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PubMed2006

Microarrays as validation strategies in clinical samples: tissue and protein microarrays.

The widespread use of DNA microarrays has led to the discovery of many genes whose expression profile may have significant clinical relevance. The translation of this data to the bedside requires that gene expression be validated as protein expression, and that annotated clinical samples be available for correlative and quantitative studies to assess clinical context and usefulness of putative biomarkers. We review two microarray platforms developed to facilitate the clinical validation of candidate biomarkers: tissue microarrays and reverse-phase protein microarrays. Tissue microarrays are arrays of core biopsies obtained from paraffin-embedded tissues, which can be assayed for histologically-specific protein expression by immunohistochemistry. Reverse-phase protein microarrays consist of arrays of cell lysates or, more recently, plasma or serum samples, which can be assayed for protein quantity and for the presence of post-translational modifications such as phosphorylation. Although these platforms are limited by the availability of validated antibodies, both enable the preservation of precious clinical samples as well as experimental standardization in a high-throughput manner proper to microarray technologies. While tissue microarrays are rapidly becoming a mainstay of translational research, reverse-phase protein microarrays require further technical refinements and validation prior to their widespread adoption by research laboratories.

Animals↗

PubMed1989

In vitro studies of human prostatic epithelial cells: attempts to identify distinguishing features of malignant cells.

Recent advances in culture techniques have enabled routine establishment and propagation of epithelial cells derived from normal and malignant tissues of the human prostate. Comparative studies of the responses of normal and cancer-derived cell populations to various growth and differentiation factors in vitro were undertaken to examine the possibility that cancer cells might respond differentially. Clonal growth assays in serum-free medium demonstrated that optimal proliferation of normal as well as cancer cell strains was generally dependent on the presence of cholera toxin, epidermal growth factor, pituitary extract, hydrocortisone, insulin, and high levels of calcium in the culture medium, and on the use of collagen-coated dishes. Only one cancer strain responded aberrantly to epidermal growth factor and hydrocortisone. Putative differentiation factors (transforming growth factor-beta and vitamin A) inhibited the growth of all normal and cancer strains. The origin of a cancer-derived cell strain that responded similarly to normal strains was verified by positive labeling with a prostate cancer-specific antibody, validating the conclusion from these studies that normal and cancer prostatic epithelial cells are not distinguishable on the basis of responses to the tested factors.

Antibodies, Neoplasm↗

PubMed1990

Quantitation of residual mouse DNA in monoclonal antibody based products.

The identification and characterization of cell substrates and testing of bulk and final products is an important issue which must be addressed by manufacturers. In view of the fact that hundreds of applications for Investigational New Drugs (IND) have been submitted over the past few years, there is an obvious need for testing of these products. Detection of DNA by molecular hybridization has been used for various applications including the quantitation and characterization of DNA in biological products. We have developed a precise assay based on hybridization for the detection and quantitation of residual genomic DNA. In order to reduce protein interference, a specific pretreatment method for isolation of DNA in monoclonal antibody based products was implemented. We have used the assay to evaluate levels of contaminating DNA in prepared lots of monoclonal antibodies. Validation experiments demonstrated a sensitivity below 10 pg DNA using nick-translated 32P-labelled genomic DNA probes. The assay allows accurate quantitation of residual DNA in biologics.

Animals↗

PubMed1999

A scale to measure attitudes about HIV-antibody testing: development and psychometric validation.

Testing for antibodies to HIV is a vital component of many AIDS prevention and intervention programs. To increase the use of HIV antibody testing, it is important to understand more about peoples' attitudes about testing so that intervention programs can focus on the specific concerns of the target population. A scale to measure attitudes about HIV antibody testing (HIV-Antibody Testing Attitude Scale, HTAS) was developed and tested for reliability and validity, using data from two samples of heterosexual college students. The HTAS is a 32-item Likert-type scale, with high internal consistency (Cronbach's alpha = .88). Scores on the HTAS correlate with self-reported testing and intention to be tested for HIV, but not with other types of self-protective behavior (e.g., condom use), indicating evidence for its discriminant validity. Preliminary factor analysis of the responses to the HTAS shows four factors: concerns about friend's responses, concerns about family's responses, beliefs about public opinion of testing, and concerns about confidentiality of tests. The HTAS is easy to administer and offers a tool to use for both designing and evaluating HIV intervention and prevention programs that emphasize HIV antibody testing.

