Synthesis and unexpected binding of monofluorinated N,N'-diacetylchitobiose and LacdiNAc to wheat germ agglutinin.

Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert them into 19F NMR-active probes. However, monofluorination of monovalent carbohydrate ligands often leads to a decreased or completely lost affinity. By chemical glycosylation, we synthesized the full series of methyl ß-glycosides of N,N'-diacetylchitobiose (GlcNAcß(1-4)GlcNAcß1-OMe) and LacdiNAc (GalNAcß(1-4)GlcNAcß1-OMe) systematically monofluorinated at all hydroxyl positions. A competitive enzyme-linked lectin assay revealed that the fluorination at the 6'-position of chitobioside resulted in an unprecedented increase in affinity to wheat germ agglutinin (WGA) by one order of magnitude. For the first time, we have characterized the binding profile of a previously underexplored WGA ligand LacdiNAc. Surprisingly, 4'-fluoro-LacdiNAc bound WGA even stronger than unmodified LacdiNAc. These observations were interpreted using molecular dynamic calculations along with STD and transferred NOESY NMR techniques, which gave evidence for the strengthening of CH/π interactions after deoxyfluorination of the side chain of the non-reducing GlcNAc. These results highlight the potential of fluorinated glycomimetics as high-affinity ligands of lectins and 19F NMR-active probes.

Pseudotyped Viruses for Influenza.

We have developed an influenza hemagglutinin (HA) pseudotype (PV) library encompassing all influenza A (IAV) subtypes from HA1-HA18, influenza B (IBV) subtypes (both lineages), representative influenza C (ICV), and influenza D (IDV) viruses. These influenza HA (or hemagglutinin-esterase fusion (HEF) for ICV and IDV) pseudotypes have been used in a pseudotype microneutralization assay (pMN), an optimized luciferase reporter assay, that is highly sensitive and specific for detecting neutralizing antibodies against influenza viruses. This has been an invaluable tool in detecting the humoral immune response against specific hemagglutinin or hemagglutinin-esterase fusion proteins for IAV to IDV in serum samples and for screening antibodies for their neutralizing abilities. Additionally, we have also produced influenza neuraminidase (NA) pseudotypes for IAV N1-N9 subtypes and IBV lineages. We have utilized these NA-PV as surrogate antigens in in vitro assays to assess vaccine immunogenicity. These NA PV have been employed as the source of neuraminidase enzyme activity in a pseudotype enzyme-linked lectin assay (pELLA) that is able to measure neuraminidase inhibition (NI) titers of reference antisera, monoclonal antibodies, and postvaccination sera. Here we show the production of influenza HA, HEF, and NA PV and their employment as substitutes for wild-type viruses in influenza serological and neutralization assays. We also introduce AutoPlate, an easily accessible web app that can analyze data from pMN and pELLA quickly and efficiently, plotting inhibition curves and calculating half-maximal concentration (IC50) neutralizing antibody titers. These serological techniques coupled with user-friendly analysis tools are faster, safer, inexpensive alternatives to classical influenza assays while also offering the reliability and reproducibility to advance influenza research and make it more accessible to laboratories around the world.

Ella versus Simoa Serum Neurofilament Assessment to Monitor Treatment Response in Highly Active Multiple Sclerosis Patients.

The measurement of serum neurofilament light chain (sNfL) is of growing importance in the field of neurology. In the management of multiple sclerosis, it can serve as a useful marker to assess disease activity and treatment response. This paper compares two available methods, namely the Single Molecule Array (Simoa) and the Ella microfluid platform, to measure longitudinal sNfL levels of 42 highly active multiple sclerosis patients treated with alemtuzumab over a period of 36 months. In order to assess the methods agreement, Bland-Altman plots and Passing-Bablok regression were analyzed. Here, we show that despite the fact that Ella measures around 24% higher values than Simoa, both are equally suitable for longitudinal sNfL monitoring.

