scholarly journals Rapid Screening of the Epidermal Growth Factor Receptor Phosphosignaling Pathway via Microplate-Based Dot Blot Assays

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Amedeo Cappione ◽  
Janet Smith ◽  
Masaharu Mabuchi ◽  
Timothy Nadler
Keyword(s):  
Large Scale ◽  
Solid Phase ◽  
Growth Factor Receptor ◽  
Cost Effective ◽  
Protein Microarrays ◽  
Rapid Screening ◽  
Dot Blot ◽  
A431 Cells ◽  
Dot Blot Assay ◽  
Multiple Samples

Expression profiling on a large scale, as is the case in drug discovery, is often accomplished through use of sophisticated solid-phase protein microarrays or multiplex bead technologies. While offering both high-throughput and high-content analysis, these platforms are often too cost prohibitive or technically challenging for many research settings. Capitalizing on the favorable attributes of the standard ELISA and slot blotting techniques, we developed a modified dot blot assay that provides a simple cost-effective alternative for semiquantitative expression analysis of multiple proteins across multiple samples. Similar in protocol to an ELISA, but based in a membrane bound 96-well microplate, the assay takes advantage of vacuum filtration to expedite the tedious process of washing in between binding steps. We report on the optimization of the assay and demonstrate its use in profiling temporal changes in phosphorylation events in the well-characterized EGF-induced signaling cascade of A431 cells.

scholarly journals Transgenic goats producing an improved version of cetuximab in milk

2020 ◽  
Author(s):  
Götz Laible ◽  
Sally Cole ◽  
Brigid Brophy ◽  
Paul Maclean ◽  
Li How Chen ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Large Scale ◽  
Growth Factor Receptor ◽  
Cost Effective ◽  
Scale Production ◽  
Cancer Treatments ◽  
Large Scale Production ◽  
Immunogenic Epitope ◽  
Dependent Cell ◽  
Transgenic Goats

ABSTRACTTherapeutic monoclonal antibodies (mAbs) represent one of the most important classes of pharmaceutical proteins to treat human diseases. Most are produced in cultured mammalian cells which is expensive, limiting their availability. Goats, striking a good balance between a relatively short generation time and copious milk yield, present an alternative platform for the cost-effective, flexible, large-scale production of therapeutic mAbs. Here, we focused on cetuximab, a mAb against epidermal growth factor receptor, that is commercially produced under the brand name Erbitux and approved for anti-cancer treatments. We generated several transgenic goat lines that produce cetuximab in their milk. Two lines were selected for detailed characterization. Both showed stable genotypes and cetuximab production levels of up to 10g/L. The mAb could be readily purified and showed improved characteristics compared to Erbitux. The goat-produced cetuximab (gCetuximab) lacked a highly immunogenic epitope that is part of Erbitux. Moreover, it showed enhanced binding to CD16 and increased antibody-dependent cell-dependent cytotoxicity compared to Erbitux. This indicates that these goats produce an improved cetuximab version with the potential for enhanced effectiveness and better safety profile compared to treatments with Erbitux. In addition, our study validates transgenic goats as an excellent platform for large-scale production of therapeutic mAbs.

scholarly journals Interlaboratory Collaboration for Optimized Screening for Urinary Tract Infection

2015 ◽  
Vol 54 (1) ◽  
pp. 93-98 ◽  
Author(s):  
Anne Russcher ◽  
Elske Kusters ◽  
Ron Wolterbeek ◽  
Ed J. Kuijper ◽  
Christa M. Cobbaert ◽  
...  
Keyword(s):  
Large Scale ◽  
Effective Means ◽  
Positive Culture ◽  
Cost Effective ◽  
Objective Evaluation ◽  
University Hospital ◽  
Rapid Screening ◽  
Gram Stain ◽  
Sample Quality ◽  
Positive Culture Result