AIDS Serodiagnosis↗

PubMed2005

Differential alterations in 5alpha-reductase type 1 and type 2 levels during development and progression of prostate cancer.

BACKGROUND: In the prostate, conversion of testosterone to dihydrotestosterone (DHT), by the enzymes 5alpha-reductase types 1 and 2 (5alphaR1, 5alphaR2) is required for normal growth and probably also for development of prostate cancer (PCa). Finasteride, a 5alphaR2 inhibitor, was shown to reduce the prevalence of PCa in the Prostate Cancer Prevention Trial. However, inhibition of both 5alphaR isoenzymes causes a greater decrease in serum DHT. The aim of this study was to assess differential expression of these enzymes at various stages of PCa development. METHODS: Immunostaining for 5alphaR1 and 5alphaR2, using specific, well-validated antibodies, was evaluated in 26 benign prostatic hyperplasia (BPH) (16 for 5alphaR2), 53 primary PCa (21 for 5alphaR2), 18 prostatic intraepithelial neoplasia (PIN), 12 primary PCa treated with neoadjuvant androgen ablation, 15 locally recurrent PCa specimens, and 18 PCa metastases. RESULTS: The mean area of moderate plus high intensity staining for 5alphaR1 increased from 4.8 +/- 2.8% of total epithelial area in BPH, to 18.9 +/- 5.7% in PIN, 17.0 +/- 3.2% in primary cancer, 38.0 +/- 7.3% in recurrent cancer, and 55.8 +/- 8.5% in PCa metastases. The mean staining area for 5alphaR2 decreased from 58.8 +/- 7.2% in BPH, to 21.1 +/- 5.5% in PIN and 34.8 +/- 6.7% in primary PCa. Staining for 5alphaR2 was increased in recurrent cancer and PCa metastases compared to primary PCa, at 58.7 +/- 5.2% and 69.2 +/- 8.7%, respectively. CONCLUSIONS: 5alphaR1 immunostaining is increased and 5alphaR2 immunostaining is decreased during development of PCa. In addition, there is increased expression of both 5alphaR isozymes in recurrent and metastatic cancers, suggesting that both isozymes may be important in the development and progression of PCa.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed2007

Current stumbling blocks in oncology drug development.

The prognosis of patients with metastatic cancer remains poor and treatment strategies including newer generations of chemotherapeutics have not significantly improved survival in most solid tumors. New approaches are required to further improve patient outcome and survival. Recently, a major leap in the understanding of the molecular mechanisms involved in signal transduction pathways that contribute to tumor growth have been identified as therapeutic targets. Particularly molecules involved in cellular proliferation (e.g., tyrosine kinases) and angiogenesis have been considered as targets for new treatment approaches. Novel therapeutics that specifically target kinase transduction pathways have shown promise as single agents and in combination with standard chemotherapy. In addition, results of recent studies with antiangiogenic monoclonal antibodies validate the use of this class of targeted therapeutics as an important new treatment modality in cancer. This review will focus on the drug development stumbling blocks of targeted treatment modalities in cancer.

Antineoplastic Agents↗

PubMed1986

Development of a sensitive, direct luminescent enzyme immunoassay for plasma estradiol-17 beta.

A rapid, sensitive, precise, chemiluminescent enzyme immunoassay for estradiol-17 beta has been developed and validated. Antibodies were produced in rabbits using estradiol-17 beta-6-(O-carboxymethyl)oxime coupled to bovine serum albumin, purified and immobilized on polystyrene beads (6.4 mm diameter). The same derivative was used to prepare the enzymatic tracer by coupling with horseradish peroxidase. The assay, direct on the serum sample, featured a 4-h binding step at 4 degrees C followed by the chemiluminescent detection using luminol/H2O2. The detection limit was 0.15 pg/tube and the assay was carried out on 20-100 microliter of sample, allowing measurement of estradiol-17 beta in plasma concentrations from 1.5 to 500 pg/ml. The method fulfills all the standard requisites of precision and accuracy and the results agree well with a radioimmunoassay procedure on extracted serum.

