Planar Protein Arrays in Microtiter Plates: Development of a New Format Towards Accurate, Automation-Friendly and Affordable (A3) Diagnostics

2012 ◽  
pp. 149-165
Author(s):  
Holger Eickhoff ◽  
Arif Malik
Keyword(s):  
Protein Arrays ◽  
Microtiter Plates

Comparison of Conventional and Electron Spectroscopic Imaging in the Fixed Beam Electron Microscope of Ordered Protein Arrays

1985 ◽  
Vol 43 ◽  
pp. 74-75
Author(s):  
E. L. Buhle ◽  
U. Aebi
Keyword(s):  
Image Processing ◽  
Electron Microscope ◽  
High Resolution ◽  
Image Plane ◽  
Electron Spectroscopic Imaging ◽  
Protein Arrays ◽  
Beam Electron ◽  
Average Unit ◽  
Fixed Beam ◽  
Energy Components

CTEM brightfield images are formed by a combination of relatively high resolution elastically scattered electrons and unscattered and inelastically scattered electrons. In the case of electron spectroscopic images (ESI), the inelastically scattered electrons cause a loss of both contrast and spatial resolution in the image. In the case of ESI imaging on the Zeiss EM902, the transmited electrons are dispersed into their various energy components by passing them through a magnetic prism spectrometer; a slit is then placed in the image plane of the prism to select the electrons of a given energy loss for image formation. The purpose of this study was to compare CTEM with ESI images recorded on a Zeiss EM902 of ordered protein arrays. Digital image processing was employed to analyze the average unit cell morphologies of the two types of images.

A pre-embedding double staining procedure used to observe the effect of fixation on the activity of intercellular adhesion molecule on T and B cells

1990 ◽  
Vol 48 (3) ◽  
pp. 378-379
Author(s):  
Jane E. Ramberg ◽  
Shigeto Tohma ◽  
Peter E. Lipsky
Keyword(s):  
B Cells ◽  
Adhesion Molecule ◽  
Colloidal Gold ◽  
Intercellular Adhesion ◽  
Intercellular Adhesion Molecule ◽  
Staining Procedure ◽  
Double Staining ◽  
T And B Cells ◽  
Microtiter Plates ◽  
Streptavidin Peroxidase

Intercellular adhesion molecule (ICAM-1) appears to be a ligand for LFA-1 dependent adhesion in T cell mediated cytotoxcity. It is found on cells of both hematopoietic and non-hematopoietic origin. While observing the activity of ICAM-1 on the surfaces of interacting T and B cells, we found that we could successfully carry out a pre-embedding double staining procedure utilizing both colloidal gold and peroxidase conjugated reagents.On 24-well microtiter plates, mitomycin-treated T4 cells were stimulated with 64.1 (anti-CD3) for one hour before the addition, in some instances, of B cells. Following a 12-48 hour incubation at 38°C, the cells were washed and then immunostained with a colloidal gold conjugated RFB-4 (anti-CD22); biotinylated R6.5 (anti-ICAM-1); followed by streptavidin/peroxidase. This method allowed us to observe two different antigens without concern about possible cross-reaction of reagents. Because we suspected ICAM-1 and R6.5 were sensitive to fixation, we tried varying concentrations of fresh paraformaldehyde before R6.5, after R6.5 and after streptavidin/peroxidase. All immunostaining and washing was done on ice with ice cold reagents.

