Managing hemolysis in serum neuron-specific enolase measurements - an automated algorithm for routine practice.

Neuron-specific enolase (NSE) derived from neurons and peripheral neuroendocrine cells is a biomarker for neuroendocrine tumors and for prognostication in comatose cardiac arrest survivors. However, as platelets and erythrocytes contain NSE, hemolysis causes falsely elevated NSE. We used native serum and hemolysate derived from the same patients to make serial dilutions, and subsequently measured NSE (mNSE) and hemolytic index (HI) in each dilution. An algorithm suitable for the laboratory information system was developed based on the mNSE, HI and the estimated gradient of hemolytic interference from 30 patients. We estimated the associated uncertainty of the corrected NSE (cNSE) results based on the observed range of the gradient and derived an equation for cNSE for samples with limited hemolysis (i.e. 5 < HI ≤ 30): cNSE = mNSE - HI × (0.34 ± 0.23) µg/L. By semi-quantitatively grading the contribution from limited hemolysis, a texted result noting the hemolysis-associated degree of uncertainty can accompany the cNSE result. The major challenge of hemolysis when using serum NSE as a biomarker can be managed using an automated algorithm for correction of NSE results based on degree of hemolysis. However, laboratorians and clinicians should be aware of the limitations associated with in vivo hemolysis.

A review of technologies for collaborative online information seeking: On the contribution of collaborative argumentation.

In everyday life, people seek, evaluate, and use online sources to underpin opinions and make decisions. While education must promote the skills people need to critically question the sourcing of online information, it is important, more generally, to understand how to successfully promote the acquisition of any skills related to seeking online information. This review outlines technologies that aim to support users when they collaboratively seek online information. Upon integrating psychological-pedagogical approaches on trust in and the sourcing of online information, argumentation, and computer-supported collaborative learning, we reviewed the literature (N = 95 journal articles) on technologies for collaborative online information seeking. The technologies we identified either addressed collaborative online information seeking as an exclusive process for searching for online information or, alternatively, addressed online information seeking within the context of a more complex learning process. Our review was driven by three main research questions: We aimed to understand whether and how the studies considered 1) the role of trust and critical questioning in the sourcing of online information, 2) the learning processes at play when information seekers engage in collaborative argumentation, and 3) what affordances are offered by technologies that support users' collaborative seeking of online information. The reviewed articles that focused exclusively on technologies for seeking online information primarily addressed aspects of cooperation (e.g., task management), whereas articles that focused on technologies for integrating the processes of information seeking into the entire learning processes instead highlighted aspects of collaborative argumentation (e.g., exchange of multiple perspectives and critical questioning in argumentation). Seven of the articles referred to trust as an aspect of seekers' sourcing strategies. We emphasize how researchers', users', and technology developers' consideration of collaborative argumentation could expand the benefits of technological support for seeking online information.

Specific antibodies and sensitive immunoassays for the human epidermal growth factor receptors (HER2, HER3, and HER4).

The use of trastuzumab in patients with breast cancer that overexpresses human epidermal growth factor receptor 2 has significantly improved treatment outcomes. However, a substantial proportion of this patient group still experiences progression of the disease after receiving the drug. Evaluation of the changes in expression of the human epidermal growth factor receptors could be of interest. Monoclonal antibodies against the extracellular domain of the human growth factor receptors, 2, 3, and 4, have been raised, and specific and sensitive immunoassays have been established. Sera from healthy individuals (Nordic Reference Interval Project and Database) were analyzed in the human epidermal growth factor receptor 2 assay (N = 805) and the human epidermal growth factor receptor 3 and 4 assays (N = 114), and reference limits were calculated. In addition, sera from 208 individual patients with breast cancer were tested in all three assays. Finally, the human epidermal growth factor receptor 2 assay was compared with a chemiluminescent immunoassay for serum human epidermal growth factor receptor 2/neu. Reference values were as follows: human epidermal growth factor receptor 2, <2.5 µg/L; human epidermal growth factor receptor 3, <2.8 µg/L; and human epidermal growth factor receptor 4, <1.8 µg/L. There were significant differences in human epidermal growth factor receptor 2 and human epidermal growth factor receptor 3 serum levels between the patients with tissue human epidermal growth factor receptor 2-positive and tissue human epidermal growth factor receptor 2-negative ( p = 0.0026, p = 0.000011) tumors, but not in the serum levels of human epidermal growth factor receptor 4 ( p = 0.054). There was good agreement between the in-house human epidermal growth factor receptor 2 assay and the chemiluminescent immunoassay. Our new specific antibodies for all the three human epidermal growth factor receptors may prove valuable in the development of novel anti-human epidermal growth factor receptor targeted therapies with sensitive immunoassays for measuring serum levels of the respective targets and in monitoring established treatment.