As the majority of urine samples submitted for culture yields a negative result, rapid screening that accurately predicts culture outcome benefits clinicians by reducing the time to result and improves the efficiency of the microbiological laboratory. Automated urinalysis using the IRIS Diagnostics iQ200 Elite (iQ200) analyzer permits just such a fast and large-scale screening. We aimed to predict and thus to reduce negative cultures with a screening algorithm based on iQ200 urinalysis in a tertiary university hospital. In parallel, we evaluated the performance of the iQ200 screen compared to that of Gram stain for sample quality. We screened 1,442 samples submitted for bacterial culture using the iQ200 analyzer; of these samples, 357 (24.8%) had a positive culture result. We identified the absence of microorganisms in the iQ200 screen as the strongest solitary predictor for a negative culture, with a sensitivity of 90.5% (323/357). The algorithm was further improved by performing logistic regression on leukocyte counts, which gave a cutoff of 65 leukocytes/μl to obtain the desired sensitivity of >95% (95.2%; 95% confidence interval [CI], 92.5 to 97.0), a negative predictive value of 97.3% (95% CI, 95.7 to 98.3), and an anticipated culture workload reduction of 44% (95% CI, 41 to 46). Concordance between sample quality based on Gram stain and iQ200 screening was only 72%, which was probably a result of interobserver effect in evaluation of the Gram stain. In conclusion, in our setting, screening by iQ200 proved to be a safe and cost-effective means to provide faster culture results, and it has the added benefit of a more objective evaluation of sample quality.

scholarly journals Novel Autoantibody Signatures in Sera of Patients with Pancreatic Cancer, Chronic Pancreatitis and Autoimmune Pancreatitis: A Protein Microarray Profiling Approach

2020 ◽  
Vol 21 (7) ◽  
pp. 2403
Author(s):  
Sahar Ghassem-Zadeh ◽  
Katrin Hufnagel ◽  
Andrea Bauer ◽  
Jean-Louis Frossard ◽  
Masaru Yoshida ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Chronic Pancreatitis ◽  
Autoimmune Pancreatitis ◽  
Large Scale ◽  
Gastrointestinal Disease ◽  
Protein Microarray ◽  
Cost Effective ◽  
Protein Microarrays ◽  
Large Scale Analysis ◽  
Diagnostic Modalities

Identification of disease-associated autoantibodies is of high importance. Their assessment could complement current diagnostic modalities and assist the clinical management of patients. We aimed at developing and validating high-throughput protein microarrays able to screen patients’ sera to determine disease-specific autoantibody-signatures for pancreatic cancer (PDAC), chronic pancreatitis (CP), autoimmune pancreatitis and their subtypes (AIP-1 and AIP-2). In-house manufactured microarrays were used for autoantibody-profiling of IgG-enriched preoperative sera from PDAC-, CP-, AIP-1-, AIP-2-, other gastrointestinal disease (GID) patients and healthy controls. As a top-down strategy, three different fluorescence detection-based protein-microarrays were used: large with 6400, intermediate with 345, and small with 36 full-length human recombinant proteins. Large-scale analysis revealed 89 PDAC, 98 CP and 104 AIP immunogenic antigens. Narrowing the selection to 29 autoantigens using pooled sera first and individual sera afterwards allowed a discrimination of CP and AIP from PDAC. For validation, predictive models based on the identified antigens were generated which enabled discrimination between PDAC and AIP-1 or AIP-2 yielded high AUC values of 0.940 and 0.925, respectively. A new repertoire of autoantigens was identified and their assembly as a multiplex test will provide a fast and cost-effective tool for differential diagnosis of pancreatic diseases with high clinical relevance.

Rapid screening of SNPs in metastatic colorectal cancer (mCRC) utilizing multiplex sequencing technology (Sequenom).

2012 ◽  
Vol 30 (4_suppl) ◽  
pp. 418-418
Author(s):  
Radhashree Maitra ◽  
Jay B. Nayak ◽  
Atrayee Basu-Mallick ◽  
Arjun Sood ◽  
Titto A Augustine ◽  
...  
Keyword(s):  
High Throughput ◽  
Direct Sequencing ◽  
Cost Effective ◽  
Rapid Screening ◽  
Accurate Identification ◽  
Multiple Snps ◽  
Fast Screening ◽  
Effective Manner ◽  
Multiplex Sequencing ◽  
Multiple Samples