Broadly Inhibiting Antineuraminidase Monoclonal Antibodies Induced by Trivalent Influenza Vaccine and H7N9 Infection in Humans.

The majority of antibodies induced by influenza neuraminidase (NA), like those against hemagglutinin (HA), are relatively specific to viruses isolated within a limited time window, as seen in serological studies and the analysis of many murine monoclonal antibodies (MAbs). We report three broadly reactive human MAbs targeting N1 NA. Two were isolated from a young adult vaccinated with trivalent influenza vaccine (TIV), which inhibited N1 NA from viruses isolated from humans over a period of a hundred years. The third antibody, isolated from a child with acute mild H7N9 infection, inhibited both group 1 N1 and group 2 N9 NAs. In addition, the antibodies cross-inhibited the N1 NAs of highly pathogenic avian H5N1 influenza viruses. These antibodies are protective in prophylaxis against seasonal H1N1 viruses in mice. This study demonstrates that human antibodies to N1 NA with exceptional cross-reactivity can be recalled by vaccination and highlights the importance of standardizing the NA antigen in seasonal vaccines to offer optimal protection.IMPORTANCE Antibodies to the influenza virus NA can provide protection against influenza disease. Analysis of human antibodies to NA lags behind that of antibodies to HA. We show that human monoclonal antibodies against NA induced by vaccination and infection can be very broadly reactive, with the ability to inhibit a wide spectrum of N1 NAs on viruses isolated between 1918 and 2018. This suggests that antibodies to NA may be a useful therapy and that the efficacy of influenza vaccines could be enhanced by ensuring the appropriate content of NA antigen.

SX-Ella Stent Danis Effectively Controls Refractory Variceal Bleed in Patients with Acute-on-Chronic Liver Failure.

BACKGROUND AND AIMS: Almost 10% of bleeding episodes are refractory to combination of vasoactive agent and endotherapy, and are associated with a mortality up to 50%. Severity of liver disease and high portal pressure are mainly responsible for it. TIPS cannot be used in these patients due to high MELD score. We aimed to evaluate the efficacy of self-expandable DE stents for control of refractory variceal bleeds in patients with ACLF. METHODS: Acute-on-chronic liver failure patients (n = 88, mean age 47.3 ± 10.9 years) with refractory variceal bleeds received either DE stent (Gr. A, n = 35) or continued with repeat endotherapy and vasoactive drug (Gr.B, n = 53). Matching by propensity risk score (PRS) was done to avoid selection bias. Competing risk Cox regression analysis was done to identify event-specific, i.e., gastrointestinal bleed-related death. RESULTS: Majority (78.4%) of patients were alcoholic with MELD score of 45.9 ± 20.1. Control of initial bleeding was significantly more in the DE stent group as compared to controls in both pre-match (89 vs. 37%; p < 0.001) and PRS-matched cohorts (73 vs. 32%; 0.007). Further, bleed-related death was also significantly lower in DE group as compared to controls in both pre-match (14 vs. 64%; p = 0.001) and PRS-matched cohorts (6 vs. 56%; p = 0.001). In a multivariate competing risk Cox model, patients who underwent DE stenting had reduced mortality in both pre-match (p = 0.04, HR 0.36, 95% CI 0.13-0.96) and PRS-matched cohorts (p < 0.001, HR 0.21, 95% CI 0.08-0.51). CONCLUSIONS: Self-expandable DE stents are very effective in control of refractory variceal bleeding and reduced mortality in patients with severe liver failure.

ß-Casein(94-123)-derived peptides differently modulate production of mucins in intestinal goblet cells.

