Therapeutic blood-brain barrier modulation and stroke treatment by a bioengineered FZD4-selective WNT surrogate in mice.

Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer's disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/ß-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we develop L6-F4-2, a non-lipidated, FZD4-specific surrogate which significantly improves subpicomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reverses neonatal retinal angiogenesis deficits, but also restores BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduces BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with potential applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.

Multi-laboratory validation of the xMAP-Food Allergen Detection Assay: A multiplex, antibody-based assay for the simultaneous detection of food allergens.

The increasing prevalence of individuals with multiple food allergies and the need to distinguish between foods containing homologous, cross-reactive proteins have made the use of single-analyte antibody-based methods (e.g., ELISAs) sometimes insufficient. These issues have resulted in the need to conduct multiple analyses and sometimes employ orthogonal methods like mass spectrometry or DNA-based methods for confirmatory purposes. The xMAP Food Allergen Detection Assay (xMAP FADA) was developed to solve this problem while also providing increased throughput and a modular design suitable for adapting to changes in analytical needs. The use of built-in redundancy provides the xMAP FADA with built-in confirmatory analytical capability by including complementary antibody bead sets and secondary analytical end points (e.g., ratio analysis and multi-antibody profiling). A measure of a method's utility is its performance when employed by analysts of varying expertise in multiple laboratory environments. To gauge this aspect, a multi-laboratory validation (MLV) was conducted with 11 participants of different levels of proficiency. The MLV entailed the analysis of incurred food samples in four problematic food matrices, meat sausage, orange juice, baked muffins, and dark chocolate. Except for a couple of instances, involving two confirmatory components in the analysis of baked muffins, the allergenic foods were detected by all participants at concentrations in the analytical samples comparable to ≤ 10 µg/g in the original food sample. In addition, despite high levels of inter-lab variance in the absolute intensities of the responses, the intra-laboratory reproducibility was sufficient to support analyses based on the calibration standards and direct comparison controls (DCCs) analyzed alongside the samples. In contrast, ratio analyses displayed inter-laboratory %CV (RSDR) values < 20%; presumably because the ratios are based on inherent properties of the antigenic elements. The excellent performance of the xMAP FADA when performed by analysts of varying proficiency indicates a reliability sufficient to meet analytical needs.

Robustness Testing of the xMAP Food Allergen Detection Assay: A Multiplex Assay for the Simultaneous Detection of Food Allergens.

ABSTRACT: The xMAP food allergen detection assay (xMAP FADA) can simultaneously detect 15 analytes (14 food allergens plus gluten) in one analysis. The xMAP FADA typically employs two antibody bead sets per analyte, providing built-in confirmation that is not available with other antibody-based assays. Before an analytical method can be used, its reliability must be assessed when conditions of the assay procedure are altered. This study was conducted to determine the effects on assay performance associated with changes in incubation temperature, amounts of the antibody bead cocktail, and concentrations of detection antibody and ß-mercaptoethanol in the reduced-denatured extraction buffer. The analysis of buffered-detergent extracts revealed lower responses at 22°C than at 37°C, but temperature had no effect on the analysis of reduced-denatured extracts. Changes in ß-mercaptoethanol and detection antibody concentrations had an effect on the detection of only milk in the reduced-denatured extracts. A slight change in the measured bead count was observed when one-fourth of the bead cocktail was used, and a large decrease in the bead count was noted when one-eighth of the recommended amount was used, but this number (≥25) was still sufficient to provide reliable results. Overall, the xMAP FADA was very robust to changes in the assay procedure, which may inadvertently occur.

Extension of xMAP Food Allergen Detection Assay To Include Sesame.