Cross Reactions↗

PubMed1983

Activation of cyclic AMP-dependent protein kinase isoenzymes: studies using specific antisera.

A novel method for rapidly determining the amount and degree of association-dissociation of the Type I and Type II cAMP-dependent protein kinases has been developed and validated. Antibodies directed against the regulatory subunits of Type I and Type II cAMP-dependent protein kinases were used. The antibodies formed complexes with holoenzymes and regulatory subunits which were precipitated by goat anti-rabbit IgG (immunoglobulin G). These complexes bound [3H]cAMP with an apparent Kb of 20 nM for protein kinase I and 80 nM for protein kinase II. Immunoprecipitated protein kinases I and II were catalytically active when incubated with cAMP, [gamma-32P]ATP, and histone H2B. When mixtures of the two kinase isoenzymes or cytosol were incubated with various amounts of [3H]cAMP and the isoenzymes were separated by precipitation with antisera specific for each isoenzyme, the amount of [3H]cAMP associated with immunoprecipitates was proportional to the concentration of [3H]cAMP. In contrast, the catalytic activity that was immunoprecipitated varied inversely with the concentration of [3H]cAMP, showing that the activation of protein kinase could be assessed by the disappearance of catalytic activity from the immunoprecipitates. In the absence of MgATP protein kinase I was activated by a 10-fold lower concentration of cAMP than protein kinase II. However, when MgATP was added to the incubation, there was no significant difference in the binding of [3H]cAMP or dissociation of catalytic subunits of the two isoenzymes. The anti-R antibodies were also used to rapidly quantitate the concentration of regulatory subunits and the relative ratio of protein kinases I and II in tissue cytosols.

Adenosine Triphosphate↗

PubMed2006

An ELISA-based method for the quantification of incorporated BrdU as a measure of cell proliferation in vivo.

In this study, we describe a new rapid and versatile method to determine the BrdU content of DNA in brain tissues dissected from BrdU-treated rats. Different to already existing BrdU ELISAs the method is suitable for the assessment of BrdU incorporation in ex vivo experiments as it is based on the analysis of tissue extracts instead of immobilized cells. The method comprises the preparation of DNA extracts from dissected tissues, the immobilization of BrdU-containing DNA with an anti-BrdU antibody and quantification of the incorporated BrdU by a peroxidase-conjugated anti-BrdU antibody. Validating the new assay in vitro, we found a clear-cut dependency of the ELISA signal from the time SKNSH neuroblastoma cells had been exposed to BrdU. Parallel studies with existing ELISAs and a parallel immunocytochemical determination of BrdU positive cells revealed comparable results. In vivo experiments showed a virtually linear relationship between the BrdU immunoreactivity in the hippocampus and the time rats have been exposed to BrdU. Repeating the determination of the BrdU content of the same set of tissue samples revealed reproducible relative differences of the ELISA signals. This was true for protocols using purified DNA as well as crude DNA extracts. For the sensitivity and reproducibility of the method heat denaturation of the DNA prior to the analysis in the ELISA was crucial. In rats treated with electroconvulsion the BrdU content of the hippocampus, determined by the new ELISA, was increased to 225% of controls. In a parallel immunohistochemical study, the number of BrdU positive cells was comparably increased to 251% of controls. The assay thus provides a rapid method to detect changes of cell proliferation in dissected brain tissues and other proliferative tissues. With appropriate protocols, the assay may also be used to assess the generation of particular cell types like neurons in neurogenic areas.

Animals↗

PubMed2003

Calpain 3 is expressed in astrocytes of rat and Microcebus brain.