418 Background: Accurate and fast screening of mutations is essential for designing individualized therapy necessary and critical for efficient disease management and better patient outcome in mCRC. Detection of hotspots by gold standard direct sequencing (DS) is time consuming and cost ineffective. Pyrosequencing (PS) technique is rapid and precisely committed towards SNP detection. Recent introduction of high throughput multiplex PCR based extension on microarray (Sequenom, SEQ) offers a robust platform capable of detecting multiple SNPs simultaneously in a rapid and cost effective manner. The current study analyzes the concordance and efficacy of the cutting edge SEQ technique to the well established DS and PS methods. Methods: DNA isolated from 122 specimens from 76 mCRC patients were sequenced by all three methods. DS and PS were performed on 4 genes at 10 hotspots. SEQ multiplexing was performed on 31 hotspots in 19 genes by 4 multiplex reactions. Results: We were able to make "calls" for all samples by DS and PS. With the multiplex system, the “calls” rate was 97.8% of successful reactions. Using PS data as our standard in the assay we calculated the percent concordance of DS and SEQ. Futhermore SEQ offered a more accurate identification of the substituted nucleotide in Kras codon 12 as compared to PS. Conclusions: The multiplexing of PCR reactions offers an excellent advantage of high throughput with strong feasibility of analyzing several samples for multiple SNPs simultaneously. The concordance rate of > 90% when compared to PS along with the ability to analyze multiple samples/ hotspots plexed together in a time effective rapid mode provided a trifold advantage of the sequenom technology. It is therefore the next generation technology for rapid genetic evaluation of cancer patients. [Table: see text]

scholarly journals Specific Ligand Binding Attributable to Individual Epitopes of Gonococcal Transferrin Binding Protein A

2002 ◽  
Vol 70 (2) ◽  
pp. 732-740 ◽  
Author(s):  
Heather P. Masri ◽  
Cynthia Nau Cornelissen
Keyword(s):  
Ligand Binding ◽  
Fusion Proteins ◽  
Iron Uptake ◽  
Solid Phase ◽  
Protein A ◽  
Dot Blot ◽  
Affinity Tags ◽  
Amino Terminal ◽  
Dot Blot Assay ◽  
Specific Ligand

ABSTRACT The gonococcal transferrin receptor complex comprises two iron-regulated proteins, TbpA and TbpB. TbpA is essential for transferrin-iron uptake and is a TonB-dependent integral outer membrane protein. TbpB is thought to increase the efficiency of iron uptake from transferrin and is lipid modified and surface exposed. To evaluate the structure-function relationships in one of the components of the receptor, TbpA, we created constructs that fused individual putative loops of TbpA with amino-terminal affinity tags. The recombinant proteins were then overexpressed in Escherichia coli, and the fusions were recovered predominately from inclusion bodies. Inclusion body proteins were solubilized, and the epitope fusions were renatured by slow dialysis. To assess transferrin binding capabilities, the constructs were tested in a solid-phase dot blot assay followed by confirmatory quantitative chemiluminescent enzyme-linked immunosorbent assays. The constructs with only loop 5 and with loops 4 and 5 demonstrated dose-dependent specific ligand binding in spite of being out of the context of the intact receptor. The immunogenicities of individual TbpA-specific epitopes were investigated by generating rabbit polyclonal antisera against the fusion proteins. Most of the fusion proteins were immunogenic under these conditions, and the resulting sera recognized full-length TbpA in immunoblots. These results suggest that individual epitopes of TbpA are both immunogenic and functional with respect to ligand binding capabilities, and the vaccine implications of these findings are discussed.

scholarly journals Bioengineered yeast-derived vacuoles with enhanced tissue-penetrating ability for targeted cancer therapy

2015 ◽  
Vol 113 (3) ◽  
pp. 710-715 ◽  
Author(s):  
Vipul Gujrati ◽  
Miriam Lee ◽  
Young-Joon Ko ◽  
Sangeun Lee ◽  
Daejin Kim ◽  
...  
Keyword(s):  
Cancer Therapy ◽  
Large Scale ◽  
Growth Factor Receptor ◽  
Drug Distribution ◽  
Cost Effective ◽  
Genetically Engineered ◽  
Tumor Xenograft ◽  
Targeted Cancer Therapy ◽  
Specific Tissue ◽  
Targeting Ability