An estimated 0.1 to 0.2% of the North American population is allergic to sesame, and deaths due to anaphylactic shock have been reported. Detecting and quantifying sesame in various food samples is critical to safeguard the allergic population by ensuring accurate ingredient labeling. Because of the modular nature of the xMAP Food Allergen Detection Assay (FADA), it was possible through method extension to add sesame as a validated additional analyte. Because raw and toasted sesame are both commonly used and the two display significantly different antigenicity, three antibodies, one monoclonal and two polyclonal, were conjugated to bead sets to ensure reliable detection. The modified xMAP FADA successfully detected sesame incurred or spiked in baked muffins, spice mix, canola oil, and in both raw and toasted sesame oils with limit of quantitation values ≤ 1.3 ppm of sesame. Canola oil, sesame oil, toasted sesame oil, and olive oil inhibited sesame detection, as did the detection of sesame incurred in foods containing oil (e.g., hummus). Despite this inhibition, the xMAP FADA was still able to reliably detect sesame at levels throughout the dynamic range of the assay (22 to 750 ng of protein per mL) in all the foods examined. Further, the high signal-to-noise ratio of the lowest calibration standard and preliminary studies conjugating the antibodies at higher concentrations indicate an ability to increase the sensitivity of the assay should the need arise.

Single-Laboratory Validation of the Multiplex xMAP Food Allergen Detection Assay with Incurred Food Samples.

An xMAP Food Allergen Detection Assay (xMAP FADA) was developed to meet analytical needs when responding to complaints by individuals with multiple food allergies and to address potential ambiguities associated with cross-reactive proteins. A single-laboratory validation (SLV) was conducted to examine the reliability of the xMAP FADA to detect 15 analytes individually or as part of a mixture at more than six concentrations in four foods. The xMAP FADA reliably detected the analytes despite the incurred dark chocolate and incurred baked muffins displaying recoveries of 10-20% and <60%, respectively. The high reliability for recoveries less than 60% in part reflects the statistical strength of the design of the xMAP FADA. Only crustacean, egg, and milk incurred in dark chocolate were not reliably detected using the PBST-buffered-detergent protocol. Following the reduced-denatured protocol, no problems were encountered in the detection of milk, although egg did not display a dynamic response in dark chocolate. The ruggedness of the xMAP FADA was ascertained by the ability of novice analysts to detect food allergens in baked rice cookies. Despite one analyst losing >80% of the beads and the count for one bead set dropping to seven, the assay displayed only a decrease in precision (increased standard deviations) and a change in the ratios between complementary antibody pairs.

Cross-reactivity by botanicals used in dietary supplements and spices using the multiplex xMAP food allergen detection assay (xMAP FADA).

Food allergies affect some 15 million Americans. The only treatment for food allergies is a strict avoidance diet. To help ensure the reliability of food labels, analytical methods are employed; the most common being enzyme-linked immunosorbent assays (ELISAs). However, the commonly employed ELISAs are single analyte-specific and cannot distinguish between false positives due to cross-reactive homologous proteins; making the method of questionable utility for regulatory purposes when analyzing for unknown or multiple food allergens. Also, should the need arise to detect additional analytes, extensive research must be undertaken to develop new ELISAs. To address these and other limitations, a multiplex immunoassay, the xMAP® food allergen detection assay (xMAP FADA), was developed using 30 different antibodies against 14 different food allergens plus gluten. Besides incorporating two antibodies for the detection of most analytes, the xMAP FADA also relies on two different extraction protocols; providing multiple confirmatory end-points. Using the xMAP FADA, the cross-reactivities of 45 botanicals used in dietary supplements and spices commercially sold in the USA were assessed. Only a few displayed cross-reactivities with the antibodies in the xMAP FADA at levels exceeding 0.0001%. The utility of the xMAP FADA was exemplified by its ability to detect and distinguish between betel nut, saw palmetto, and acai which are in the same family as coconut. Other botanicals examined included allspice, amchur, anise seed, black pepper, caraway seed, cardamom, cayenne red pepper, sesame seed, poppy seed, white pepper, and wheat grass. The combination of direct antibody detection, multi-antibody profiling, high sensitivity, and a modular design made it possible for the xMAP FADA to distinguish between homologous antigens, provide multiple levels of built-in confirmatory analysis, and optimize the bead set cocktail to address specific needs.

Cross-reactivity profiles of legumes and tree nuts using the xMAP® multiplex food allergen detection assay.

