-approved diagnostics for evaluation of PD-L1 expression on tumor or immune cells across a range of tumor types and algorithms., Methods: Literature searches were conducted in MEDLINE (via PubMed) and EMBASE to identify studies published between January 1, 2010, and March 31, 2019, that evaluated analytical concordance between two or more assays based on antibodies from FDA-approved assays. Proceedings of key oncology and pathology congresses that took place between January 2016 and March 2019 were searched for abstracts of studies evaluating PD-L1 assay concordance., Results: A total of 42 studies across a range of tumor types met the selection criteria. Concordance between 28-8-, 22C3-, and SP263-based assays in lung cancer, urothelial carcinoma, and squamous cell carcinoma of the head and neck was high when used to assess PD-L1 expression on tumor cells (TCs). SP142-based assays had overall low concordance with other approved assays when used to assess PD-L1 expression on TCs. Analytical concordance for assessment of PD-L1 expression on immune cells was variable and generally lower than for PD-L1 expression on TCs., Conclusion: A large body of evidence supports the potential interchangeability of 28-8-, 22C3-, and SP263-based assays for the assessment of PD-L1 expression on TCs in lung cancer. Further studies are required in tumor types for which less evidence is available., Competing Interests: Robin Edwards Employment: Bristol Myers Squibb Stock and Other Ownership Interests: Bristol Myers Squibb/Celgene Patents, Royalties, Other Intellectual Property: I am an inventor on pending patent applications filed for work related to my employment at Bristol Myers Squibb. The patent applications are assigned to Bristol Myers Squibb. Travel, Accommodations, Expenses: Bristol Myers Squibb/Celgene No other potential conflicts of interest were reported.Robin Edwards Employment: Bristol Myers Squibb Stock and Other Ownership Interests: Bristol Myers Squibb/Celgene Patents, Royalties, Other Intellectual Property: I am an inventor on pending patent applications filed for work related to my employment at Bristol Myers Squibb. The patent applications are assigned to Bristol Myers Squibb. Travel, Accommodations, Expenses: Bristol Myers Squibb/Celgene No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

Standard-of-care infliximab dosing regimens were developed prior to the routine use of therapeutic drug monitoring and identification of target concentrations. Not surprisingly, subtherapeutic infliximab concentrations in pediatric Crohn's disease (CD) are common. The primary aim was to conduct a real-world pharmacokinetic (PK) evaluation to discover blood biomarkers of rapid clearance, identify exposure targets, and a secondary aim to translate PK modeling to the clinic. In a multicenter observational study, 671 peak and trough infliximab concentrations from 78 patients with CD were analyzed with a drug-tolerant assay (Esoterix; LabCorp, Calabasas, CA). Individual area under the curve (AUC) estimates were generated as a measure of drug exposure over time. Population PK modeling (nonlinear mixed-effect modeling) identified serum albumin, antibody to infliximab, erythrocyte sedimentation rate (ESR), and neutrophil CD64 as biomarkers for drug clearance. Week 14 and week 52 biochemical remitters (fecal calprotectin < 250 µg/g) had higher infliximab exposure (AUC) throughout induction. The optimal infliximab AUC target during induction for week 14 biochemical remission was 79,348 µg*h/mL (area under the receiver operating characteristic curve (AUROC) 0.77, [0.63-0.90], 85.7% sensitive, and 64.3% specific) with those exceeding the AUC target more likely to achieve a surgery-free week 52 biochemical remission (OR 4.3, [1.2-14.6]). Pretreatment predictors for subtherapeutic week 14 AUC included neutrophil CD64 > 6 (OR 4.5, [1.4-17.8]), ESR > 30 mm/h (OR 3.8, [1.4-11]), age < 10 years old (OR 4.2, [1.2-20]), and weight < 30 kg (OR 6.6, [2.1-25]). We created a decision-support PK dashboard with an iterative process and embedded the modeling program within the electronic health record. Model-informed precision dosing guided by real-world PKs is now available at the bedside in real-time., (© 2020 The Authors. Clinical Pharmacology & Therapeutics © 2020 American Society for Clinical Pharmacology and Therapeutics.)

Dysregulated autoantibodies and cytokines were deemed to provide important cues for potential illnesses, such as various carcinomas and autoimmune diseases. Increasing biotechnological approaches have been applied to screen and identify the specific alterations of these biomolecules as distinctive biomarkers in diseases, especially autoimmune diseases. As a versatile and robust platform, protein microarray technology allows researchers to easily profile dysregulated autoantibodies and cytokines associated with autoimmune diseases using various biological specimens, mainly serum samples. Here, we summarize the applications of protein microarrays in biomarker discovery for autoimmune diseases. In addition, the key issues in the process of using this approach are presented for improving future studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Song, Bai, Song, Zhao, Liu and Hu.)