Prevalence and Induction of Circulating Antibodies against Recombinant Staphylokinase

1994 ◽  
Vol 71 (01) ◽  
pp. 129-133 ◽  
Author(s):  
P J Declerck ◽  
S Vanderschueren ◽  
J Billiet ◽  
H Moreau ◽  
D Collen
Keyword(s):  
Intravenous Administration ◽  
Human Subjects ◽  
Microtiter Plates ◽  
Staphylococcus Aureus Bacteremia ◽  
Alternative Agent ◽  
Antibody Titers ◽  
Potential Alternative ◽  
Circulating Antibodies ◽  
Low Levels ◽  
Antibody Levels

SummaryStreptokinase (SK) is a routinely used thrombolytic agent but it is immunogenic and allergenic; staphylokinase (STA) is a potential alternative agent which is under early clinical evaluation. The comparative prevalence of antibodies against recombinant STA (STAR) and against SK was studied in healthy subjects and their induction with intravenous administration in small groups of patients.Enzyme-linked immunosorbent assays, using microtiter plates coated with STAR or SK and calibration with affinospecific human antibodies, revealed 2.1 to 65 μg/ml (median 11 μg/ml) anti-STAR antibodies and 0.9 to 370 μg/ml (median 18 μg/ml) anti-SK antibodies (p <0.001 vs anti-STAR antibodies) in plasma from 100 blood donors, with corresponding values of 0.6 to 100 μg/ml (median 7.1 μg/ml) and 0.4 to 120 μg/ml (median 7.3 μg/ml), respectively, in 104 patients with angina pectoris. Three out of 17 patients with Staphylococcus aureus bacteremia had significantly increased anti-STAR antibody levels (150, 75 and 75 μg/ml), and STAR neutralizing activities (2.2, 3.6 and 4.1 μg STAR neutralized per ml plasma, respectively). In 6 patients with acute myocardial infarction, given 10 mg STAR intravenously over 30 min, median anti-STAR antibody levels were 3.5 μg/ml at baseline, 2.9 μg/ml at 6 to 8 days and 1.2 μg/ml at 2 to 9 weeks, with median corresponding titers of STAR neutralizing activity at 2 to 9 weeks of 42 μg/ml plasma. Conversely, in 5 patients treated with 1,500,000 units SK over 60 min, median anti-SK antibodies increased from 2.9 μg/ml at baseline to 360 μg/ml at 5 to 10 days, with corresponding median SK neutralizing activities of 13 μg/ml. Antibodies against STAR did not cross-react with SK and vice versa.Plasma from human subjects contains low levels of circulating antibodies against recombinant staphylokinase, and intravenous administration of this compound boosts antibody titers. These antibodies do however not cross-react with streptokinase, whereby the use of these two immunogenic thrombolytic agents would not be mutually exclusive.

Fully Automated Parallel Oligonucleotide Synthesizer

2001 ◽  
Vol 66 (8) ◽  
pp. 1299-1314 ◽  
Author(s):  
Michal Lebl ◽  
Christine Burger ◽  
Brett Ellman ◽  
David Heiner ◽  
Georges Ibrahim ◽  
...  
Keyword(s):  
Solid Phase ◽  
Building Blocks ◽  
Solid Support ◽  
Continuous Operation ◽  
Gear Pump ◽  
Oligonucleotide Synthesis ◽  
Center Of Rotation ◽  
Microtiter Plates ◽  
The Individual ◽  
Design And Construction

Design and construction of automated synthesizers using the tilted plate centrifugation technology is described. Wash solutions and reagents common to all synthesized species are delivered automatically through a 96-channel distributor connected to a gear pump through two four-port selector valves. Building blocks and other specific reagents are delivered automatically through banks of solenoid valves, positioned over the individual wells of the microtiterplate. These instruments have the following capabilities: Parallel solid-phase oligonucleotide synthesis in the wells of polypropylene microtiter plates, which are slightly tilted down towards the center of rotation, thus generating a pocket in each well, in which the solid support is collected during centrifugation, while the liquid is expelled from the wells. Eight microtiterplates are processed simultaneously, providing thus a synthesizer with a capacity of 768 parallel syntheses. The instruments are capable of unattended continuous operation, providing thus a capacity of over two millions 20-mer oligonucleotides in a year.

scholarly journals High-Throughput Peptide Derivatization toward Supramolecular Diversification in Microtiter Plates