The homology between proteins in legumes and tree nuts makes it common for individuals with food allergies to be allergic to multiple legumes and tree nuts. This propensity for allergenic and antigenic cross-reactivity means that commonly employed commercial immunodiagnostic assays (e.g., dipsticks) for the detection of food allergens may not always accurately detect, identify, and quantitate legumes and tree nuts unless additional orthogonal analytical methods or secondary measures of analysis are employed. The xMAP® Multiplex Food Allergen Detection Assay (FADA) was used to determine the cross-reactivity patterns and the utility of multi-antibody antigenic profiling to distinguish between legumes and tree nuts. Pure legumes and tree nuts extracted using buffered detergent displayed a high level of cross-reactivity that decreased upon dilution or by using a buffer (UD buffer) designed to increase the stringency of binding conditions and reduce the occurrence of false positives due to plant-derived lectins. Testing for unexpected food allergens or the screening for multiple food allergens often involves not knowing the identity of the allergen present, its concentration, or the degree of modification during processing. As such, the analytical response measured may represent multiple antigens of varying antigenicity (cross-reactivity). This problem of multiple potential analytes is usually unresolved and the focus becomes the primary analyte, the antigen the antibody was raised against, or quantitative interpretation of the content of the analytical sample problematic. The alternative solution offered here to this problem is the use of an antigenic profile as generated by the xMAP FADA using multiple antibodies (bead sets). By comparing the antigenic profile to standards, the allergen may be identified along with an estimate of the concentration present. Cluster analysis of the xMAP FADA data was also performed and agreed with the known phylogeny of the legumes and tree nuts being analyzed. Graphical abstract The use of cluster analysis to compare the multi-antigen profiles of food allergens.

Current industrial practices and regulatory requirements to assess analyte and reagent stability using ligand-binding assays.

Specific guidelines on bioanalytical method validation for drug development support are recommended by regulatory agencies. Regarding stability assessment, US FDA states that 'Stability procedures should evaluate the stability of the analytes during sample collection and handling, after long-term (frozen at the intended storage temperature) and short-term (bench-top, room temperature) storage, and after going through freeze and thaw cycles and the analytical process'. Additional regulatory considerations are discussed including topics such as analyte and reagent stability. This article reviews the regulatory requirements as issued by the USA (FDA), Europe (EMA) and Japan (MHLW), for stability studies where bioanalytical methods are used to support drug development programs and summarizes the current industry standard for conducting stability studies when utilizing ligand-binding assays.

Multiplex detection of food allergens and gluten.

To help safeguard the food supply and detect the presence of undeclared food allergens and gluten, most producers and regulatory agencies rely on commercial test kits. Most of these are ELISAs with a few being PCR-based. These methods are very sensitive and analyte specific, requiring different assays to detect each of the different food allergens. Mass spectrometry offers an alternative approach whereby multiple allergens may be detected simultaneously. However, mass spectrometry requires expensive equipment, highly trained analysts, and several years before a quantitative approach can be achieved. Using multianalyte profiling (xMAP®) technology, a commercial multiplex test kit based on the use of established antibodies was developed for the simultaneous detection of up to 14 different food allergens plus gluten. The assay simultaneously detects crustacean seafood, egg, gluten, milk, peanut, soy, and nine tree nuts (almond, Brazil nut, cashew, coconut, hazelnut, macadamia, pine nut, pistachio, and walnut). By simultaneously performing multiple tests (typically two) for each analyte, this magnetic bead-based assay offers built-in confirmatory analyses without the need for additional resources. Twenty-five of the assays were performed on buffer extracted samples, while five were conducted on samples extracted using reduced-denatured conditions. Thus, complete analysis for all 14 allergens and gluten requires only two wells of a 96-well microtiter plate. This makes it possible to include in a single analytical run up to 48 samples. All 30 bead sets in this multiplex assay detected 5 ng/mL of food allergen and gluten with responses greater than background. In addition, 26 of the bead sets displayed signal/noise ratios of five or greater. The bead-based design makes this 30-plex assay expandable to incorporate new antibodies and capture/detector methodologies by ascribing these new detectors to any of the unassigned bead sets that are commercially available.

Recommendations on biomarker bioanalytical method validation by GCC.