Despite the appreciable success of synthetic nanomaterials for targeted cancer therapy in preclinical studies, technical challenges involving their large-scale, cost-effective production and intrinsic toxicity associated with the materials, as well as their inability to penetrate tumor tissues deeply, limit their clinical translation. Here, we describe biologically derived nanocarriers developed from a bioengineered yeast strain that may overcome such impediments. The budding yeastSaccharomyces cerevisiaewas genetically engineered to produce nanosized vacuoles displaying human epidermal growth factor receptor 2 (HER2)-specific affibody for active targeting. These nanosized vacuoles efficiently loaded the anticancer drug doxorubicin (Dox) and were effectively endocytosed by cultured cancer cells. Their cancer-targeting ability, along with their unique endomembrane compositions, significantly enhanced drug penetration in multicellular cultures and improved drug distribution in a tumor xenograft. Furthermore, Dox-loaded vacuoles successfully prevented tumor growth without eliciting any prolonged immune responses. The current study provides a platform technology for generating cancer-specific, tissue-penetrating, safe, and scalable biological nanoparticles for targeted cancer therapy.

scholarly journals Nonisotopic Detection and Typing of Human Papillomavirus DNA in Genital Samples by the Line Blot Assay

1999 ◽  
Vol 37 (6) ◽  
pp. 1852-1857 ◽  
Author(s):  
François Coutlée ◽  
Patti Gravitt ◽  
Harriet Richardson ◽  
Catherine Hankins ◽  
Eduardo Franco ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Large Scale ◽  
Epidemiological Studies ◽  
Dot Blot ◽  
Hpv Dna ◽  
Amplification Method ◽  
Dot Blot Assay ◽  
Hpv Types ◽  
Cervicovaginal Lavage ◽  
Papillomavirus Dna

The line blot assay, a gene amplification method that combines PCR with nonisotopic detection of amplified DNA, was evaluated for its ability to detect human papillomavirus (HPV) DNA in genital specimens. Processed samples were amplified with biotin-labeled primers for HPV detection (primers MY09, MY11, and HMB01) and for β-globin detection (primers PC03 and PC04). Amplified DNA products were hybridized by a reverse blot method with oligonucleotide probe mixtures fixed on a strip that allowed the identification of 27 HPV genotypes. The line blot assay was compared to a standard consensus PCR test in which HPV amplicons were detected with radiolabeled probes in a dot blot assay. Two hundred fifty-five cervicovaginal lavage specimens and cervical scrapings were tested in parallel by both PCR tests. The line blot assay consistently detected 25 copies of HPV type 18 per run. The overall positivity for the DNA of HPV types detectable by both methods was 37.7% (96 of 255 samples) by the line blot assay, whereas it was 43.5% (111 of 255 samples) by the standard consensus PCR assay. The sensitivity and specificity of the line blot assay reached 84.7% (94 of 111 samples) and 98.6% (142 of 144 samples), respectively. The agreement for HPV typing between the two PCR assays reached 83.9% (214 of 255 samples). Of the 37 samples with discrepant results, 33 (89%) were resolved by avoiding coamplification of β-globin and modifying the amplification parameters. With these modifications, the line blot assay compared favorably to an assay that used radiolabeled probes. Its convenience allows the faster analysis of samples for large-scale epidemiological studies. Also, the increased probe spectrum in this single hybridization assay permits more complete type discrimination.

scholarly journals Identification of Group B Streptococcus Capsule Type by Use of a Dual Phenotypic/Genotypic Assay

2017 ◽  
Vol 55 (9) ◽  
pp. 2637-2650 ◽  
Author(s):  
Areej Alhhazmi ◽  
Armaan Pandey ◽  
Gregory J. Tyrrell
Keyword(s):  
Sialic Acid ◽  
Large Scale ◽  
Capsular Polysaccharide ◽  
Group B Streptococcus ◽  
Epidemiological Studies ◽  
Dot Blot ◽  
Content Type ◽  
Dot Blot Assay ◽  
Important Virulence Factor ◽  
Group B