ACS Nano ◽  
2021 ◽  
Author(s):  
Yiyang Lin ◽  
Matthew Penna ◽  
Christopher D. Spicer ◽  
Stuart G. Higgins ◽  
Amy Gelmi ◽  
...  
Keyword(s):  
High Throughput ◽  
Microtiter Plates ◽  
Peptide Derivatization

scholarly journals Tracking the Antibody Immunome in Sporadic Colorectal Cancer by Using Antigen Self-Assembled Protein Arrays

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2718
Author(s):  
María González-González ◽  
José María Sayagués ◽  
Luis Muñoz-Bellvís ◽  
Carlos Eduardo Pedreira ◽  
Marcello L. R. de Campos ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Clinical Utility ◽  
Genetic Alterations ◽  
Western World ◽  
Sporadic Colorectal Cancer ◽  
Protein Arrays ◽  
Humoral Immune ◽  
Cellular Processes ◽  
Healthy Donors ◽  
Associated Proteins

Sporadic Colorectal Cancer (sCRC) is the third leading cause of cancer death in the Western world, and the sCRC patients presenting with synchronic metastasis have the poorest prognosis. Genetic alterations accumulated in sCRC tumor cells translate into mutated proteins and/or abnormal protein expression levels, which contribute to the development of sCRC. Then, the tumor-associated proteins (TAAs) might induce the production of auto-antibodies (aAb) via humoral immune response. Here, Nucleic Acid Programmable Protein Arrays (NAPPArray) are employed to identify aAb in plasma samples from a set of 50 sCRC patients compared to seven healthy donors. Our goal was to establish a systematic workflow based on NAPPArray to define differential aAb profiles between healthy individuals and sCRC patients as well as between non-metastatic (n = 38) and metastatic (n = 12) sCRC, in order to gain insight into the role of the humoral immune system in controlling the development and progression of sCRC. Our results showed aAb profile based on 141 TAA including TAAs associated with biological cellular processes altered in genesis and progress of sCRC (e.g., FSCN1, VTI2 and RPS28) that discriminated healthy donors vs. sCRC patients. In addition, the potential capacity of discrimination (between non-metastatic vs. metastatic sCRC) of 7 TAAs (USP5, ML4, MARCKSL1, CKMT1B, HMOX2, VTI2, TP53) have been analyzed individually in an independent cohort of sCRC patients, where two of them (VTI2 and TP53) were validated (AUC ~75%). In turn, these findings provided novel insights into the immunome of sCRC, in combination with transcriptomics profiles and protein antigenicity characterizations, wich might lead to the identification of novel sCRC biomarkers that might be of clinical utility for early diagnosis of the tumor. These results explore the immunomic analysis as potent source for biomarkers with diagnostic and prognostic value in CRC. Additional prospective studies in larger series of patients are required to confirm the clinical utility of these novel sCRC immunomic biomarkers.

scholarly journals Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Tobias Habicher ◽  
Tobias Klein ◽  
Jacqueline Becker ◽  
Andreas Daub ◽  
Jochen Büchs
Keyword(s):  
Controlled Release ◽  
Bacillus Licheniformis ◽  
Protease Activity ◽  
Microtiter Plate ◽  
Fed Batch ◽  
Batch Mode ◽  
Screening Process ◽  
Microtiter Plates ◽  
Batch Fermenter ◽  
Production Conditions