Aim: To evaluate the diagnostic value of anti-citrullinated α-enolase peptide 1 (anti-CEP 1) antibody in patients with rheumatoid arthritis (RA) by conducting a systematic review and meta-analysis., Methods: The PubMed, Web of Science, Embase, Scopus, and Cochrane Library databases were searched for relevant studies published until September 23, 2020. A bivariate mixed-effects model was used to calculate the diagnostic indices from primary data of eligible studies. We performed meta-regression and subgroup analysis to explore the sources of heterogeneity., Results: Twenty-four articles, with a total of 17 380 patients with RA and 7505 control participants, met the criteria for inclusion in the meta-analysis. The pooled sensitivity, specificity, and positive and negative likelihood ratios for the anti-CEP 1 antibody were 44% (95% CI: 38%-51%), 97% (95% CI: 96%-98%), and 14.81 (95% CI: 10.66-20.57) and 0.57 (95% CI: 0.52-0.64), respectively. The pooled positive and negative predictive values were 0.96 (95% CI: 0.95-0.97) and 0.53 (95% CI: 0.43-0.63), respectively. The area under the summary receiver operating characteristic curve was 0.86. Meta-regression indicated that the anti-CEP 1 antibody detection method may be a source of heterogeneity. The subgroup analysis of the group in which the anti-CEP 1 antibody was detected by using a commercial enzyme-linked immunosorbent assay (ELISA) kit had a sensitivity of 59% (95% CI: 50%-68%) and a specificity of 93% (95% CI: 85%-97%)., Conclusions: The anti-CEP 1 antibody had moderate RA diagnostic value with relatively low sensitivity and high specificity. An ELISA may increase the RA diagnostic sensitivity., (© 2021 The Authors. International Journal of Rheumatic Diseases published by Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.)

Pancreatic intraepithelial neoplasia (PanIN) is a microscopic precursor lesion to pancreatic ductal adenocarcinoma (PDAC); however, there are few biomarkers that segregate high-grade PanIN/PDAC from low-grade PanIN lesions. mAb Das-1 is a monoclonal antibody against a colonic epithelial antigen that is reactive to premalignant conditions of the upper gastrointestinal tract including Barrett's esophagus, incomplete-type gastric intestinal metaplasia, and intraductal papillary mucinous neoplasm of the pancreas at high risk of malignancy. We sought to examine a role for Das-1 expression in differentiating high-grade PanIN/PDAC from low-grade PanIN lesions. We examined surgical specimens from 86 patients and 2 autopsied pancreata (74 with and 14 without PDAC) with 107 distinct PanIN lesions, 74 PDAC cases, and 32 associated lymph node metastases, with internal controls of normal pancreatic ducts observed in 56 cases. All of the normal pancreatic duct controls (0/56) and low-grade PanIN (0/95) lesions were nonreactive to Das-1. Das-1 expression among high-grade PanIN (7/12, 58%), PDAC (55/74, 74%), and lymph node metastasis (21/32, 66%) cases was significantly higher (p < 0.0001). Clinicopathologically, Das-1 reactivity was significantly correlated with nodal metastasis (p = 0.021). Overall, the sensitivity, specificity, and accuracy of Das-1 in segregating high-grade PanIN/PDAC from low-grade PanIN lesions and normal ducts were 72%, 100%, and 90%, respectively. Thus, mAb Das-1 reacts with high specificity with high-grade PanIN and PDAC and may help in preoperative diagnosis and/or clinical risk stratification., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Automated immunoassays are extensively used in routine laboratory diagnostics of endocrine disorders because of their advantages, such as high sensitivity, precision, and specificity. However, these methods are limited by the susceptibility of the immunochemical reaction to various interferences. They may present interferences related to the assay's design, for example, the endogenous presence of anti-streptavidin antibodies (ASA) in platforms that use the biotin-streptavidin interaction. To date, there have been few reports in the literature of interference from endogenous ASA. However, such antibodies would potentially lead to falsely decreased or increased results of hormones that can lead to incorrect diagnoses. We report six patients with unusual thyroid function tests, incongruent to their clinical findings. They present elevated concentrations of total T3 and T4 and TSH values within the reference range when measured at Cobas 8000® e801 module (Roche Diagnostics®). Neither patient had been taking biotin; however, all demonstrated the presence of ASA causing falsely high results on competitive assays and also falsely low results on sandwich assays. The hormone panel was also analyzed in the same samples using a different platform available in our laboratory: Cobas 6000® e601 module (Roche Diagnostics®). Nine samples were sent to an external laboratory to be measured with the chemiluminescent method: ADVIA Centaur® (Siemens® Healthcare Diagnostics). The interference seems to affect e801 module and competitive assays the most without affecting results obtained by this chemiluminescent method. This interference could potentially affect other assays performed on the same platform, such as ATPO and estradiol. Finally, laboratories should suspect the presence of interference when there is no correlation between the hormone profile and the patient's clinic. The biotin neutralization protocol demonstrated its effectiveness to eliminate ASA interference., (Copyright © 2021 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Currently, screening cut point (CP) calculated from an assay validation with replicates are applied to an immunogenicity study with nonreplicates, for which the antidrug antibodies rate is determined. IID treats the replicate of a sample as coming from another independent sample. AVE uses average results from each sample across runs but inter-assay variability is reduced. Therefore, we propose a random effect model (REM) for calculating CP. Materials & method: We investigate impact of noncompatibility design between validation and immunogenicity studies on CP and compare these methods. Conclusion: IID may not fit for use when replicates' variability dominates all sources of uncertainty. REM considers covariance structure of repeated measurements. CP by REM is smaller than that by IID but larger than that by AVE.