The 5th GCC in Barcelona (Spain) and 6th GCC in San Antonio (TX, USA) events provided a unique opportunity for CRO leaders to openly share opinions and perspectives, and to agree upon recommendations on biomarker bioanalytical method validation.

The impact of decreased bead count to determine concentrations of amyloid beta1-42, total-tau, and phosphorylated-tau181 in human cerebrospinal fluid using xMAP technology.

Alzheimer's disease is the leading cause of human dementia. The lack of diagnostic tests and limited therapeutic options has driven the search for endogenous biomarkers. The INNO-BIA AlzBio3 assay is a multiplex flow-based immunoassay measuring Aß42, tau, and p-tau in cerebrospinal fluid (CSF). This study assesses assays performance under varying bead count (BC) parameters. Original method validation parameters at 100 BC were acceptable. Reanalyses performed at 3, 10, 25, and 50 BCs were compared to 100 BC data by ANOVA, Bland-Altman analysis, evaluation of concordance correlation coefficients, and frequency distribution of coefficient of variation (CV) ranges. Method validation characteristics were acceptable with 100 BCs. Equivalency for 25 and 50 versus 100 BCs was demonstrated, but not for 3 and 10 BCs. A general trend of decreasing agreement between decreasing BCs and the 100 BC reference resulted in decreases in concordance coefficients ρ(c) . The frequency of CV values greater than 20% increased with decreasing BCs, and CV values of 5% or less decreased with decreased BCs. Statistical analyses demonstrate that BCs of 3 and 10 are not equivalent with the reference and should not be used as a basis for determination of Aß42, tau, and p-tau concentration in human CSF.

The Alzheimer's Association external quality control program for cerebrospinal fluid biomarkers.

BACKGROUND: The cerebrospinal fluid (CSF) biomarkers amyloid ß (Aß)-42, total-tau (T-tau), and phosphorylated-tau (P-tau) demonstrate good diagnostic accuracy for Alzheimer's disease (AD). However, there are large variations in biomarker measurements between studies, and between and within laboratories. The Alzheimer's Association has initiated a global quality control program to estimate and monitor variability of measurements, quantify batch-to-batch assay variations, and identify sources of variability. In this article, we present the results from the first two rounds of the program. METHODS: The program is open for laboratories using commercially available kits for Aß, T-tau, or P-tau. CSF samples (aliquots of pooled CSF) are sent for analysis several times a year from the Clinical Neurochemistry Laboratory at the Mölndal campus of the University of Gothenburg, Sweden. Each round consists of three quality control samples. RESULTS: Forty laboratories participated. Twenty-six used INNOTEST enzyme-linked immunosorbent assay kits, 14 used Luminex xMAP with the INNO-BIA AlzBio3 kit (both measure Aß-(1-42), P-tau(181P), and T-tau), and 5 used Meso Scale Discovery with the Aß triplex (AßN-42, AßN-40, and AßN-38) or T-tau kits. The total coefficients of variation between the laboratories were 13% to 36%. Five laboratories analyzed the samples six times on different occasions. Within-laboratory precisions differed considerably between biomarkers within individual laboratories. CONCLUSIONS: Measurements of CSF AD biomarkers show large between-laboratory variability, likely caused by factors related to analytical procedures and the analytical kits. Standardization of laboratory procedures and efforts by kit vendors to increase kit performance might lower variability, and will likely increase the usefulness of CSF AD biomarkers.

Insights in regulated bioanalysis of human insulin and insulin analogs by immunoanalytical methods.

Despite the long and illustrious history of insulin and insulin analogs as important biotherapeutics, the regulated bioanalysis (in this article, regulated bioanalysis refers to the formalized process for generating bioanalytical data to support pharmacokinetic and toxicokinetic assessments intended for development of insulin and insulin analogs as biotherapeutics, as opposed to the analytical process used for measuring insulin as a biomarker) of these peptides remains a challenging endeavor for a number of reasons. Paramount is the fact that the therapeutic concentrations are often low in serum/plasma and not too dissimilar from the endogenous level, particularly in patients with insulin resistance, such as Type 2 diabetes mellitus. Accordingly, this perspective was written to provide helpful background information for the design and conduct of immunoassays to support regulated bioanalysis of insulin and insulin analogs. Specifically, it highlights the technical challenges for determination of insulin and insulin analogs by immunoanalytical methods that are intended to support evaluations of pharmacokinetics and toxicokinetics. In a broader sense, this perspective describes the general bioanalytical issues that are common to regulated bioanalysis of peptides and articulates some of the bioanalytical differences between conventional monoclonal antibodies and peptide therapeutics.

