Hepatitis B screening in a New York City obstetrics service.

A cross-sectional chart review study was performed of hepatitis B virus (HBV) surface antigen screening of 532 women admitted to a New York City hospital obstetrics service from 1984 to 1985. Comparison of serologic results to risk factors for hepatitis B revealed that women born outside the United States and those with a positive test for or history of a sexually transmitted disease were more likely to be infected with HBV. Obstetric clinics with large immigrant populations should consider screening all patients for HBV.     

Generation and comparative analysis of approximately 3.3 Mb of mouse genomic sequence orthologous to the region of human chromosome 7q11.23 implicated in Williams syndrome.

Williams syndrome is a complex developmental disorder that results from the heterozygous deletion of a approximately 1.6-Mb segment of human chromosome 7q11.23. These deletions are mediated by large (approximately 300 kb) duplicated blocks of DNA of near-identical sequence. Previously, we showed that the orthologous region of the mouse genome is devoid of such duplicated segments. Here, we extend our studies to include the generation of approximately 3.3 Mb of genomic sequence from the mouse Williams syndrome region, of which just over 1.4 Mb is finished to high accuracy. Comparative analyses of the mouse and human sequences within and immediately flanking the interval commonly deleted in Williams syndrome have facilitated the identification of nine previously unreported genes, provided detailed sequence-based information regarding 30 genes residing in the region, and revealed a number of potentially interesting conserved noncoding sequences. Finally, to facilitate comparative sequence analysis, we implemented several enhancements to the program, including the addition of links from annotated features within a generated percent-identity plot to specific records in public databases. Taken together, the results reported here provide an important comparative sequence resource that should catalyze additional studies of Williams syndrome, including those that aim to characterize genes within the commonly deleted interval and to develop mouse models of the disorder.     

Accelerating Regulated Bioanalysis for Biotherapeutics: Case Examples Using a Microfluidic Ligand Binding Assay Platform

The Gyrolab? xP is a microfluidic platform for conducting ligand binding assays (LBAs) and is recognized for its utility in discovery bioanalysis. However, few reports have focused on the technology for regulated bioanalysis. This technology has the advantage of low reagent consumption, low sample volume, and automated ligand binding methods. To improve bioanalysis testing timelines and increase the speed at which biotherapeutics are delivered to patients, we evaluated the technology for its potential to deliver high-quality data at reduced testing timelines for regulated bioanalysis. Six LBA methods were validated to support bioanalysis for GLP toxicokinetic or clinical pharmacokinetic studies. Validation, sample analysis, and method transfer are described. In total, approximately 4000 samples have been tested for regulated bioanalysis to support 6 GLP toxicology studies and approximately 1000 samples to support 2 clinical studies. Gyrolab? xP had high run pass rates (≥83%) and high incurred sample reanalysis (ISR) pass rates (>94%). The maximum total error observed across all QC levels for a given assay was <30% for all six LBAs. High instrument response precision (CV ≤5%) was observed across compact discs (CDs), and methods were validated to use a single standard curve across multiple CDs within a Gyrolab? xP run. Reduced bioanalysis timelines were achieved compared to standard manual plate-based methods, and methods were successfully transferred across testing labs, paving the way for this platform for use in late-stage clinical development.     

Bioanalytical Method Validation: Concepts,Expectations and Challenges in Small Molecule and Macromolecule—A Report of PITTCON 2013 Symposium

The concepts, importance, and implications of bioanalytical method validation has been discussed and debated for a long time. The recent high profile issues related to bioanalytical method validation at both Cetero Houston and former MDS Canada has brought this topic back in the limelight. Hence, a symposium on bioanalytical method validation with the aim of revisiting the building blocks as well as discussing the challenges and implications on the bioanalysis of both small molecules and macromolecules was featured at the PITTCON 2013 Conference and Expo. This symposium was cosponsored by the American Chemical Society (ACS)—Division of Analytical Chemistry and Analysis and Pharmaceutical Quality (APQ) Section of the American Association of Pharmaceutical Scientists (AAPS) and featured leading speakers from the Food & Drug Administration (FDA), academia, and industry. In this symposium, the speakers shared several unique examples, and this session also provided a platform to discuss the need for continuous vigilance of the bioanalytical methods during drug discovery and development. The purpose of this article is to provide a concise report on the materials that were presented.     