We recently reported the identification of a peptide from yoghurts with promising potential for intestinal health: the sequence (94-123) of bovine ß-casein. This peptide, composed of 30 amino acid residues, maintains intestinal homoeostasis through production of the secreted mucin MUC2 and of the transmembrane-associated mucin MUC4. Our study aimed to search for the minimal sequence responsible for the biological activity of ß-CN(94-123) by using several strategies based on (i) known bioactive peptides encrypted in ß-CN(94-123), (ii) in silico prediction of peptides reactivity and (iii) digestion of ß-CN(94-123) by enzymes of intestinal brush border membranes. The revealed sequences were tested in vitro on human intestinal mucus-producing HT29-MTX cells. We demonstrated that ß-CN(108-113) (an ACE-inhibitory peptide) and ß-CN(114-119) (an opioid peptide named neocasomorphin-6) up-regulated MUC4 expression whereas levels of the secreted mucins MUC2 and MUC5AC remained unchanged. The digestion of ß-CN(94-123) by intestinal enzymes showed that the peptides ß-CN(94-108) and ß-CN(117-123) were present throughout 1·5 to 3 h of digestion, respectively. These two peptides raised MUC5AC expression while ß-CN(117-123) also induced a decrease in the level of MUC2 mRNA and protein. In addition, this inhibitory effect was reproduced in airway epithelial cells. In conclusion, ß-CN(94-123) is a multifunctional molecule but only the sequence of 30 amino acids has a stimulating effect on the production of MUC2, a crucial factor of intestinal protection.

A novel biomarker assay qualification strategy for rare human matrices - a case study of biomarkers in aqueous humor.

Background: Measuring pharmacodynamic biomarkers near the therapeutic site of action presents considerable challenges for sites with limited matrix volume or difficult access. Bioanalytical method qualification requires the use of numerous matrix samples, which is problematic for rare matrices. The aim of this study was to design and implement a streamlined, fit-for-purpose strategy for qualification of biomarker assays in rare matrices. Materials & methods: A multiplexed biomarker immunoassay was developed in human aqueous humor. Results: Our strategy was successfully implemented, providing characterization of assay performance while reducing number of samples in assay qualification. Our assay was used in clinical trial support for an ophthalmic drug candidate. Conclusion: Our results indicate this approach can be applied to other early stage drug development programs facing similar challenges.

Bioanalytical assay development and qualification requires the use of numerous matrix samples, which is problematic for rare matrices. We designed and successfully implemented a streamlined strategy for qualification of biomarker assays in rare matrices.

Analytical assessment and validation of the ProteinSimple ELLA serum B-cell maturation antigen assay.

Objectives: Soluble B-Cell Maturation Antigen (sBCMA) is a degradation product of plasma cell-bound BCMA found in serum. Serum sBCMA concentrations correlate with bone marrow plasma cellularity, making it an attractive biomarker for monitoring plasma cell disorders, such as multiple myeloma. Here we evaluated the automated BCMA immunoassay for the ProteinSimple ELLA, for the analysis of sBCMA. Design & methods: Inter and intra-run precision was assessed through replicate sBCMA measurements at 3 different concentration levels. Linearity was determined through serial dilution of a high sBMCA patient sample. Accuracy was assessed through split specimen analysis on two separate lots of reagents. Stability was assessed at 3 temperature levels over 14 days. Cross-reactivity was assessed on BCMA targeting and non-targeting chemotherapeutics. A reference range was established through the analysis of 146 healthy donor samples. The effect of endogenous interferents was assessed through spiking and recovery studies. Results: Inter and intra-run precision studies afforded CVs of <10% at all three concentration levels. Analytical measurement range was confirmed from 0.1 to 7 ng/mL. Accuracy studies afforded a slope of 0.976, intercept of 1.22, R2 of 0.996. Assayed sBCMA values were unaffected by endogenous interferents and non-BMCA targeting antibodies. BCMA targeting therapeutics negatively affected assayed sBCMA concentrations. The reference range was established at 19-58 ng/mL sBCMA is analytically stable. Conclusions: The ProteinSimple ELLA sBCMA assay shows acceptable performance for the clinical assessment of sBCMA. The assay was highly affected by BCMA targeting therapeutics, thereby patients undergoing this therapy should not have their sBCMA levels assessed by this method.

Emergent rescue transjugular intrahepatic portosystemic shunt within 8 h improves survival in patients with refractory variceal bleed.