Characterization of a new highly sensitive immunometric assay for thyroglobulin with reduced interference from autoantibodies.

Measurements of serum thyroglobulin (Tg) with sensitive immunoassays are of great importance for the management of patients with differentiated thyroid carcinomas. However, interference of circulating autoantibodies to Tg (hTgAb) hampers the usefulness of most assays. We have produced a panel of monoclonal antibodies (mAbs) selected to bind Tg in the presence of Tg autoantibodies and developed a sensitive immunoassay for Tg with minor interference by hTgAbs. The antibodies were characterized by cross-inhibition and immunoassay combination studies, as well as affinity estimation. The within-run and total imprecision of the assay were determined with 2664 samples in 60 separate runs. The most sensitive assay combination with superior protection against autoantibodies consisted of two solid phase mAbs and two tracer mAbs with distinct binding sites. The assay was linear and displayed a wide dynamic range up to 1342 µg/l with a functional sensitivity of 0.1 µg/l and a total imprecision of less than 10 %. There was good agreement between the new high sensitive immunofluorometric assay (IFMA) and two well-established Tg assays from Brahms Kryptor and Roche Diagnostics. Mean difference between the new IFMA and the Kryptor assay was 0.059 µg/l with a 95 % confidence interval of -0.032 to 0.151 µg/l, whereas the mean difference between the new IFMA and the Roche assay was -0.80 µg/l with a 95 % confidence interval of -1.24 to -0.35 µg/l.

Dual-immuno-MS technique for improved differentiation power in heterodimeric protein biomarker analysis: determination and differentiation of human chorionic gonadotropin variants in serum.

If the biomarker potential of intact heteromers and their free subunits is different, differentiation between these forms may reveal important clinical information. Such differentiation may however be analytically challenging. One possible way of circumventing this challenge is by performing a dual-immuno-MS approach. In the present paper, a two-step immunoaffinity sample preparation step is succeeded by digestion and subsequent LC-MS analysis to provide high-sensitivity quantification and differentiation between the heterodimer human chorionic gonadotropin (hCG) and its free ß-subunit in serum. Intact and free variants are captured in two separate immunoextraction steps in order to increase the differentiation power of the method. Intact heterodimer variants were depleted prior to free subunit variants in order to incorporate a method quality control. The method was optimized for serum samples. A fully validated immuno-MS method was used as foundation, and partial validation according to the European Medicines Agency's (EMA) guidelines on validation of bioanalytical methods was performed for the dual approach. An accelerated digestion step was incorporated making batch processing of samples within 1 day possible (approx. 3.5 h of sample preparation including digestion). Acceptable linearity (R (2) ≥ 0.990 for four variants and R (2) of 0.920 and 0.966 for the remaining two) and specificity were demonstrated, and the method was robust toward varying levels of intact heterodimer versus free subunit. The method was also successfully tested on realistic samples, demonstrating both the differences in total hCG and the distribution between intact hCG and its free ß-subunit in real samples. Graphical abstract Schematic overview of the dual immuno-MS process.

Evident or doubtful? How lexical hints in written information influence laypersons' understanding of influenza.

There are clear differences in the way written information on health issues presents research findings. In some cases, the source of a piece of information (e.g. "expert professor") is highlighted to emphasize its credibility and relevance. In other cases, the impact of a certain argument is stressed by avoiding hints on tentativeness such as "mostly" or "up to now." This article examines whether and how far such differences influence laypersons' comprehension of the contents provided. In an experimental setting, 157 laypersons were asked to read an online article on a new approach to preventing influenza. The texts manipulated whether there were (a) hints on the source of information and (b) lexical hints on the tentativeness of the information (hedges). After reading the text, participants were asked to write an essay reporting their opinion on the topic. Their argumentation on vaccination was assessed with content analysis and their attitudes toward vaccination were surveyed with a questionnaire. Results indicated that when lexical hints were given, tentativeness led participants to focus more on the actual information in the text. Additionally, decisions more strongly favored the direction implied in the text when the source of the medical information was not reported. Consequences for the way health information should be presented to laypersons are discussed.

