In vitro evaluation of concentration, labeling effectiveness and stability for 131 I-labeled radioimmunoassay ligand using real-time detection technology.

Radioimmunoassay belongs to the analytical method enabling highly specific and sensitive quantification of molecules. The verification of the real-time radioimmunoassay technology usefulness for ligand-quality characteristics evaluation such as concentration, influence of radiolabeling on binding affinity and stability was estimated. The anti-epidermal growth factor receptor antibody 131 I-cetuximab was employed as the ligand antibody. The concentration of 131 I-cetuximab was derived from the shape of binding curves coming from the ligand-receptor interaction. The binding curves also allowed the estimation of 131 I-cetuximab binding affinity for different radiolabeling procedures (incubation times 1, 5, and 10 minutes) in stability testing up to 96 hours at 4°C. The stability testing also included comparative analysis by size exclusion high-performance liquid chromatography. The assessment of cetuximab concentrations using real-time method showed acceptable accordance between real and calculated values. The real-time method revealed that 1-minute radiolabeling proved to be the optimal incubation time for direct radioiodination of cetuximab. Stability testing showed the significant change in radioligand affinity by one order at the longest incubation times (72 and 96 hours). Characterization of stability and binding behavior of radiolabeled monoclonal antibodies by the verified real-time method before use in other assays may be employed to eliminate variability and suboptimal antibody performance.

Systematic verification of bioanalytical similarity between a biosimilar and a reference biotherapeutic: committee recommendations for the development and validation of a single ligand-binding assay to support pharmacokinetic assessments.

For biosimilar drug development, it is critical to demonstrate similar physiochemical characteristics, efficacy, and safety of the biosimilar product compared to the reference product. Therefore, pharmacokinetic (PK) and immunogenicity (antidrug antibody, ADA) assays that allow for the demonstration of biosimilarity are critical. Under the auspices of the American Association of Pharmaceutical Scientists (AAPS) Ligand-Binding Assay Bioanalytical Focus Group (LBABFG), a Biosimilars Action Program Committee (APC) was formed in 2011. The goals of this Biosimilars APC were to provide a forum for in-depth discussions on issues surrounding the development and validation of PK and immunogenicity assays in support of biosimilar drug development and to make recommendations thereof. The Biosimilars APC's recommendations for the development and validation of ligand-binding assays (LBAs) to support the PK assessments for biosimilar drug development are presented here. Analytical recommendations for the development and validation of LBAs to support immunogenicity assessments will be the subject of a separate white paper.

Development of NMR spectroscopic methods for dynamic detection of acetylcholine synthesis by choline acetyltransferase in hippocampal tissue.

Choline acetyltransferase (ChAT) is the key enzyme for acetylcholine (ACh) synthesis and constitutes a reliable marker for the integrity of cholinergic neurons. Cortical ChAT activity is decreased in the brain of patients suffering from Alzheimer's and Parkinson's diseases. The standard method used to measure the activity of ChAT enzyme relies on a very sensitive radiometric assay, but can only be performed on post-mortem tissue samples. Here, we demonstrate the possibility to monitor ACh synthesis in rat brain homogenates in real time using NMR spectroscopy. First, the experimental conditions of the radiometric assay were carefully adjusted to produce maximum ACh levels. This was important for translating the assay to NMR, which has a low intrinsic sensitivity. We then used (15) N-choline and a pulse sequence designed to filter proton polarization by nitrogen coupling before (1) H-NMR detection. ACh signal was resolved from choline signal and therefore it was possible to monitor ChAT-mediated ACh synthesis selectively over time. We propose that the present approach using a labeled precursor to monitor the enzymatic synthesis of ACh in rat brain homogenates through real-time NMR represents a useful tool to detect neurotransmitter synthesis. This method may be adapted to assess the state of the cholinergic system in the brain in vivo in a non-invasive manner using NMR spectroscopic techniques.

Changes in measurement procedure from a radioassay to a microbiologic assay necessitate adjustment of serum and RBC folate concentrations in the U.S. population from the NHANES 1988-2010.