ABSTRACTThe group B streptococcus (GBS) capsular polysaccharide (CPS) is an important virulence factor which is also used for GBS typing. There are 10 CPS types (Ia, Ib, and II to IX). GBS that do not phenotypically type are considered nontypeable. All genes required for CPS synthesis are found on the GBScpsoperon, which contains a highly variable CPS-determining region (cpsG-cpsK). The objective of this study was development of an assay to detect sialic acid on the GBS cell surface, followed by a genotypic PCR CPS typing assay. Sialic acid is located at the terminal end of the side chain of all known GBS CPS types. Sialic acid can be bound to commercially available lectins such as slugLimax flavuslectin. BiotinylatedL. flavus-streptavidin-peroxidase complex was used in an enzyme immunoassay and dot blot assay to detect sialic acid. This was followed by a PCR typing scheme that was developed to target the serotype-determining region of thecpslocus for Ia, Ib, and II to IX. Sialic acid from the CPS types Ia, Ib, and II to IX was detectable on the GBS cell surfaces of all previously identified CPS-typed GBS strains assayed. This was followed by the real-time PCR typing assay which successfully identified CPS Ia, Ib, and II to IX types. The combination of phenotypic and genotypic assays provides an accurate tool for detection of CPS expression and assignment of CPS typing. These assays have the potential to be used for CPS typing in large-scale epidemiological studies.

A simple Dot Blot Assay for population scale screening of DNA methylation

2018 ◽  
Author(s):  
Nelia Luviano ◽  
Sayuri Diaz-Palma ◽  
Céline Cosseau ◽  
Christoph Grunau
Keyword(s):  
Dna Methylation ◽  
Large Scale ◽  
General Rule ◽  
Enzyme Linked Immunosorbent Assay ◽  
Global Dna Methylation ◽  
Epigenetic Changes ◽  
Dot Blot ◽  
Wide Range ◽  
Dot Blot Assay ◽  
Experimental Populations

AbstractThe study of epigenetic changes in natural and experimental populations has increased the need to find a cost-effective and high throughput method to analyze multiple samples to effectuate a population-wide screening to study epigenetic changes triggered by biotic or abiotic stress. One of the most studied epigenetic marks is global DNA methylation, its measurement is used as a first step to differentiate methylation between individuals. There is a wide range of methods designed to detect genome-wide 5 methyl-cytosine (5mC) that differ in sensitivity, price, level of expertise required, but as a general rule, require large amounts of DNA and are relatively expensive. This is a limit for the analysis of 5mC in a large number of individuals as a prerequisite to population-wide testing of methylation markers. In this work, we evaluated a method based on antibody recognition of 5mC to measure the DNA methylation level of individuals of the species Biomphalaria glabrata, the intermediate host of schistosomiases, a neglected tropical disease. We validated the method to complete a large screening in the genome of B. glabrata snails treated with a chemical inhibitor of DNA methylation; however, the method can be applied to any species containing 5mC. The dot blot assay is a suitable method to perform a large-scale screening of global DNA methylation to compare 5mC levels between individuals from different natural or experimental populations. The dot blot method compares favorably with methods with an equivalent sensitivity such as the Enzyme Linked Immunosorbent Assay (ELISA) kit since it requires a smaller amount of DNA (30 ng) is less expensive and allows many more samples to be analyzed.

scholarly journals A Ponceau Staining based Dot-Blot Assay for reliable and cost-effective Protein Quantification

2019 ◽  
Author(s):  
Dario-Lucas Helbing ◽  
Leopold Böhm ◽  
Yan Cui ◽  
Leonie Karoline Stabenow ◽  
Helen Morrison
Keyword(s):  
Protein Extraction ◽  
Cost Effective ◽  
Protein Quantification ◽  
Dot Blot ◽  
Effective Manner ◽  
Dot Blot Assay ◽  
Ponceau S ◽  
Reliable Quantification ◽  
Complete Protein ◽  
Bca Assay

AbstractReliable quantification of protein extracts from tissues can be a challenge e.g. due to interference of the high fat content in tissues of the nervous system. Further problems like under- or overerstimation of protein concentrations in protein quantification kits like the bicinchoninic acid (BCA) assay can occur. In addition, common lysis buffers such as RIPA buffer are known to be unable to solubilize a large amount of proteins (~10-30%) leading to unsatisfactory and unreliable experimental results with techniques such as immunoblotting. In this work, we have developed a Ponceau S staining based protein quantification assay. This assay is compatible with tissues or cells directly lysed in 2x SDS gel loading buffer, containing bromophenolblue, leading to more complete protein extraction. Protein concentrations of several samples can be determined in a fast and cost-effective manner and subsequent experiments (e.g. Western blot) can be performed without loss of proteins. The presented protein quantification method is highly reliable, fast and economical. Using this method, it is possible to save between 2300 to 3200€ per 1000 lysates as compared to the costs of a commercial BCA kit.

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