Multiplexing determination of small cell lung cancer biomarkers and their isovariants in serum by immunocapture LC-MS/MS.

A multiplex method for the determination of the small cell lung cancer (SCLC) markers progastrin releasing peptide (ProGRP) and neuron specific enolase (NSE) is presented, which involves coextraction by immunoaffinity (IA) beads and codetermination by selected reaction monitoring (SRM). The performance was compared with two IA SRM methods which were recently validated for individual marker determination. The multiplexing method reduces sample volume, handling time per sample, and reagent consumption and shows good linearity, recovery, quantitative measurements, and sensitivity with lower limit of detection (LLOD) values of 7.2 pM (=90 pg/mL) and 4.5 pM (=210 pg/mL) and lower limit of quantitation (LLOQ) values of 24 pM (=300 pg/mL) and 15 pM (=700 pg/mL), for total ProGRP and γ-NSE, respectively. The novel aspect of this approach is the multiplexing of ProGRP and NSE with the additional ability to perform fingerprinting by the selective determination of ProGRP isoform 1, ProGRP isoform 3, and total ProGRP, as well as the α- and the γ-subunit of NSE isoenzymes. Six serum samples from patients with SCLC were analyzed to demonstrate the methods feasibility to simultaneously differ between and individually quantify ProGRP, NSE, and their isoform and isoenzyme variants, respectively. Both the presence of and variation between all the isoforms and isoenzymes, as well as covarying results with the conventional immunometric assays for total ProGRP and γ-NSE, were seen in the analyses of patient serum samples.

Epitope analysis and detection of human chorionic gonadotropin (hCG) variants by monoclonal antibodies and mass spectrometry.

Human chorionic gonadotropin (hCG) is an important marker for pregnancy, pregnancy-related disorders, and various cancers. Different molecular forms of hCG occur in different clinical conditions, and these can be distinguished with immunoassays using well-characterized monoclonal antibodies. Exact knowledge of the epitopes of the antibodies used is crucial for the design of assays with desired specificity. The epitopes of many hCG antibodies have been determined by comparing their reactivity with six 1st International Reference Reagents (IRRs) for hCG, but the specificity of some antibodies remains to be exactly defined. We have therefore studied the reactivity of 30 monoclonal antibodies (mAbs) with the six 1st IRRs for hCG, and variants were investigated using immunoaffinity extraction combined with liquid chromatography-mass spectrometry (LC-MS/MS) for the detection of hCG variants by specific tryptic signature peptides. Each of the mAbs had previously been characterized with regard to epitope specificity in the 2nd Tissue Differentiation Workshop on hCG of the International Society of Oncology and BioMarkers (ISOBM). Simultaneous identification of different hCG variants by LC-MS/MS confirmed that two standards used for mAb characterization, nicked hCG (hCGn, 1st IRR 99/642) and nicked ß subunit of hCG (hCGßn, 1st IRR 99/692), are heterogeneous, being composed of two major variants each: hCGn44/45 and hCGn47/48 as well as hCGßn44/45 and hCGß47/48. Furthermore, MS revealed cross-contamination by non-nicked hCG of the 1st IRR hCGn (99/642) standard. This information enabled fine-tuning of the previous epitope classifications of mAbs specific for heterodimeric hCG (c-mAbs). LC-MS/MS confirmed that c2-mAbs and most c1-mAbs did not recognize hCGn as the observed response in radioimmunoassays obviously resulted from the contamination of hCGn with hCG. Thus, c1 and c2 epitopes are partially dependent on hCGß peptide loop 2. c3-mAbs recognized both hCG and hCGn. It appeared that c-mAbs cannot discriminate between hCGn44/45 and hCGn47/48 as they either recognize both or neither variant. For most mAbs directed against hCGß, epitope specificity determined by LC-MS/MS was highly concordant with that obtained using standard immunological methods. In analogy to c-mAbs, hCGß-mAbs cannot discern between hCGßn44/45, hCGßn47/48, or intact hCGß as all 15 mAbs recognizing hCGß also recognized both nicked variants irrespective of which of the three major hCGß antigenic domains their epitopes were located within: on the caps of peptide loops 1 and 3, around the cystine knot, or along the hCGßCTP. LC-MS/MS confirmed that their epitopes were not located on hCGß peptide loop 2. Thus, LC-MS/MS provided in-depth information on hCG variant composition of hCGn (99/642) and hCGßn (99/692) and hCG variant specificity profiles and facilitated precise classification of the epitopes of anti-hCG mAbs. This has impact on the design of selective immunoassays.