The calcium-dependent protease calpain is involved in numerous functions, including the control of cell survival, plasticity and motility. Whereas the isoforms calpain 1 and 2 have been described as ubiquitously expressed enzymes, calpain 3 has been called "muscle-specific", although trace amounts of calpain 3 mRNA have been detected by Northern blot in brain homogenates. In this study, we validated antibodies raised either against the peptides that were specific for a given isoform or the peptides present in all the three isoforms. We then used the anti-calpain 3 antibodies together with antibodies directed against cell-type-specific proteins to determine by double- and triple-labelling immunocytochemistry if the protease is expressed in specific cell populations of rat as well as lesser mouse lemur (Microcebus murinus) brain. Calpain 3 was almost exclusively found in cells displaying astrocyte morphology. These cells, most of which co-expressed glial fibrillary acidic protein, were particularly numerous close to the striatal subventricular zone (where numerous neurones forming the rostral migratory stream (RMS) towards the olfactory bulbs are generated) and the RMS itself. Other immunoreactive cells were found close to the pial surface of the forebrain, in the corpus callosum and in the dentate gyrus. Calpain 3 may be involved in astrocyte plasticity and/or motility.

Animals↗

PubMed1990

Invalidation of antiglobulin tests by a high thermal amplitude cryoglobulin.

A multiply transfused patient was referred for evaluation of a transfusion reaction. The direct and indirect antiglobulin tests (DAT, IAT) for alloantibody were negative. However, IgG-coated control cells failed to agglutinate in the negative reactions, casting doubt on their validity. At 4 degrees C, the patient's serum exhibited a large cryoprecipitate (2.9 mg/mL), made up predominantly of an IgG kappa paraprotein and having trace amounts of IgM and C3. Clear serum separated at 37 degrees C became cloudy within 10 minutes at room temperature (RT); within 4 hours, approximately 60 percent of the total precipitable cryoprotein had precipitated. Red cells (RBCs) incubated in fresh serum that had cooled to RT or RBCs obtained from RT or refrigerated samples contained cryoprecipitate that sedimented with the RBCs during washing with RT saline. On resuspension, enough IgG cryoglobulin redissolved to neutralize completely the commercial anti-IgG reagents. If the patient's samples were maintained at 37 degrees C, cryoprecipitate did not form, and RBCs washed four times at 37 degrees C gave valid DAT and IAT reactions. The removal of all cryoprecipitate from the patient's serum by centrifugation after overnight incubation at 4 degrees C also made possible valid antibody screening and compatibility tests.

Blood Grouping and Crossmatching↗

PubMed2006

Construction and optimization of a CC49-based scFv-beta-lactamase fusion protein for ADEPT.

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).

Amino Acid Sequence↗

PubMed2002

Bcl-2 overexpression in human melanoma cells increases angiogenesis through VEGF mRNA stabilization and HIF-1-mediated transcriptional activity.

The aim of this paper was to study the molecular mechanisms by which bcl-2 increases hypoxia-induced vascular endothelial growth factor (VEGF) expression. We demonstrated that bcl-2 overexpression in M14 human melanoma cell line enhances hypoxia-induced VEGF mRNA stability and promoter activation. In particular, the half-life of the message was longer in bcl-2 transfectants (approximately 330 min) than in control cells (approximately 180 min). In addition, bcl-2 overexpression increased VEGF promoter activity through the hypoxia-inducible factor-1 (HIF-1) transcription factor. Increased HIF-1a protein expression and DNA binding activity were detected in bcl-2 overexpressing cells compared with control cells. An enhanced functional activity of secreted VEGF was found both in in vitro and in vivo angiogenic assays, and the use of VEGF specific antibodies validated the role of VEGF on bcl-2-induced angiogenesis. Taken together our results indicate that bcl-2 plays an important role in melanoma angiogenesis, and that VEGF mRNA stabilization and HIF-1-mediated transcriptional activity are two important control points in bcl-2/hypoxia-induced VEGF expression.