The NHANES measured serum and RBC folate concentrations by using a radioassay during prefortification (1988-1994) and postfortification (1999-2006) periods followed by the use of a microbiologic assay (MBA) from 2007-2010. The MBA produces higher concentrations than does the radioassay and is considered to be more accurate. To allow for accurate long-term trending (1988-2010), we evaluated different regression models (linear, piecewise linear, and fractional polynomial) to assay-adjust the radioassay results to be comparable to the MBA results. The data used to derive the regression models originated from 2 crossover studies in which the 2 assays were applied to a set of 325 serum and 171 whole-blood samples. Fractional polynomial regression of logarithmically transformed data provided the best fit for serum folate. Linear regression of logarithmically transformed whole-blood data provided an equally good fit compared with the other models and was the simplest to apply for RBC folate. Prefortification serum and RBC folate geometric mean concentrations increased after adjustment from 13.0 to 16.7 nmol/L and from 403 to 747 nmol/L, respectively. Postfortification serum folate concentrations increased from ~30 to ~43 nmol/L, and RBC folate concentrations increased from ~600 to ~1100 nmol/L after adjustment, with some variation across survey cycles. The presented regression equations allow the estimation of more accurate prevalence estimates and long-term trends in blood folate concentrations in the U.S. population by using results that are equivalent to the MBA. This information will be useful to public health officials in the United States who are dealing with folic acid fortification issues.

Radioligand binding analysis as a tool for quality control of GPCR production for structural characterization: adenosine A(2a)R as a template for study.

Functional characterization of G protein-coupled receptors is essential to ascertain the suitability of a protein target for downstream studies and to help develop optimal expression and isolation procedures. Radioligand binding analysis is a well-established technique, which allows direct measurement of the amount of functional receptor in a sample. It can be readily applied to both membrane-bound and soluble receptor samples and is an ideal method for monitoring the amount of functional protein at each stage in the expression and isolation process. This unit presents protocols for the radioligand binding analysis of the human adenosine A(2a) receptor and provides examples of how these assays can be used at several stages to help optimize expression, solubilization, and isolation procedures.

Monitoring of anti-transglutaminase autoantibodies in pediatric celiac disease using a sensitive radiobinding assay.

OBJECTIVES: The diagnosis of celiac disease (CD) is based on the histological identification of gluten-sensitive enteropathy and detection of anti-tissue transglutaminase antibodies (tTGA) and/or endomysial antibodies. Serial measurements of tTGA are now recommended as a follow-up strategy to monitor compliance with a gluten-free diet (GFD). We evaluated the performances of a quantitative radiobinding assay (RBA) of tTGA immunoglobulin A at diagnosis and during monitoring of GFD in pediatric CD. METHODS: Eighty children with confirmed CD were selected. Levels of serum tTGA measured by RBA and a commercial enzyme-linked immunosorbent assay (ELISA) were compared at diagnosis. The relation between RBA-tTGA levels and histological damage was analyzed, as well as the time course of tTGA clearance during GFD. RESULTS: Both RBA and ELISA showed high sensitivity and specificity for tTGA detection at diagnosis. There was no relation between RBA-tTGA levels at diagnosis and severity of mucosal damage. Upon initiation of GFD, the rate of RBA-tTGA positivity declined slower than that of endomysial antibodies positivity, with >50% of the children still tTGA positive at year 5; however, tTGA levels decreased rapidly during the first year of GFD and more slowly thereafter. Children who seroreverted had lower tTGA levels at diagnosis (2080±1554 cpm) than those who remained tTGA positive throughout follow-up (3688±1435 cpm). CONCLUSIONS: The high sensitivity of RBA is likely responsible for higher tTGA positivity rates during GFD than previously reported with ELISA. A decreasing trend for tTGA levels may represent a better surrogate marker of compliance with GFD than absolute normal tTGA levels.

Estimation of the dissociation rate of unlabelled ligand-receptor complexes by a 'two-step' competition binding approach.