Determining ProGRP and isoforms in lung and thyroid cancer patient samples: comparing an MS method with a routine clinical immunoassay.

This paper compares two methods to determine the tumor marker progastrin-releasing peptide (ProGRP): as routine assay, the automated time-resolved immunofluorometric assay (TR-IFMA), which allows total ProGRP determination; and the immunocapture liquid chromatography selected reaction monitoring mass spectrometry (LC-SRM-MS) method, which additionally allows isoform differentiation. The investigation included 60 serum samples from patients suffering from various cancer diseases which may cause elevated ProGRP levels (small cell lung carcinoma; SCLC, non-small cell lung carcinoma; NCLC; and medullary thyroid cancer; MTC, as well as some with unspecific diagnosis). The two methods showed good correlation (R (2) = 0.887). However, the MS method determines the total ProGRP concentration systematically approximately 30 % lower than the TR-IFMA, implying that the absolute values generated by the methods are not interchangeable. The MS method gives additional information about isoform levels in the samples, providing novel insight into isoform expression on the protein level.

Digging deeper into the field of the small cell lung cancer tumor marker ProGRP: a method for differentiation of its isoforms.

In this paper, we have used a newly developed immunocapture and LC-MS method to demonstrate for the first time the presence of protein isoforms 1 and 3 of the small cell lung cancer (SCLC) marker progastrin-releasing peptide (ProGRP) in sera. In addition, the method allows for indirect determination of the relative presence of the other known isoform of ProGRP, also known as ProGRP isoform 2. This new method is able to determine total ProGRP as a marker in sera at clinically relevant levels and to differentiate between isoforms at the low-pM level through combining selective sample preparation by immunoextraction, tryptic digestion, and separation followed by detection with LC-SRM-MS of the signature peptides, NLLGLIEAK (total ProGRP), LSAPGSQR (ProGRP isoform 1), and DLVDSLLQVLNVK (ProGRP isoform 3), with accuracies ≤ 25% for lower limit of quantification (LLOQ) and precisions ≤ 33%. By analyzing serum samples from four patients diagnosed with SCLC using the here described new and fully validated method, the ability is shown to both determine total ProGRP concentration and to differentiate between ProGRP isoforms 1 and 3 in one single run. Quantification of various ProGRP isoforms in one single run may be helpful for further understanding of the underlying biochemical processes in SCLC and differentiation of small cell lung cancer.

Sports drug testing using immuno-MS: clinical study comprising administration of human chorionic gonadotropin to males.

The applicability of a mass spectrometry (MS)-based method for determination of various forms of human chorionic gonadotropin (hCG) in doping analysis was demonstrated. A clinical study involving the hCG-containing pharmaceuticals Pregnyl and Ovitrelle was carried out, comprising a single injection of one pharmaceutical per participant to a total of 24 healthy male voluntaries. Hereafter, serum and urine samples were collected over a period of 14 days. The analysis of the samples using immuno-MS demonstrated elimination profiles of intact hCG for both pharmaceuticals, with last day of detection following administration at day 7 in serum, and at day 10 in urine, at limit of detections as defined by the World Anti-Doping Agency. Furthermore, the method allowed detection and differentiation of the various forms of hCG known to be present in serum and urine as a function of metabolism. For both pharmaceuticals, only the intact hCG was detected in serum, whereas in urine the injection of Pregnyl as hCG source (containing urinary hCG, i.e., most hCG variants) was shown to generate a more complex hCG variant pattern compared to Ovitrelle (contains only intact hCG). By detecting hCG using this MS-based approach in doping analysis, strong analytical evidence is provided minimizing the risk of false-positive and false-negative results.

Digital studying in times of COVID-19: teacher- and student-related aspects of learning success in german higher education.

