TY  - JOUR
A1  - Schmidt, Carsten
A1  - Schierack, Peter
A1  - Gerber, Ulrike
A1  - Schroeder, Christian
A1  - Choi, Youngeun
A1  - Bald, Ilko
A1  - Lehmann, Werner
A1  - Rödiger, Stefan
T1  - Streptavidin homologues for applications on solid surfaces at high temperatures
JF  - Langmuir
N2  - One of the most commonly used bonds between two biomolecules is the bond between biotin and streptavidin (SA) or streptavidin homologues (SAHs). A high dissociation constant and the consequent high-temperature stability even allows for its use in nucleic acid detection under polymerase chain reaction (PCR) conditions. There are a number of SAHs available, and for assay design, it is of great interest to determine as to which SAH will perform the best under assay conditions. Although there are numerous single studies on the characterization of SAHs in solution or selected solid phases, there is no systematic study comparing different SAHs for biomolecule-binding, hybridization, and PCR assays on solid phases. We compared streptavidin, core streptavidin, traptavidin, core traptavidin, neutravidin, and monomeric streptavidin on the surface of microbeads (10-15 mu m in diameter) and designed multiplex microbead-based experiments and analyzed simultaneously the binding of biotinylated oligonucleotides and the hybridization of oligonucleotides to complementary capture probes. We also bound comparably large DNA origamis to capture probes on the microbead surface. We used a real-time fluorescence microscopy imaging platform, with which it is possible to subject samples to a programmable time and temperature profile and to record binding processes on the microbead surface depending on the time and temperature. With the exception of core traptavidin and monomeric streptavidin, all other SA/SAHs were suitable for our investigations. We found hybridization efficiencies close to 100% for streptavidin, core streptavidin, traptavidin, and neutravidin. These could all be considered equally suitable for hybridization, PCR applications, and melting point analysis. The SA/SAH-biotin bond was temperature sensitive when the oligonucleotide was mono-biotinylated, with traptavidin being the most stable followed by streptavidin and neutravidin. Mono-biotinylated oligonucleotides can be used in experiments with temperatures up to 70 degrees C. When oligonucleotides were bis-biotinylated, all SA/SAH-biotin bonds had similar temperature stability under PCR conditions, even if they comprised a streptavidin variant with slower biotin dissociation and increased mechanostability.
Y1  - 2020
U6  - https://doi.org/10.1021/acs.langmuir.9b02339
SN  - 0743-7463
VL  - 36
IS  - 2
SP  - 628
EP  - 636
PB  - American Chemical Society
CY  - Washington
ER  - 
TY  - JOUR
A1  - Roggenbuck, Dirk
A1  - Goihl, Alexander
A1  - Hanack, Katja
A1  - Holzloehner, Pamela
A1  - Hentschel, Christian
A1  - Veiczi, Miklos
A1  - Schierack, Peter
A1  - Reinhold, Dirk
A1  - Schulz, Hans-Ulrich
T1  - Serological diagnosis and prognosis of severe acute pancreatitis by analysis of serum glycoprotein 2
JF  - Clinical chemistry and laboratory medicine : journal of the Forum of the European Societies of Clinical Chemistry - the European Branch of the International Federation of Clinical Chemistry and Laboratory Medicine
N2  - To better understand emerging adults’ perceptions of family interactions and value transmission to the next generation, we examined Hmong American emerging adults’ reflections on their parents’ parenting. Participants discussed what parenting practices they would do differently and others they hoped to emulate with their future adolescent children. Thirty Hmong American emerging adults (18-25 years; M = 21.2 years; 50% female) participated in interviews that focused retrospectively on the parent–adolescent relationship. Results revealed that emerging adults wanted to parent differently in three ways: less pressure about education, fewer restrictions, and more open communication. Emerging adults imagined being a similar
parent in four ways: promoting education, promoting life values, giving
guidance, and offering love and support. The findings highlight parenting practices that Hmong American emerging adults plan on transmitting (and not transmitting) to their own children, offering a glimpse into the type of parents the emerging adults may become.