Development of a Generic Anti-PEG Antibody Assay Using BioScale’s Acoustic Membrane MicroParticle Technology

Immunogenicity testing for PEGylated biotherapeutics should include methods to detect both anti-protein and anti-PEG antibodies (anti-PEG). Although some methods have been published for the detection of anti-PEG antibodies, the information is incomplete and, in some cases, reagents used (such as Tween-20) are known to interfere with detection. This rapid communication describes the use of BioScale’s Acoustic Membrane MicroParticle (AMMP®) technology using the ViBE® Workstation to measure anti-PEG antibodies in human serum samples. Briefly, a sample spiked with monoclonal human IgG anti-PEG antibody is diluted in buffer and incubated with paramagnetic beads coated with linear chain mPEG to capture anti-PEG antibodies. The complex is then captured on an acoustic membrane coated with Protein A. The change in mass on the membrane caused by the binding of the complex to the membrane results in a signal proportional to the mass of anti-PEG antibodies. The data indicate that an assay with a sensitivity of less than 1000 ng/mL for IgG is achievable. This level of sensitivity is better than current published reports on IgG anti-PEG antibody detection.KEY WORDS: acoustic membrane microparticle technology, anti-peg antibodies, emerging technology, immunogenicity assays, pegylated biotherapeutics     

Human monoclonal antibodies to thyroid antigens derived by hybridization of lymphocytes from a diabetic patient

Human monoclonal antibodies to human endocrine cells have been obtained following the generation of immunoglobulin-secreting interspecies lymphocyte hybridomas. Peripheral blood lymphocytes from an adult patient presenting with acute onset, Type I, diabetes mellitus were fused in vitro with mouse myeloma cells of the NS1 cell line. Initial selection of resulting hybridomas was made by their ability to proliferate in HAT medium. Those hybridomas secreting human immunoglobulins were identified by radioimmunoassay and, thereafter, cloned at frequent intervals to ensure continued antibody production. Human monoclonal antibodies selected in this manner are being employed to identify those epitopes which are common antigenic targets during initial stages of autoimmune-mediated diabetes mellitus and associated multiple endocrinopathies. Of these antibodies, one (HML 3.22) recognizes an epitope present on the human TSH receptor and a second (HML 3.21) identifies a component of thyroglobulin. The potential value of human monoclonal antibodies as probes for analyzing autoimmune-mediated endocrine diseases is discussed.     

Comparative proteomic profiling of murine skin

Mammalian skin is regularly exposed to different environmental stresses, each of which results in specific compensatory changes in protein expression that can be assessed by proteomic analysis. We have established a reference proteome map of BALB/c murine skin allowing the resolution of greater than 500 protein spots in a single two-dimensional polyacrylamide gel. Forty-four protein spots, corresponding to 28 different cutaneous proteins, were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Mascot online database searching algorithm. Twenty-five proteins were expressed at higher levels in the epidermis, whereas only nine were found predominantly in the subepidermal tissues. A subset of protein spots exhibited strain-specific expression. Proteins of diverse function were identified, including those involved in stress response, apoptosis, growth inhibition, the maintenance of structural integrity, translational control, energy metabolism, calcium binding, cholesterol transport, and the scavenging of free radicals. Prohibitin expression was detected cutaneously, with more abundant protein and mRNA levels in the epidermis. Five molecular chaperones including protein di-sulfide isomerase, 78 kDa glucose-regulated protein precursor, heat shock protein 60 (HSP60), HSP70, and HSP27 were also identified. Of these, HSP27 expression was confined mainly to the epidermis, and expression of protein disulfide isomerase was found primarily in the subepidermal tissues. Proteomic analysis of skin following heat or cold shock resulted in increased levels of HSP27, HSP60, and HSP70 suggesting involvement of these chaperones in the cutaneous response mechanism to temperature stress. These data establish numerous reference markers within the proteome map of murine skin and provide an important framework for future efforts aimed at characterization of the epidermal and subepidermal responses to environmental changes.     

Ground scratching and preferred leg use in domestic chicks: changes in motor control in the first two weeks post-hatching

Lateralisation of a variety of visual functions: food discrimination, fear response, copulation, and performance of topographical and other tasks, such as olfactory and auditory functions, have been described in the domestic chick, Gallus gallus domesticus. A bias to left hemisphere control on day 8 and to the right on day 11 has also been demonstrated in the domestic chick. In this study we show that motor control as to foot preference in initiating a scratching bout and a tape-removing task is lateralised in both adults and chicks. There was a preference for the right leg to initiate a bout of ground scratching in both male and female adult birds. Second, foot preference is also affected by the changes in shifts of bias on day 8 and day 11. The right leg preference in initiating a ground scratching bout observed on day 5 is reversed to a left leg preference on day 8. This then reverts to the right leg preference after day 11. Hence it is postulated that the hemisphere that is not activated due to the bias of age controls the first leg to be used in initiating routine movements such as ground scratching. For the tape-removing task the right leg was used to remove a tape adhered to the beak of the chick for the trained group on day 8; but there was no preference in the naive group. Similarly, on day 11 a left foot bias was observed for the trained group and right foot bias for the naive group. To remove a tape the activated hemisphere on days of bias is used; whereas in a novel situation the foot use is reversed. Thus, footedness is affected by age, type of task, and changing hemispheric dominance.