Animals↗

PubMed2025

Automated chromatin profiling with spa-ChIP-seq uncovers the impacts of condition variations.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is widely used to study the genomic localization of DNA-associated proteins. However, conventional protocols include multiple manual steps that can introduce inconsistency and limit scalability, thereby restricting the inclusion of appropriate replicates and controls. Although the introduction of liquid handling platforms has improved reproducibility, most existing efforts have automated only a subset of the workflow, and extending automation to efficiently map non-histone proteins, such as chromatin regulators, remains challenging. Here, we present a fully automated implementation of our previously developed single-pot ChIP-seq protocol (Texari et al. 2021), named spa-ChIP-seq, which enables scalable processing of 8 to 96 ChIP-seq samples from crosslinked cells to sequencing-ready library in approximately three days with an estimated cost of $70 per sample. Benchmarking spa-ChIP-seq against manual ChIP-seq performed in parallel demonstrates comparable signal-to-noise ratio between the two workflows. Using spa-ChIP-seq, we systematically evaluate multiple parameters including shearing and crosslinking conditions, buffer compositions, and the ratio of antibody to cell-number. We find, for the first time to our knowledge, that weaker genomic localization signals are sensitive to changing the antibody to cell-number ratio, whereas the stronger signals remain unaffected. This finding underscores the importance of maintaining consistent antibody-to-cell-number ratio for comparative studies, such as treatment responses or chromatin-QTL mapping. The spa-ChIP-seq protocol is publicly available, including deck setups, operational parameters, and scripts. We envision that this robust, cost-efficient protocol will facilitate high-throughput, reproducible ChIP-seq analyses, supporting large-scale studies of antibody validation, compound screening, population genomics, and diagnostic frameworks.

Journal Article↗

PubMed1998

Report of the 1997 Asia Pacific Consensus Conference on the management of Helicobacter pylori infection.

While European and United States guidelines for the management of Helicobacter pylori infection have been developed, there are no guidelines for the Asian Pacific. International experts and recognised local authorities met in Singapore in 1997 to develop appropriate guidelines, taking into account the high background prevalence of infection, high incidence rates of gastric cancer and resource limitations. Recommendations were made based on randomised controlled trials or where this was not possible, they were based on the current best available evidence or on good clinical practice. A number of acceptable diagnostic tests for infection are available throughout the region. The non-endoscopic methods of choice are the urea breath test or a locally validated antibody test. If endoscopy was to be performed, a biopsy urease test was recommended as the test of first choice, with histology recommended only if this was negative. Post treatment testing was not recommended for all patients; a urea breath test was considered the test of choice if available. All gastric and duodenal ulcer patients who are infected with H. pylori should be treated for H. pylori whether the ulcer is active or in remission. Patients requiring long term non-steroidal anti-inflammatory drug therapy who have a current or recent history of dyspepsia, patients with early gastric cancer or low grade gastric mucosa associated lymphoid tissue lymphoma, and patients with a family history of gastric cancer should be treated. However, it was concluded that there wasn't sufficient evidence that cure of H. pylori infection reduces the risk or prevents the development of gastric adenocarcinoma. Many patients with dyspepsia in the region will request or require early upper endoscopy because of an inherent fear of gastric cancer. However, where endoscopy is not available or is too costly, alternative acceptable approaches were recommended in high risk cancer regions. While evidence is inconclusive to support treatment of H. pylori infection in non-ulcer dyspepsia, it was agreed that treatment be offered to patients with documented infection on a case-by-case basis. Treatment regimens need to attain an eradication rate of 90% or greater by per protocol analysis and 80% or greater by intention-to-treat analysis. A number of 7-day regimens were recommended based on available evidence. These regimens were considered likely to maximize the chances of successful eradication with one course of treatment, thereby reducing the risk of acquired antibiotic resistance and leading to long term cost savings.

Algorithms↗

PubMed2006

Proteomic analysis of malignant ovarian cancer effusions as a tool for biologic and prognostic profiling.