In the wake of COVID-19, study conditions in Europe have changed dramatically. To limit contact between students and teachers, since March 2020 teaching has largely taken place digitally (remotely via digital means) and in private. Because the success of digital learning likely relies on many factors beyond good digital infrastructure conditions, this article focuses on which aspects, at both the teacher and the student levels, promote digital learning success. The large-scale student survey "Studying in Times of the Corona Pandemic" conducted at German universities and universities of applied sciences in the summer semester of 2020 offers data on how COVID-19 has affected several aspects of university studying in Germany. Here, we consider this data within the theoretical framework "theory of transactional distance" introduced by Moore (in: Moore (ed) Handbook of distance education, Routledge, 2018), according to which the success of digital teaching is influenced by dialogue, structure, and learner autonomy. Based on various regression analyses, our results show that several (digital) framework conditions must be created on both the teacher and student levels to achieve sufficient digital learning success. In this sense, our findings provide guidance on which aspects institutions of higher education should focus on when developing or updating their digitalization strategies. In accordance with collaborative learning approaches a key factor for learning success appears to be enabling peer-to-peer interactions. This finding supports our prediction that the possibility of engaging in interactive learning activities is crucial for students' learning experience, as it might reduce the perception of transactional distance and allow for social exchange. The strongest predictor of students' learning success turned out to be the (perceived) digital competencies of the teachers. This finding clearly emphasizes that teachers must be qualified to address the very specific challenges of teaching in digital contexts and indicates that universities may need to implement more teacher qualification programs. Supplementary Information: The online version contains supplementary material available at 10.1186/s41239-023-00382-w.

Immuno-MS based targeted proteomics: highly specific, sensitive, and reproducible human chorionic gonadotropin determination for clinical diagnostics and doping analysis.

The human chorionic gonadotropin (hCG) proteins constitute a diverse group of molecules that displays biomarker value in pregnancy detection and cancer diagnostics, as well as in doping analysis. For the quantification of hCGß and qualitative differentiation between other hCG variants in a selective, sensitive, and reproducible manner, the targeted proteomics approach based on mass spectrometric (MS) selected reaction monitoring (SRM) detection was exploited. By optimizing immunoaffinity extraction using monoclonal antibodies coated to magnetic beads, access was granted for the MS to the low-abundance target proteins, ensuring proper sensitivity with limits of detection (LODs) of 2 and 5 IU/L, respectively, for urine and serum samples. Validation according to key elements and recommendations defined by the European Medicines Agency in Guideline on Validation of Bioanalytical Methods was performed. For both matrixes this demonstrated good within-day precision results (within 20% for the lowest concentration, and within 15% for the medium and high concentration), good accuracy results (within 15% for all concentrations), and proper linearity, >0.997 for serum and of 0.999 for urine, in the concentration range up to 5000 IU/L. The method's application in clinical diagnostics was tested on samples from a pregnant woman and from patients previously diagnosed with testicular cancer. For doping analysis, samples from one man having received injection of the hCG-containing pharmaceutical Pregnyl were analyzed. The method proved to be quantitatively accurate with indisputable identification specificity, reducing risks of false positive and false negative results. The successfully validated method advocates thus for more extended use of MS in routine analysis.

The new molecular markers DDIT3, STT3A, ARG2 and FAM129A are not useful in diagnosing thyroid follicular tumors.

Preoperative characterization of thyroid follicular lesions is challenging. Fine-needle aspiration specimens cannot differentiate follicular carcinomas from benign follicular neoplasias. Recently, promising markers have been detected using modern molecular techniques. We conducted a retrospective study to confirm the usefulness of immunohistochemical staining for the protein markers, DDIT3, STT3A (ITM1), ARG2 and FAM129A (C1orf24) in separating benign and malignant thyroid follicular lesions. Formalin-fixed, paraffin-embedded thyroid tissue from 30 in-house cases (15 follicular carcinomas and 15 follicular adenomas), as well as 8 follicular carcinomas and 21 follicular adenomas on tissue microarray slides were stained immunohistochemically for DDIT3, STT3A, ARG2 and FAM129A expression. Control tissue consisted of thyroid parenchyma adjacent to the tumors and 11 separate cases of normal thyroid parenchyma. All in-house cases of follicular adenomas, follicular carcinomas and adjacent normal thyroid tissue showed positive immunostaining with anti-DDIT3 and anti-STT3A. Anti-ARG2 and anti-FAM129A polyclonal antibodies showed positive staining in 20 and 60% of in-house follicular adenomas, and 40 and 87% of in-house follicular carcinomas, respectively. Monoclonal anti-FAM129A demonstrated positive staining in 13 and 33% of in-house follicular adenomas and follicular carcinomas, respectively. Polyclonal anti-DDIT3, -STT3A and -FAM129A antibodies showed positive staining in all tissue microarray slides of follicular carcinoma and in 76, 85 and 81% of the follicular adenomas, respectively. Monoclonal anti-STT3A stained 81% of the follicular adenoma cores. Anti-ARG2 stained positive in 13% of follicular carcinomas and 10% of follicular adenomas on the tissue microarray slides. In conclusion, DDIT3, STT3A, ARG2 and FAM129A immunohistochemistry does not appear to be useful in the diagnosis of thyroid follicular neoplasias, as they do not reliably distinguish follicular thyroid carcinoma from follicular thyroid adenoma.