BACKGROUND: Transjugular-intrahepatic portosystemic-shunt (TIPS) and SX-Ella stent Danis (DE stent) are available rescue therapies for refractory variceal bleeding in cirrhosis. Any delay in appropriate therapy is associated with high mortality. Determining the best timing for rescue TIPS is crucial and largely unknown. METHODS: Cirrhotic patients with refractory variceal bleed (n = 121) who underwent rescue TIPS within 24-h (n = 66) were included. Their early rebleed (upto 42 days) rate, 6-week and 1-year survival were compared with matched patients who underwent rescue DE stent (n = 55). Outcomes based on timing of TIPS (within 8-h/8-24 h) were also analyzed. RESULTS: At baseline, patients who received rescue DE stent were sicker with higher MELD score (27.6 ± 8.3 vs. 22.3 ± 7.9; p = 0.001), active bleeding at endoscopy (54.5% vs. 34.8%; p = 0.03) compared to TIPS-group. After propensity score matching, adjusting for MELD-Na score and non-bleed complications, DE patients (n = 34) had higher mortality at 6-week (17/34; 50%) and 1-year (29/34; 85.3%) compared to TIPS-group (20.6% and 38.2%, respectively; both p < 0.02), with higher rebleeding rate (10/34; 29.4% vs. 1/34; 2.9%, p = 0.003). Rescue TIPS placed within 8-h compared with 8-24 h had lower 6-week (48.6% vs. 12.9%; p = 0.003) and 1-year mortality (62.9% vs. 16.1%, p = 0.001) despite comparable rebleed rates (2/31; 6.5% vs. 2/35;5.7%; p = 0.90). Post-TIPS Portal pressure gradient at 6-weeks and 1-year was comparable between survivors and non-survivors. Active bleeding at endoscopy [HR = 11.8; 95% CI 2.96-47.53], presence of AKI [HR = 5.8; 95% CI 1.92-17.41], MELD-Na > 24 [HR = 1.1; 95% CI 1.0-1.17], mean arterial pressure > 64.5 mmHg [HR = 0.8; 95% CI 0.75-0.92] independently predicted 6-week mortality in rescue TIPS-group. CONCLUSIONS: Rescue TIPS placement preferably within 8-h of refractory variceal bleed improves short- and long-term survival. It provides better outcome than DE stent for control of bleeding and prevention of rebleeding, even in patients with high MELD-Na score.

Differential recognition of influenza A virus H1N1 neuraminidase by DNA vaccine-induced antibodies in pigs and ferrets.

Neuraminidase (NA) accounts for approximately 10-20% of the total glycoproteins on the surface of influenza viruses. It cleaves sialic acids on glycoproteins, which facilitates virus entry into the airways by cleaving heavily glycosylated mucins in mucus and the release of progeny virus from the surface of infected cells. These functions make NA an attractive vaccine target. To inform rational vaccine design, we define the functionality of influenza DNA vaccine-induced NA-specific antibodies relative to antigenic sites in pigs and ferrets challenged with a vaccine-homologous A/California/7/2009(H1N1)pdm09 strain. Sera collected pre-vaccination, post-vaccination and post-challenge were analyzed for antibody-mediated inhibition of NA activity using a recombinant H7N1CA09 virus. Antigenic sites were further identified with linear and conformational peptide microarrays spanning the full NA of A/California/04/2009(H1N1)pdm09. Vaccine-induced NA-specific antibodies inhibited the enzymatic function of NA in both animal models. The antibodies target critical sites of NA such as the enzymatic site, second sialic binding site and framework residues, shown here by high-resolution epitope mapping. New possible antigenic sites were identified that potentially block the catalytic activity of NA, including an epitope recognized solely in pigs and ferrets with neuraminidase inhibition, which could be a key antigenic site affecting NA function. These findings show that our influenza DNA vaccine candidate induces NA-specific antibodies that target known critical sites, and new potential antigenic sites of NA, inhibiting the catalytic activity of NA.