PURPOSE: Malignant epithelial ovarian cancer effusions are important in disease dissemination and clinical outcome. The identification of biochemical events active in effusions may improve our identification and application of targeted therapeutics. EXPERIMENTAL DESIGN: Archival effusion samples for which outcome information was known were studied. Clinical variables were comparable between these groups. Two cohorts of patients with malignant effusion were assessed: those with effusion at presentation (Tap1) or at first recurrence (Tap2). Expression and activated fraction of selected signaling proteins were quantitated on serial protein microarrays using validated antibodies. Proteomic results and clinical variables were analyzed by univariate analysis followed by Cox proportional hazards model analysis. RESULTS: Malignant effusions (>80% malignant cells) were distinguished from benign effusions by higher expression of AKT, activated extracellular signal-regulated kinase, activated (P < or = 0.001) and total cAMP-responsive element binding protein (P = 0.01), and JNK (P = 0.03). Malignant pleural effusions could not be differentiated from ascites by signaling profiles. Both had signal expression clusters for survival, proliferation and metastasis, and injury pathways. Cox proportional hazards model analysis revealed high p38 and pEGFR/EGFR ratio as jointly associated with poor survival in Tap1 cases (both P < or = 0.002). Phospho-JNK quantity was associated with worse outcome in Tap2 patients (P = 0.004), when taking other factors into consideration. CONCLUSIONS: Proliferation, survival, and apoptosis signaling dysregulation can be identified in ovarian cancer effusion samples. Biochemical characterization of clinical effusions may provide either predictive and/or correlative information on patient outcome from which to further understand the mechanisms of effusion development and target clinical intervention.

Apoptosis↗

PubMed1998

Further validation of antibody-capture and antigen-capture enzyme-linked immunosorbent assays for determining exposure of cattle to bovine coronavirus.

OBJECTIVE: To further validate an antibody-capture ELISA for measuring bovine coronavirus (BCV) exposure (antibody seroresponse) in cattle and to explain the apparent loss of sensitivity of a BCV antigen-capture ELISA when testing feces from adult versus neonatal cattle. ANIMALS: 98 adult cows from herds with and without winter dysentery; 10 gnotobiotic or colostrum-deprived calves. PROCEDURES: Results of an antibody-capture ELISA for BCV and a plaque reduction virus neutralization assay performed on paired serum samples from 24 cattle were compared with each other and with results of immunoelectron microscopy (IEM) of feces for BCV. For samples from 98 cattle, results of antibody-capture ELISA were compared with results of IEM. Calves were inoculated with feces ELISA-positive or IEM-positive for BCV and monitored for BCV infection. An ELISA was developed to detect BCV antigen-antibody complexes in feces and results were compared with results of an antigen-capture ELISA and IEM. RESULTS: Antibody-capture ELISA results correlated with neutralization assay results, but agreed more closely with results of IEM. Calves became infected with BCV following inoculation with either ELISA-positive or ELISA-negative but IEM-positive feces. Results of the antigen-antibody ELISA correlated with results of IEM and the antigen-capture ELISA. CLINICAL IMPLICATIONS: In adult cattle, testing of paired serum samples by use of an antibody-capture ELISA may be a better indicator of recent BCV exposure than results of virus neutralization tests. Antigen-antibody binding in feces may interfere with results of antigen-capture ELISA for BCV.

Animals↗

PubMed2003

Design and peptide-based validation of phage display antibodies for proteomic biochips.

To validate potential application of phage display-antibody arrays for high-throughput screening on a novel proteomics biochip, we examined the epitopes versus the full protein of glucose-6-phosphate-dehydrogenase (G6PD) from yeast. In a predictive approach, we used the Hopp-Woods method and compared the results with antibodies directed against the entire enzyme. In total, 16 peptides of a length of 11 amino acids each fulfilling the desired criteria were identified and synthesized. Subsequently, antibodies against G6PD were raised using a phage display library. Selective interaction of the antibodies with certain peptides facilitated the identification of epitopes predicted by the hydropathic profile. The setup was adapted to a novel biochip system based on surface-enhanced absorption for direct CCD-camera based screening.

Amino Acid Sequence↗