Investigation of nonspecific cross-reacting antigen 2 as a prognostic biomarker in bone marrow plasma from colorectal cancer patients.

Carcinoembryonic antigen (CEA) is still the only routinely used biomarker in colorectal cancer (CRC), but its utility is hampered by poor specificity and sensitivity, and the search for novel biomarkers is highly warranted. The nonspecific cross-reacting antigen 2 (NCA-2), a truncated CEA species molecule which is transcribed from the same gene, has been suggested as an alternative biomarker to CEA. In the present work, specific immunofluorometric assays were used for detection of NCA-2 and full-length CEA in bone marrow plasma from 277 CRC patients to assess their value as prognostic biomarkers, and detection was also performed in tumor tissue and a CRC cell line. Elevated plasma CEA was associated with advanced tumor stage at diagnosis and adverse patient outcome, while for NCA-2, although the same trends were observed, no additional prognostic information was gained. While specific detection of NCA-2 was clearly achieved in plasma samples, cross-reactivity with full-length CEA was observed when the antigen was exposed to common fixation chemicals. The results from this study indicate that NCA-2 is probably not a prognostic biomarker in CRC and, furthermore, underline the issue of antibody specificity when investigating CEA species molecules.

Characteristics and clinical validity of two immunoassays for ProGRP.

Progastrin-releasing peptide (proGRP) is a promising serum tumor marker for small cell lung cancer (SCLC). We have tested assay specificity and performed a correlation study between a recently developed time-resolved immunofluorometric assay (TR-IFMA) for proGRP and the established Advanced Life Science Institute (ALSI) ELISA method. Between-method correlation and comparison of clinical performance were studied in 481 individuals, among them, 178 lung cancers, 84 benign diseases of the lung, and 219 healthy controls. Follow-up time >6 years was observed for 89 patients with SCLC. The two assays had quite different epitope specificities where the TR-IFMA recognized a considerable smaller proGRP fragment than the ALSI ELISA. However, the correlation between the two methods for elevated proGRP values (>85 ng/l) was good (ρ = 0.948). Both assays displayed good discrimination between benign lung diseases and SCLC. The cut-off values for positive classification of SCLC versus non-small cell lung cancers and benign lung diseases at >95% specificity were 85 ng/l for the TR-IFMA and 42 ng/l for the ALSI ELISA. Both proGRP assays showed good clinical validity. However, due to differences in the recommended cut-off values, switching methods is not recommended. There was a significant difference in survival of patients with TR-IFMA proGRP values over the cut-off (85 ng/l) compared with patients with values under the cut-off, p = 0.0002. In contrast, the ALSI ELISA assay failed to provide statistically significant prognostic information, p = 0.066.

TD-12 workshop report: characterization of monoclonal antibodies to neuron-specific enolase.

Twelve antibodies to neuron-specific enolase (NSE) have been evaluated by four working groups. Human brain γγ-enolase, neuroblastoma-derived αγ-enolase, and recombinant γγ-enolase were used to determine antibody specificity and binding kinetics. All antibodies were found to be specific for the γ-isoform. It was possible to assign 11 of the antibodies to at least five epitope groups based on cross-inhibition experiments, QCM and SPR technology, and immunoassay combinations. Antibodies 9601 and 9602 showed the highest affinity for both native and recombinant γγ-enolase. Immunometric assays for both γγ- and αγ-enolase could be made by pairing 9601 with most of the other ISOBM antibodies. Antibodies differed in their ability to recognize native αγ-enolase, native γγ-enolase, and recombinant γγ-enolase. Some immunometric assay combinations appear to favor the detection of heterodimeric αγ-enolase over the homodimeric γγ-enolase. Although the majority of the antibodies failed to detect human NSE or recombinant NSE in Western blots, mAb 9601 recognized both, while E17 and 18E5 were specific for human NSE.