KW  - acute pancreatitis
KW  - chronic pancreatitis
KW  - GP2 isoform alpha
KW  - pancreatic neoplasms
KW  - severe acute pancreatitis
KW  - zymogen granule membrane glycoprotein GP2
Y1  - 2017
U6  - https://doi.org/10.1515/cclm-2016-0797
SN  - 1434-6621
SN  - 1437-4331
VL  - 55
SP  - 854
EP  - 864
PB  - De Gruyter
CY  - Berlin
ER  - 
TY  - JOUR
A1  - Nawaz, Shiza
A1  - Khan, Muhammad Moman
A1  - Noack, Jonas
A1  - Awan, Asad Bashir
A1  - Schiebel, Juliane
A1  - Roggenbuck, Dirk
A1  - Schierack, Peter
A1  - Sarwar, Yasra
A1  - Ali, Aamir
T1  - Rapid detection of biofilm formation by zoonotic serovars of Salmonella enterica and avian pathogenic E. coli isolates from poultry
JF  - Pakistan veterinary journal
N2  - Biofilms are complex, sessile microbial communities that are problematic in clinical settings due to their association with survival and pathogenicity of bacteria. The biofilm formation supporting conditions for zoonotic serovars of Salmonella and avian pathogenic E. coli (APEC) from poultry have not been well studied yet. Clinical isolates of zoonotic Salmonella and APEC from poultry were evaluated for biofilm formation in four media at 37 degrees C and 40 degrees C after incubation of 48 and 72 hrs. The biofilms formed in 96 well plates were visualized and quantified with a new module of Aklides system using fluorescence microscope coupled with automated VideoScan Technology. After 72 hrs, brain heart infusion at 40 degrees C and Rappaport-Vassiliadis Soya broth at 37 degrees C were found most suitable for APEC and Salmonella biofilm formations respectively. The new information will be useful for further biofilm associated studies particularly for evaluation of antibiofilm compounds and contribute in infection control. (C) 2020 PVJ. All rights reserved
KW  - APEC
KW  - biofilm formation
KW  - Salmonella
KW  - video scan technology
Y1  - 2020
U6  - https://doi.org/10.29261/pakvetj/2020.066
SN  - 0253-8318
SN  - 2074-7764
VL  - 40
IS  - 4
SP  - 527
EP  - 530
PB  - University of Agriculture, Faculty of Veterinary Science
CY  - Faisalabad
ER  - 
TY  - JOUR
A1  - Schmidt, Carsten
A1  - Roediger, Stefan
A1  - Gruner, Melanie
A1  - Moncsek, Anja
A1  - Stohwasser, Ralf
A1  - Hanack, Katja
A1  - Schierack, Peter
A1  - Schroeder, Christian
T1  - Multiplex localization of sequential peptide epitopes by use of a planar microbead chip
JF  - Analytica chimica acta : an international journal devoted to all branches of analytical chemistry
N2  - Epitope mapping is crucial for the characterization of protein-specific antibodies. Commonly, small overlapping peptides are chemically synthesized and immobilized to determine the specific peptide sequence. In this study, we report the use of a fast and inexpensive planar microbead chip for epitope mapping. We developed a generic strategy for expressing recombinant peptide libraries instead of using expensive synthetic peptide libraries. A biotin moiety was introduced in vivo at a defined peptide position using biotin ligase. Peptides in crude Escherichia coli lysate were coupled onto streptavidin-coated microbeads by incubation, thereby avoiding tedious purification procedures. For read-out we used a multiplex planar microbead chip with size- and fluorescence-encoded microbead populations. For epitope mapping, up to 18 populations of peptide-loaded microbeads (at least 20 microbeads per peptide) displaying the primary sequence of a protein were analyzed simultaneously. If an epitope was recognized by an antibody, a secondary fluorescence-labeled antibody generated a signal that was quantified, and the mean value of all microbeads in the population was calculated. We mapped the epitopes for rabbit anti-PA28 gamma (proteasome activator 28 gamma) polyclonal serum, for a murine monoclonal antibody against PA28 gamma, and for a murine monoclonal antibody against the hamster polyoma virus major capsid protein VP1 as models. In each case, the identification of one distinct peptide sequence out of up to 18 sequences was possible. Using this approach, an epitope can be mapped multiparametrically within three weeks. (C) 2016 Elsevier B.V. All rights reserved.