Influenza A (N1-N9) and Influenza B (B/Victoria and B/Yamagata) Neuraminidase Pseudotypes as Tools for Pandemic Preparedness and Improved Influenza Vaccine Design.

To better understand how inhibition of the influenza neuraminidase (NA) protein contributes to protection against influenza, we produced lentiviral vectors pseudotyped with an avian H11 hemagglutinin (HA) and the NA of all influenza A (N1-N9) subtypes and influenza B (B/Victoria and B/Yamagata). These NA viral pseudotypes (PV) possess stable NA activity and can be utilized as target antigens in in vitro assays to assess vaccine immunogenicity. Employing these NA PV, we developed an enzyme-linked lectin assay (pELLA) for routine serology to measure neuraminidase inhibition (NI) titers of reference antisera, monoclonal antibodies and post-vaccination sera with various influenza antigens. We also show that the pELLA is more sensitive than the commercially available NA-Fluor™ in detecting NA inhibition in these samples. Our studies may lead to establishing the protective NA titer that contributes to NA-based immunity. This will aid in the design of superior, longer lasting and more broadly protective vaccines that can be employed together with HA-targeted vaccines in a pre-pandemic approach.

Validation of a Harmonized Enzyme-Linked-Lectin-Assay (ELLA-NI) Based Neuraminidase Inhibition Assay Standard Operating Procedure (SOP) for Quantification of N1 Influenza Antibodies and the Use of a Calibrator to Improve the Reproducibility of the ELLA-NI With Reverse Genetics Viral and Recombinant Neuraminidase Antigens: A FLUCOP Collaborative Study.

Current vaccination strategies against influenza focus on generating an antibody response against the viral haemagglutination surface protein, however there is increasing interest in neuraminidase (NA) as a target for vaccine development. A critical tool for development of vaccines that target NA or include an NA component is available validated serology assays for quantifying anti-NA antibodies. Additionally serology assays have a critical role in defining correlates of protection in vaccine development and licensure. Standardisation of these assays is important for consistent and accurate results. In this study we first validated a harmonized enzyme-linked lectin assay (ELLA)- Neuraminidase Inhibition (NI) SOP for N1 influenza antigen and demonstrated the assay was precise, linear, specific and robust within classical acceptance criteria for neutralization assays for vaccine testing. Secondly we tested this SOP with NA from influenza B viruses and showed the assay performed consistently with both influenza A and B antigens. Third, we demonstrated that recombinant NA (rNA) could be used as a source of antigen in ELLA-NI. In addition to validating a harmonized SOP we finally demonstrated a clear improvement in inter-laboratory agreement across several studies by using a calibrator. Importantly we showed that the use of a calibrator significantly improved agreement when using different sources of antigen in ELLA-NI, namely reverse genetics viruses and recombinant NA. We provide a freely available and detailed harmonized SOP for ELLA-NI. Our results add to the growing body of evidence in support of developing biological standards for influenza serology.

Inherent Serum Inhibition of Influenza Virus Neuraminidases.

Influenza virus vaccines have been designed for human and veterinary medicine. The development for broadly protective influenza virus vaccines has propelled the vaccine field to investigate and include neuraminidase (NA) components into new vaccine formulations. The antibody-mediated protection induced by NA vaccines is quantified by inhibition of sialic acid cleavage. Non-immune inhibitors against influenza viruses naturally occur in varying proportions in sera from different species. In this brief report, the inherent ability of raw animal sera to inhibit a panel of influenza virus NA was determined. Raw sera from the same species inhibited more than 50% of influenza viruses tested from four different subtypes, but the breadth of inhibiting NA activity depended on the source of sera. Furthermore, different influenza viruses were inhibited by different sources of sera. Overall, additional studies are needed to ensure that scientific methods are consistent across studies in order to compare NA inhibition results. Through future investigation into the differences between sera from different animal species and how they influence NA inhibition assays, there can be effective development of a broadly protective influenza virus vaccines for veterinary and human use.