BACKGROUND AND PURPOSE: Because the in vivo effectiveness of ligands may also be determined by the rate by which they dissociate from their target receptors, drug candidates are being increasingly screened for this kinetic property. The dissociation rate of unlabelled ligand-receptor complexes can be estimated indirectly from their ability to slow the association of subsequently added radioligand molecules. EXPERIMENTAL APPROACH: We used the 'two-step competition' binding approach consisting of pre-incubating the receptor preparation with a wide range of ligand concentrations, washing off free ligand molecules, adding radioligand and monitoring its receptor binding after a fixed time. Based on the rationale that binding of both ligands is mutually exclusive and that they bind according to the law of mass action to a single class of sites, the unlabelled ligand's dissociation rate can be estimated from the upward shift that the competition curve experiences after washing. KEY RESULTS: The relevance of the 'two-step competition' approach was explored by computer simulations and by comparing the dissociation behaviour of unlabelled D(2) dopamine and CB(1) cannabinoid receptor antagonists in this and alternative approaches. Besides providing satisfactory estimations of dissociation rates, the method also detects the ability of the unlabelled ligand molecules to be released from 'sinks' such as the cell membrane. CONCLUSIONS AND IMPLICATIONS: As the 'two-step competition' requires rapid intermediate washing steps and needs radioligand binding to be measured at only one time point, this approach is particularly suited for binding studies on intact plated cells. LINKED ARTICLES: This article is part of a themed section on Analytical Receptor Pharmacology in Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2010.161.issue-6.

Assessment and interpretation of circulating 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D in the clinical environment.

The unique cis-triene structure of vitamin D and related metabolites makes it susceptible to oxidation, ultraviolet (UV) light-induced conformational changes, heat-induced conformational changes, and attacks by free radicals. Vitamin D(2) is much less bioactive than vitamin D(3) in humans. Metabolic activation and inactivation of vitamin D are well characterized and result in a plethora of metabolites, of which only 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH)(2)D) provide any clinically relevant information. 25(OH)D(2) and 25(OH)D(3) are commonly known as calcifediol and the 1,25(OH)(2)D metabolites as calcitriol. In this review the current state of the science on the clinical assessment of circulating 25(OH)D and 1,25(OH)(2)D is described.

Ligand binding assays at equilibrium: validation and interpretation.

The focus of this review paper is factors affecting data interpretation in ligand binding assays under equilibrium conditions. Protocols for determining K(d) (the equilibrium dissociation constant) and K(dA) (the equilibrium inhibitor constant) for receptor ligands are discussed. The basic theory describing the interaction of a radiotracer and an unlabelled competitor ligand with a receptor is developed. Inappropriate experimental design may result in ligand depletion and non-attainment of equilibrium, distorting the calculation of K(d) and K(dA) . Strategies, both theoretical and practical, will be given to avoid and correct such errors, thus leading to the determination of reliable values for these constants. In determining K(dA) from competition binding studies, two additional concepts are discussed. First, the necessity to measure an adequate specific binding signal from the bound radiotracer ligand limits the range of affinity constants that can be measured: a particular set of assay conditions may lead to an upper limit on the apparent affinity of unlabelled ligands. Second, an extension of the basic assay methodology can indicate whether the interaction between the tracer and a test ligand is mediated by a competitive or an allosteric mechanism. Finally, the review ends with a discussion of two factors that are often overlooked: buffer composition and the temperature at which the assay is conducted, and the impact these can have on affinity measurements and the understanding of drug interactions.

A report on the International Transglutaminase Autoantibody Workshop for Celiac Disease.

OBJECTIVES: Measurement of transglutaminase autoantibodies (TGAA) is considered to be the most efficient single serologic test for celiac disease (CD) by the American Gastroenterological Association Institute. We hypothesized that a large international collaborative effort toward improving and standardizing TGAA measurement is both feasible and necessary. The primary aim of this workshop is to compare TGAA assays among various research and clinical laboratories to examine assay concordance and improve (and eventually standardize) the TGAA assay. METHODS: A total of 20 laboratories (5 commercial laboratories, 15 research and clinical laboratories) participated that included enzyme-linked immunosorbent assay (ELISA) and radiobinding assays. A total of 150 serum samples were distributed to each laboratory, with each laboratory receiving an equal aliquot that was coded and blinded, composed of 100 healthy control sera and 50 CD sera. RESULTS: Laboratory sensitivity ranged from 69% to 93% and specificity ranged from 96% to 100%. By receiver operator characteristic analysis, the area under the curve (C index) ranged from 0.9488 to 0.9904. When analyzing for linear correlation, r-squared was as high as 0.8882 but as low as 0.4244 for the celiac samples between different laboratories performing ELISA. CONCLUSIONS: This transglutaminase autoantibody workshop allows for larger-scale international participation for the purposes of improving and eventually standardizing the TGAA assay with subsequent workshops.

Validation of the reference tissue model for estimation of dopaminergic D2-like receptor binding with [18F](N-methyl)benperidol in humans.