Unique challenges of providing bioanalytical support for biological therapeutic pharmacokinetic programs.

Regulatory recommendations for providing bioanalytical support for biological therapeutics have co-evolved with the increasing success of these unique pharmaceuticals. Immunoassays have been used to quantify biological macromolecules for more than 50 years. These assays rely on the use of antigen-specific antibodies. More recently, LC-MS methods have being adapted to quantitate biologics. LC-MS has attributes that complement the limitations encountered by immunoassays. Whether employing immunoassay or LC-MS methods, compared with traditional chemical-based therapeutics, biological therapeutics present unique analytical challenges to analysts. In this article, we review bioanalytical strategies for supporting biologics and discuss the regulatory and analytical challenges that must be met.

Systematic analytical validation of commercial kits for the determination of novel biomarkers for clinical drug development.

The use of biomarkers during clinical drug-development programs may expedite pipeline decision making by adding critical information about the pharmacological mechanism and efficacy of a potential therapeutic agent. Currently, advice for laboratorians conducting method development and analytical validation of biomarker methods is provided by published White Paper recommendations from industry thought leaders. The adaptation of commercial test kits to generate biomarker data to support regulated studies offers unique challenges and limitations. In this perspective, we address these issues, including factors to consider when identifying a kit manufacturer and adapting commercial test kits for use in regulated studies. We offer a logical and systematic approach for defining the extent of analytical validation needed for application of commercial kits based upon the intended use of the biomarker data.

Best practices during bioanalytical method validation for the characterization of assay reagents and the evaluation of analyte stability in assay standards, quality controls, and study samples.

Characterization of the stability of analytes in biological samples collected during clinical studies together with that of critical assay reagents, including analyte stock solutions, is recognized as an important component of bioanalytical assay validation. Deficiencies in these areas often come to light during regulatory inspections. Best practices, based on current regulatory guidance, for the assessment of these issues as they pertain to ligand binding and chromatographic assays are covered in this review. Additionally, consensus recommendations reached during the recent AAPS/FDA Workshop on bioanalytical assay validation are highlighted.

Evaluation of an assay for serum 1,5-anhydroglucitol (GlycoMark) and determination of reference intervals on the Hitachi 917 analyzer.

BACKGROUND: 1,5-Anhydroglucitol (1,5-AG) is a glucose analogue, which is decreased in hyperglycemic individuals. We report the technical performance of an assay (GlycoMark) on a chemistry analyzer, evaluation of analyte stability and determination of reference intervals for 1,5-AG in a non-diabetic US population. METHODS: NCCLS protocols were followed to evaluate the reagent on a Hitachi 917 chemistry analyzer. RESULTS: Intra- and interassay imprecision ranged from 1.3% to 3.8% and 0.79% to 3.7%, respectively. The assay was linear to 110 microg/ml. Interference from triglyceride, hemoglobin and bilirubin was <10% to concentrations of 12.6 mmol/l, 12.1 and 911.4 micromol/l, respectively. Correlation coefficients between lot numbers on the Hitachi 917 and between analyses on the Hitachi 917 and the Hitachi 7170 analyzers were >0.99. The lowest limit of detection was 0.49 microg/ml (mean+/-2 S.D.). 1,5-AG was stable at 4 degrees C for 7 days, at 22 degrees C for 5 days, at -80 degrees C for 14 days and for three freeze-thaw cycles at -80 degrees C. The US reference intervals (nonparametric 2.5th-97.5th percentiles) were 10.2-33.8 microg/ml (males) and 5.9-31.8 microg/ml (females). CONCLUSIONS: The performance of the GlycoMark assay for the measurement of 1,5-AG was acceptable on the Hitachi 917 analyzer.