Lectin-based assay for the determination of the inhibition activity of small molecule inhibitors of neuraminidases.

Small molecules inhibitors of neuraminidases (NAs) are ones of the most prospective molecules proposed for the treatment of influenza viruses. The determination of their inhibition activity in vitro is an important step during the development of antiviral drugs. However, the analytical methods typically used for the evaluation of NA activity and inhibition (fluorescence-based assays using MUNANA substrate or thiobarbituric acid assay, TBA) may suffer from interferences caused by tested inhibitors as signal quenching or self-fluorescence, moreover in TBA are used toxic and carcinogenic reagents. The determination of the NA activity can be effectively performed by alternative methods based on lectin - glycan recognition, usually as enzyme-linked lectin assay (ELLA). We have adapted the ELLA assay to a lectin-based assay in a microplate format with fluorescence detection for determination of NA inhibitory activity. We optimized our protocol and the developed method was tested using four different small molecule NA inhibitors or potential NA inhibitors (DANA, zanamivir, quercetin and α-mangostin) with three bacterial NAs (from Clostridium perfringens, Vibrio cholerae and Arthrobacter ureafaciens), and the IC50 values for NA inhibitors were determined. The inhibition effect of DANA was observed for all 3 tested NAs (IC50 = 10.1 µM for V. cholerae, 13.4 µM for C. perfringens and 402.9 µM for A. ureafaciens, respectively) and of Zanamivir only for NA from V. cholerae (IC50 = 101.9 µM). For both quercetin and α-mangostin, no inhibition effect to the tested NAs was observed. The main advantages of herein described method are good sensitivity due to fluorescent signal detection, the absence of the interference caused by fluorescent signal quenching by tested inhibitors, the use of natural substrates (glycoproteins) and the avoiding the use of toxic reagents.

A Novel Automated Immunoassay Platform to Evaluate the Association of Adiponectin and Leptin Levels with Breast Cancer Risk.

Adiponectin and leptin are adipokines secreted by the adipose tissue that are associated with several chronic diseases including cancer. We aimed to compare the immunoassay platform ELLA with an enzyme-linked immunosorbent assay (ELISA) kit and to assess whether the results of the association analyses with breast cancer risk were dependent on the assay used. We measured adiponectin and leptin with ELLA and ELISA on baseline serum samples of 116 Italian postmenopausal women enrolled in two international breast cancer prevention trials. Results were compared with Deming, Passing-Bablok regression and Bland-Altman plots. Disease-free survival was analyzed with the Cox model. There was a good correlation between the methods for adiponectin and leptin (r > 0.96). We found an increased breast cancer risk for very low adiponectin levels (HR for ELLA = 3.75; 95% CI: 1.37;10.25, p = 0.01), whereas no significant association was found for leptin levels. The disease-free survival curves were almost identical for values obtained with the two methods, for both biomarkers. The ELLA platform showed a good concordance with ELISA for adiponectin and leptin measurements. Our results support the association of very low adiponectin levels with postmenopausal breast cancer risk, irrespective of the method used. The ELLA platform is a time-saving system with high reproducibility, therefore we recommend its use for biomarker assessment.

AutoPlate: Rapid Dose-Response Curve Analysis for Biological Assays.

The emergence of COVID-19 has emphasised that biological assay data must be analysed quickly to develop safe, effective and timely vaccines/therapeutics. For viruses such as SARS-CoV-2, the primary way of measuring immune correlates of protection is through assays such as the pseudotype microneutralisation (pMN) assay, thanks to its safety and versatility. However, despite the presence of existing tools for data analysis such as PRISM and R the analysis of these assays remains cumbersome and time-consuming. We introduce an open-source R Shiny web application and R library (AutoPlate) to accelerate data analysis of dose-response curve immunoassays. Using example data from influenza studies, we show that AutoPlate improves on available analysis software in terms of ease of use, flexibility and speed. AutoPlate (https://philpalmer.shinyapps.io/AutoPlate/) is a tool for the use of laboratories and wider scientific community to accelerate the analysis of biological assays in the development of viral vaccines and therapeutics.