Positron emission tomography measurements of dopaminergic D2-like receptors may provide important insights into disorders such as Parkinson's disease, schizophrenia, dystonia and Tourette's syndrome. The positron emission tomography (PET) radioligand [18F](N-methyl)benperidol ([18F]NMB) has high affinity and selectivity for D2-like receptors and is not displaced by endogenous dopamine. The goal of this study is to evaluate the use of a graphical method utilizing a reference tissue region for [18F]-NMB PET analysis by comparisons to an explicit three-compartment tracer kinetic model and graphical method that use arterial blood measurements. We estimated binding potential (BP) in the caudate and putamen using all three methods in 16 humans and found that the three-compartment tracer kinetic method provided the highest BP estimates while the graphical method using a reference region yielded the lowest estimates (P<.0001 by repeated-measures ANOVA). However, the three methods yielded highly correlated BP estimates for the two regions of interest. We conclude that the graphical method using a reference region still provides a useful estimate of BP comparable to methods using arterial blood sampling, especially since the reference region method is less invasive and computationally more straightforward, thereby simplifying these measurements.

Consensus nomenclature for in vivo imaging of reversibly binding radioligands.

An international group of experts in pharmacokinetic modeling recommends a consensus nomenclature to describe in vivo molecular imaging of reversibly binding radioligands.

Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays.

The Third AAPS/FDA Bioanalytical Workshop, entitled "Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays" was held on May 1-3, 2006 in Arlington, VA. The format of this workshop consisted of presentations on bioanalytical topics, followed by discussion sessions where these topics could be debated, with the goal of reaching consensus, or identifying subjects where addition input or clarification was required. The discussion also addressed bioanalytical validation requirements of regulatory agencies, with the purpose of clarifying expectations for regulatory submissions. The proceedings from each day were reviewed and summarized in the evening sessions among the speakers and moderators of the day. The consensus summary was presented back to the workshop on the last day and was further debated. This communication represents the distillate of the workshop proceedings and provides the summary of consensus reached and also contains the validation topics where no consensus was reached.

Combined testing of antibody titer and affinity improves insulin autoantibody measurement: Diabetes Antibody Standardization Program.

The aim of the workshop was to assess whether four laboratories could reproducibly measure insulin autoantibody (IAA) affinity in coded sera from non-diabetic relatives of patients with type 1 diabetes, newly diagnosed patients, and healthy blood donors, and whether combining affinity with autoantibody titer could improve concordance and performance of IAA assays. IAA affinity was measured by competitive binding using constant amounts of Tyr14A [125I] human insulin and increasing quantities of unlabeled human insulin. There was high concordance between laboratories in distinguishing high, moderate, and low affinity IAA, although IAA binding to insulin varied with assay format. Multiple islet autoantibody-positive and patient sera were identified by high affinity IAA regardless of laboratory-designated IAA status. Combining affinity and titer significantly improved sensitivity, specificity, and concordance of IAA measurement. This workshop has demonstrated that different laboratories are able to reproduce IAA affinity results and that considering IAA affinity is likely to improve the diagnostic performance of IAA assays.

A semi-automated method for measuring the potential for protein covalent binding in drug discovery.

INTRODUCTION: Covalent protein binding of metabolically reactive intermediates of drugs has been implicated in drug toxicity including the occurrence of idiosyncratic drug toxicity. Investigators therefore would prefer to avoid developing compounds that produce significant amounts of reactive metabolites. By incubating the radiolabeled drug of interest with liver microsomes it is possible to evaluate the propensity of a drug candidate to covalently bind to proteins. METHODS: Here we present a semi-automated method in which a Brandel cell harvester is used to collect and wash proteins that have been incubated with radiolabeled drug. This method utilizes glass fiber filter paper to capture precipitated protein, rather than the more traditional exhaustive extraction/centrifugation approach. Using model compounds (including [14C]diclofenac, [3H]imipramine, [14C]naphthalene, and [14C]L-746530) we compare the covalent binding results obtained using this method to results generated using the traditional method and we performed cross-laboratory testing of assay reproducibility. RESULTS: It was found that results from new method correlated highly with the traditional method (R2=0.89). The cross-laboratory testing of the method showed an average interlaboratory coefficient of variation of only 18.4%. DISCUSSION: This method provides comparable results to the more traditional centrifugation-based method with considerable time and labor savings.