TD-11 workshop report: characterization of monoclonal antibodies to S100 proteins.

Fourteen monoclonal antibodies with specificity against native or recombinant antigens within the S100 family were investigated with regard to immunoreactivity. The specificities of the antibodies were studied using ELISA tests, Western blotting epitope mapping using competitive assays, and QCM technology. The mimotopes of antibodies against S100A4 were determined by random peptide phage display libraries. Antibody specificity was also tested by IHC and pair combinations evaluated for construction of immunoradiometric assays for S100B. Out of the 14 antibodies included in this report eight demonstrated specificity to S100B, namely MAbs 4E3, 4D2, S23, S53, 6G1, S21, S36, and 8B10. This reactivity could be classified into four different epitope groups using competing studies. Several of these MAbs did display minor reactivity to other S100 proteins when they were presented in denatured form. Only one of the antibodies, MAb 3B10, displayed preferential reactivity to S100A1; however, it also showed partial cross-reactivity with S100A10 and S100A13. Three antibodies, MAbs 20.1, 22.3, and S195, were specific for recombinant S100A4 in solution. Western blot revealed that MAb 20.1 and 22.3 recognized linear epitopes of S100A4, while MAb S195 reacted with a conformational dependent epitope. Surprisingly, MAb 14B3 did not demonstrate any reactivity to the panel of antigens used in this study.

"You Are Not Alone"-Opportunities and Challenges for University Students' Collaborative Engagement When Dealing With Online Information About COVID-19.

In view of the COVID-19 pandemic, students had to cope with the challenging situation of handling a vast amount of potentially conflicting online information while staying informed. Reading conflicting scientific information has been shown to require cognitive effort for one to integrate it successfully, but reading such information during a crisis-such as the COVID-19 pandemic-may cause additional emotional stress, as students also had to cope with critical aspects of the pandemic (e.g., physical distancing and uncertainty). Different studies have indicated that in crises, stress can be relieved by seeking online social support (as a coping strategy). Similarly, working together (as collaborative learning) can also help people more critically discuss information on a cognitive level. Based on the approaches of online collaborative learning and online social support seeking, we were interested in whether an individual vs. collaborative communication setting would lead to any differences in students' cognitive as well as emotional engagement with conflicting information about COVID-19. In a 2 × 2 mixed design, N = 109 education science students were exposed to two conflicting texts regarding COVID-19 testing that contained current scientific information. The online experiment was conducted in Germany in April 2020, which was the beginning of lockdown in that country. After reading the two texts, participants were asked to reflect on their engagement with the conflicting information either individually (individual group, n = 49) or via chat collaboratively (collaboration group, n = 60 in 30 dyads). With respect to participants' written reflections (content-analyzed regarding cognitive as well as emotional engagement), participants in the collaborative group, compared to those in the individual group, more often discussed the pandemic in general and less often engaged emotionally when discussing the evidence from texts. All participants reported higher perceived information overload, lower self-efficacy in sourcing information about COVID-19, and higher active coping strategies after the reflection task compared to before reading the information, with no significant differences between the collaborative and individual groups. We discuss these findings regarding any opportunities and challenges that arise in online collaboration between students for cognitive and emotional engagement when handling conflicting information about COVID-19.

Liquid chromatography mass spectrometry based characterization of epitope configurations.

Here we evaluate a quick and easy tool for determination of epitope configuration using immunocapture and liquid chromatography mass spectrometry (LC-MS) subsequent to pre-treatment of the target protein to disrupt its three-dimensional structure. The approach can be a valuable screening tool to identify antibodies that can be used in peptide capture by anti-protein antibodies. The experimental set-up was established using seven monoclonal antibodies (mAbs) with known linear or conformational epitope recognition. The mAbs were developed to target either of the two biomarkers, progastrin releasing peptide (ProGRP) or human chorionic gonadotropin (hCG). Best coherence with established epitope configuration was seen when using both denaturation, reduction and alkylation as pre-treatment method of the proteins (≥70% reduction in MS signal intensity compared to control) prior to immunocapture and LC-MS determination. The final method was used to determine the epitope configuration of four anti-thyroglobulin mAbs with unknown epitope configuration; all four mAbs showed configurational epitope recognition. These results were also supported by western blots of native, and reduced and alkylated protein using three of the evaluated mAbs, and by analysis native, and reduced and alkylated protein in a routine immunofluorometric assay employing the four evaluated antibodies.