KW  - Epitope mapping
KW  - In vivo biotinylation
KW  - Multiplexed assays
KW  - Microbeads
KW  - VideoScan technology
Y1  - 2016
U6  - https://doi.org/10.1016/j.aca.2015.12.030
SN  - 0003-2670
SN  - 1873-4324
VL  - 908
SP  - 150
EP  - 160
PB  - Elsevier
CY  - Amsterdam
ER  - 
TY  - JOUR
A1  - Schiebel, Juliane
A1  - Boehm, Alexander
A1  - Nitschke, Joerg
A1  - Burdukiewicz, Michal
A1  - Weinreich, Joerg
A1  - Ali, Aamir
A1  - Roggenbuck, Dirk
A1  - Roediger, Stefan
A1  - Schierack, Peter
T1  - Genotypic and Phenotypic Characteristics Associated with Biofilm Formation by Human Clinical Escherichia coli Isolates of Different Pathotypes
JF  - Applied and environmental microbiology
N2  - Bacterial biofilm formation is a widespread phenomenon and a complex process requiring a set of genes facilitating the initial adhesion, maturation, and production of the extracellular polymeric matrix and subsequent dispersal of bacteria. Most studies on Escherichia coli biofilm formation have investigated nonpathogenic E. coli K-12 strains. Due to the extensive focus on laboratory strains in most studies, there is poor information regarding biofilm formation by pathogenic E. coli isolates. In this study, we genotypically and phenotypically characterized 187 human clinical E. coli isolates representing various pathotypes (e.g., uropathogenic, enteropathogenic, and enteroaggregative E. coli). We investigated the presence of biofilm-associated genes ("genotype") and phenotypically analyzed the isolates for motility and curli and cellulose production ("phenotype"). We developed a new screening method to examine the in vitro biofilm formation ability. In summary, we found a high prevalence of biofilm-associated genes. However, we could not detect a biofilm-associated gene or specific phenotype correlating with the biofilm formation ability. In contrast, we did identify an association of increased biofilm formation with a specific E. coli pathotype. Enteroaggregative E. coli (EAEC) was found to exhibit the highest capacity for biofilm formation. Using our image-based technology for the screening of biofilm formation, we demonstrated the characteristic biofilm formation pattern of EAEC, consisting of thick bacterial aggregates. In summary, our results highlight the fact that biofilm-promoting factors shown to be critical for biofilm formation in nonpathogenic strains do not reflect their impact in clinical isolates and that the ability of biofilm formation is a defined characteristic of EAEC. IMPORTANCE Bacterial biofilms are ubiquitous and consist of sessile bacterial cells surrounded by a self-produced extracellular polymeric matrix. They cause chronic and device-related infections due to their high resistance to antibiotics and the host immune system. In nonpathogenic Escherichia coli, cell surface components playing a pivotal role in biofilm formation are well known. In contrast, there is poor information for their role in biofilm formation of pathogenic isolates. Our study provides insights into the correlation of biofilm-associated genes or specific phenotypes with the biofilm formation ability of commensal and pathogenic E. coli. Additionally, we describe a newly developed method enabling qualitative biofilm analysis by automated image analysis, which is beneficial for high-throughput screenings. Our results help to establish a better understanding of E. coli biofilm formation.
KW  - biofilm formation
KW  - Escherichia coli
KW  - pathotypes
KW  - VideoScan
Y1  - 2017
U6  - https://doi.org/10.1128/AEM.01660-17
SN  - 0099-2240
SN  - 1098-5336
VL  - 83
PB  - American Society for Microbiology
CY  - Washington
ER  - 
TY  - JOUR
A1  - Schlör, Anja
A1  - Holzlöhner, Pamela
A1  - Listek, Martin
A1  - Grieß, Cindy
A1  - Butze, Monique
A1  - Micheel, Burkhard
A1  - Hentschel, Christian
A1  - Sowa, Mandy
A1  - Roggenbuck, Dirk
A1  - Schierack, Peter
A1  - Füner, Jonas
A1  - Schliebs, Erik
A1  - Goihl, Alexander
A1  - Reinhold, Dirk
A1  - Hanack, Katja
T1  - Generation and validation of murine monoclonal and camelid recombinant single domain antibodies specific for human pancreatic glycoprotein 2
JF  - New biotechnology
N2  - Pancreatic secretory zymogen-granule membrane glycoprotein 2 (GP2) has been identified as a major autoantigenic target in Crohn’s disease patients. It was reported recently that a long (GP2a) and a short (GP2b) isoform of GP2 exist and that in the outcome of inflammatory bowel diseases (IBD) GP2-specific autoantibodies probably appear as new serological markers for diagnosis and therapeutic monitoring. To investigate this further and in order to establish diagnostic tools for the discrimination of both GP2 isoforms, a set of different murine monoclonal and camelid recombinant single domain antibodies (camelid VHH) was generated and validated in various enzyme-linked immunosorbent assay (ELISA) formats, immunofluorescence on transgenic cell lines and immunohistochemistry on monkey pancreas tissue sections. Out of six binders identified, one was validated as highly specific for GP2a. This murine monoclonal antibody (mAb) was used as capture antibody in construction of a sandwich ELISA for the detection of GP2a. Camelid VHHs or a second murine mAb served as detection antibodies in this system. All antibodies were also able to stain GP2a or GP2b on transgenic cell lines as well as on pancreatic tissue in immunohistochemistry. The KD values measured for the camelid VHHs were between 7 nM and 23pM. This set of specific binders will enable the development of suitable diagnostic tools for GP2-related studies in IBD.