Detection by radioligand assay of antibodies against Borna disease virus in patients with various psychiatric disorders.

Using a radioligand assay, which preserves the natural form of the antigen, antibodies against Borna disease virus nucleoprotein and phosphoprotein were detected in 11 and 19 sera of 171 psychiatric patients, respectively. Compared with results by Western blotting, three and nine sera were concordantly positive, respectively. The four sera showing the highest levels of antibodies by radioligand assay were all negative by Western blotting; however, dilution and inhibition tests supported the positive results. Our results suggest the importance of conformational structure to detect human anti-Borna disease virus antibodies.

Development and validation of fluorescent receptor assays based on the human recombinant estrogen receptor subtypes alpha and beta.

This article describes the development and validation of two fluorescent receptor assays for the hRec-estrogen receptor subtypes alpha and beta. As a labelled ligand an autofluorescent phyto-estrogen (coumestrol) has been used. The estrogen receptor (ER) belongs to the nuclear receptor family, a class of soluble DNA binding proteins, mainly present in the cytoplasm of the cell, that act as ligand-activated enhancer factors. It consists of two different forms, expressed as ER-alpha (66 kDa) and ER-beta (59 kDa). The ER-alpha is mainly located in the uterus and the ER-beta can be found in vascular tissue. Detection and identification of compounds having estrogenic effects is of importance in drug discovery programmes within the pharmaceutical industry for their search for ER-subtype selective (ant)agonists which may prove to be of therapeutic value in treating a variety of estrogen-linked pathologies (breast cancer, osteoporosis, cardiovascular disease, type II diabetes and Alzheimer disease). Furthermore, interactions of (xeno-)estrogens with the endogenous hormonal system of the exposed organism can affect embryos, gonads, and reproductive behaviour. The latter can eventually lead to reduced reproduction and deterioration of a population. For that reason, monitoring of (xeno-)estrogens in food products and in the environment, attracts considerable attention by health councils throughout the world. The following characteristics were obtained for the human recombinant (hRec) estrogen receptor-beta assay, which is suitable for ER subtype selective drug-discovery purposes (IC50 values for 17-beta-estradiol and genistein were 5.1 nM and 25 nM, respectively): goodness of fit (R2) was always > 0.98 (x = 0.9933, n = 10). LLOQ of the assay is typically > or = 500 picomolar, whereas the ULOQ of the assay is < or = 20.0 nanomolar. For the hRec-estrogen receptor-alpha assay, which is suitable for monitoring of (xeno-)estrogens (IC50 values for 17-beta-estradiol and genistein were 0.68 nM and 65 nM, respectively) the following characteristics were obtained: goodness of fit (R2) was always > 0.96 (x = 0.9838, n = 10). LLOQ of the assay is typically > or = 200 picomolar, whereas the ULOQ of the assay is < or = 5.0 nanomolar.

Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules.

PURPOSE: With this publication a subcommittee of the AAPS Ligand Binding Assay Bioanalytical Focus Group (LBABFG) makes recommendations for the development, validation, and implementation of ligand binding assays (LBAs) that are intended to support pharmacokinetic and toxicokinetic assessments of macromolecules. METHODS: This subcommittee was comprised of 10 members representing Pharmaceutical, Biotechnology, and the contract research organization industries from the United States, Canada, and Europe. Each section of this consensus document addresses a specific analytical performance characteristic or aspect for validation of a LBA. Within each section the topics are organized by an assay's life cycle, the development phase, pre-study validation, and in-study validation. Because unique issues often accompany bioanalytical assays for macromolecules, this document should be viewed as a guide for designing and conducting the validation of ligand binding assays. RESULTS: Values of +/- 20% (25% at the lower limit of quantification [LLOQ]) are recommended as default acceptance criteria for accuracy (% relative error [RE], mean bias) and interbatch precision (%coefficient of variation [CV]). In addition, we propose as secondary criteria for method acceptance that the sum of the interbatch precision (%CV) and the absolute value of the mean bias (%RE) be less than or equal to 30%. This added criterion is recommended to help ensure that in-study runs of test samples will meet the proposed run acceptance criteria of 4-6-30. Exceptions to the proposed process and acceptance criteria are appropriate when accompanied by a sound scientific rationale. CONCLUSIONS: In this consensus document, we attempt to make recommendations that are based on bioanalytical best practices and statistical thinking for development and validation of LBAs.