Detection and Titration of Influenza A Virus Neuraminidase Inhibiting (NAI) Antibodies Using an Enzyme-Linked Lectin Assay (ELLA).

The neuraminidase (NA) of influenza A viruses (IAV) is a structurally and antigenically important envelope glycoprotein. There are eleven known subtypes of NA of which two, N1 and N2, circulate in swine. The sialidase activity of NA is required for the release of nascent virus particles from infected cell membranes and inhibition of NA enzymatic activity can significantly reduce virus titers and duration of infection. Efforts to improve IAV vaccine technology in humans have focused on the generation of neuraminidase inhibiting (NAI) antibodies and should be considered in swine as well. The enzyme-linked lectin assay (ELLA) conducted in 96-well plates has enabled high-throughput analysis of serum samples for NAI antibody titers. Through the use of reverse genetics, custom antigen panels and antisera can be generated to encompass the antigenically diverse population of NA that circulate in swine. The ELLA is a robust method to assess NAI antibody titers and characterize the antigenic difference between NA antigens.

Expression, Purification, and Applications of the Recombinant Lectin PVL from Psathyrella velutina Specific for Terminal N-Acetyl-Glucosamine.

The lectin PVL from the mushroom Psathyrella velutina is the founding member of novel family of fungal lectins. It adopts a seven bladed ß-propeller presenting six binding sites specific for the recognition of non-reducing terminal N-acetyl-glucosamine (GlcNAc). The latest can be mainly found in glycoconjugates presenting truncated glycans where aberrant ß-GlcNAc terminated glycans represent tumor markers. It can also be found in O-GlcNAcylated proteins where disruption of the O-GlcNAcylation homeostasis is associated with many physiopathological states. The recombinant PVL lectin proved to be a very powerful tool for labelling terminal GlcNAc antigens displayed by extracellular glycoconjugates but also by O-GlcNAcylated proteins found in the cytoplasm and nucleus. This chapter will describe how to produce and purify recombinant PVL and several applications for rPVL as probe for the detection of terminal O-GlcNAc.

Glycosylation markers in cancer.

Alteration of glycosylation, a hallmark of cancer, results in the production of tumor-associated glycans or glycoproteins. These molecules are subsequently secreted or membrane-shed into the blood stream and thus serve as tumor-associated markers. Increased glycosylation in cancer is triggered by overexpression of glycoproteins that carry certain specific glycans, increase or decrease of nucleotide sugar donors and altered expression of glycosyltransferase and glycosidase enzymes. In this chapter, the biochemistry and function of glycoprotein, glycan and enzyme markers are reviewed. These glycosylation markers, applicable for detection and monitoring of cancer, include CA19-9, CA125, CEA, PSA and AFP. Because of their specific affinity to distinct sugar moieties, lectins are useful for developing assays to detect these tumor associated glycans and glycoproteins in clinical samples. As such, various enzyme-linked lectin assays (ELLA) have been developed for diagnosis, monitoring and prognosis. Because glycosylation changes occur early in cancer, the detection of tumor associated glycosylation markers using lectin based assays is an effective strategy to improve diagnosis and treatment resulting better outcomes clinically.

Use of Ella® to facilitate drug quantification and antidrug antibody detection in preclinical studies.

Aim: To explore the usability of the Ella® platform for preclinical analysis of therapeutic antibodies and antidrug antibodies (ADA). Experimental: Two well-established ELISAs for the measurement of human IgG and ADA were transferred to the Ella platform. ELISA and the Ella platform were compared using assay qualification data and results of preclinical sample analysis. Results: The performance and results of both assays on the Ella platform were comparable to those of ELISA. The Ella platform had several advantages, including time efficiency, low sample consumption and a high degree of automation. ADA were assessed on Ella for the first time. Conclusion: The Ella platform is a promising tool for the analysis of preclinical samples.