Abstract Background Substrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in industrial production processes. In contrast, screening processes are usually performed in microtiter plates operated in batch mode. This leads to a different physiological state of the production organism in early screening and can misguide the selection of potential production strains. To close the gap between screening and production conditions, new techniques to enable fed-batch mode in microtiter plates have been described. One of these systems is the ready-to-use and disposable polymer-based controlled-release fed-batch microtiter plate (fed-batch MTP). In this work, the fed-batch MTP was applied to establish a glucose-limited fed-batch screening procedure for industrially relevant protease producing Bacillus licheniformis strains. Results To achieve equal initial growth conditions for different clones with the fed-batch MTP, a two-step batch preculture procedure was developed. Based on this preculture procedure, the standard deviation of the protease activity of glucose-limited fed-batch main culture cultivations in the fed-batch MTP was ± 10%. The determination of the number of replicates revealed that a minimum of 6 parallel cultivations were necessary to identify clones with a statistically significant increased or decreased protease activity. The developed glucose-limited fed-batch screening procedure was applied to 13 industrially-relevant clones from two B. licheniformis strain lineages. It was found that 12 out of 13 clones (92%) were classified similarly as in a lab-scale fed-batch fermenter process operated under glucose-limited conditions. When the microtiter plate screening process was performed in batch mode, only 5 out of 13 clones (38%) were classified similarly as in the lab-scale fed-batch fermenter process. Conclusion The glucose-limited fed-batch screening process outperformed the usual batch screening process in terms of the predictability of the clone performance under glucose-limited fed-batch fermenter conditions. These results highlight that the implementation of glucose-limited fed-batch conditions already in microtiter plate scale is crucial to increase the precision of identifying improved protease producing B. licheniformis strains. Hence, the fed-batch MTP represents an efficient high-throughput screening tool that aims at closing the gap between screening and production conditions.

A receptor-antibody sandwich assay for teicoplanin.

1987 ◽  
Vol 33 (9) ◽  
pp. 1615-1618 ◽  
Author(s):  
A Corti ◽  
L Cavenaghi ◽  
E Giani ◽  
G Cassani
Keyword(s):  
Detection Limit ◽  
Dose Response ◽  
Response Curve ◽  
Synthetic Analog ◽  
Sandwich Complexes ◽  
Sandwich Assay ◽  
Receptor Antibody ◽  
Microtiter Plates ◽  
Analytical Recovery ◽  
Biological Target

Abstract We have developed a new method for quantifying teicoplanin in complex matrixes, a receptor-antibody sandwich assay (RASA). The method is based on bioselective adsorption of teicoplanin onto microtiter plates coated with albumin-epsilon-aminocaproyl-D-alanyl-D-alanine, a synthetic analog of its biological target, and reaction with anti-teicoplanin antibodies. The sandwich complexes are detected by incubation with peroxidase-labeled goat antibodies to rabbit IgGs and chromogenic reaction with o-phenylenediamine. The dose-response curve was linear for teicoplanin concentrations in the range from 0 to 0.15 mg/L. We used the assay to measure teicoplanin concentrations in various biological matrixes. Analytical recovery from serum was 99.5%, the interassay CV was 5.1%, and the detection limit was 30 micrograms/L (P less than 0.01). Mean analytical recoveries from other biological specimens were 98% from ascitic fluid, 100% from pleuric liquid, 104.8% from prostate homogenate, and 98.5% from bronchial expectorate.

scholarly journals Establishing a Fed‐Batch Process for Protease Expression with Bacillus licheniformis in Polymer‐Based Controlled‐Release Microtiter Plates

2019 ◽  
Vol 15 (2) ◽  
pp. 1900088 ◽  
Author(s):  
Tobias Habicher ◽  
Edward K. A. Rauls ◽  
Franziska Egidi ◽  
Timm Keil ◽  
Tobias Klein ◽  
...  
Keyword(s):  
Controlled Release ◽  
Bacillus Licheniformis ◽  
Batch Process ◽  
Fed Batch ◽  
Microtiter Plates ◽  
Protease Expression

scholarly journals Mouse protein arrays from a TH1 cell cDNA library for antibody screening and serum profiling

Genomics ◽  
2005 ◽  
Vol 85 (3) ◽  
pp. 285-296 ◽  
Author(s):  
Claudia Gutjahr ◽  
Derek Murphy ◽  
Angelika Lueking ◽  
Andrea Koenig ◽  
Michal Janitz ◽  
...  
Keyword(s):  
Cdna Library ◽  
Th1 Cell ◽  
Protein Arrays ◽  
Antibody Screening ◽  
Serum Profiling ◽  
Mouse Protein
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