KW  - glycoprotein GP2
KW  - Monoclonal antibodies
KW  - Camelid single domain antibodies
Y1  - 2018
U6  - https://doi.org/10.1016/j.nbt.2018.03.006
SN  - 1871-6784
SN  - 1876-4347
VL  - 45
SP  - 60
EP  - 68
PB  - Elsevier
CY  - Amsterdam
ER  - 
TY  - JOUR
A1  - Deutschmann, Claudia
A1  - Roggenbuck, Dirk
A1  - Schierack, Peter
A1  - Rödiger, Stefan
T1  - Autoantibody testing by enzyme-linked immunosorbent assay-a case in which the solid phase decides on success and failure
JF  - Heliyon
N2  - Background: The enzyme-linked immunosorbent assay (ELISA) is an indispensable tool for clinical diagnostics to identify or differentiate diseases such as autoimmune illnesses, but also to monitor their progression or control the efficacy of drugs. One use case of ELISA is to differentiate between different states (e.g. healthy vs. diseased). Another goal is to quantitatively assess the biomarker in question, like autoantibodies. Thus, the ELISA technology is used for the discovery and verification of new autoantibodies, too. Of key interest, however, is the development of immunoassays for the sensitive and specific detection of such biomarkers at early disease stages. Therefore, users have to deal with many parameters, such as buffer systems or antigen-autoantibody interactions, to successfully establish an ELISA. Often, fine-tuning like testing of several blocking substances is performed to yield high signal-to-noise ratios. <br /> Methods: We developed an ELISA to detect IgA and IgG autoantibodies against chitinase-3-like protein 1 (CHI3L1), a newly identified autoantigen in inflammatory bowel disease (IBD), in the serum of control and disease groups (n = 23, respectively). Microwell plates with different surface modifications (PolySorp and MaxiSorp coating) were tested to detect reproducibility problems. <br /> Results: We found a significant impact of the surface properties of the microwell plates. IgA antibody reactivity was significantly lower, since it was in the range of background noise, when measured on MaxiSorp coated plates (p < 0.0001). The IgG antibody reactivity did not differ on the diverse plates, but the plate surface had a significant influence on the test result (p = 0.0005). <br /> Conclusion: With this report, we want to draw readers' attention to the properties of solid phases and their effects on the detection of autoantibodies by ELISA. We want to sensitize the reader to the fact that the choice of the wrong plate can lead to a false negative test result, which in turn has serious consequences for the discovery of autoantibodies.
KW  - biochemistry
KW  - coatings
KW  - surface chemistry
KW  - immunology
KW  - proteins
KW  - laboratory medicine
KW  - clinical research
KW  - enzyme-linked immunosorbent
KW  - assay
KW  - biomarker discovery
KW  - reproducibility
KW  - solid-phase
KW  - autoantibody
Y1  - 2020
U6  - https://doi.org/10.1016/j.heliyon.2020.e03270
SN  - 2405-8440
VL  - 6
IS  - 1
PB  - Elsevier
CY  - London [u.a.]
ER  - 
TY  - JOUR
A1  - Awan, Asad Bashir
A1  - Schiebel, Juliane
A1  - Boehm, Alexander
A1  - Nitschke, Joerg
A1  - Sarwar, Yasra
A1  - Schierack, Peter
A1  - Ali, Aamir
T1  - Association of biofilm formation and cytotoxic potential with multidrug resistance in clinical isolates of pseudomonas  aeruginosa
JF  - EXCLI Journal
N2  - Multidrug resistant (MDR) Pseudomonas aeruginosa having strong biofilm potential and virulence factors are a serious threat for hospitalized patients having compromised immunity In this study, 34 P. aeruginosa isolates of human origin (17 MDR and 17 non-MDR clinical isolates) were checked for biofilm formation potential in enriched and minimal media. The biofilms were detected using crystal violet method and a modified software package of the automated VideoScan screening method. Cytotoxic potential of the isolates was also investigated on HepG2, LoVo and T24 cell lines using automated VideoScan technology. Pulse field gel electrophoresis revealed 10 PFGE types in MDR and 8 in non-MDR isolates. Although all isolates showed biofilm formation potential, strong biofilm formation was found more in enriched media than in minimal media. Eight MDR isolates showed strong biofilm potential in both enriched and minimal media by both detection methods. Strong direct correlation between crystal violet and VideoScan methods was observed in identifying strong biofilm forming isolates. High cytotoxic effect was observed by 4 isolates in all cell lines used while 6 other isolates showed high cytotoxic effect on T24 cell line only. Strong association of multidrug resistance was found with biofilm formation as strong biofilms were observed significantly higher in MDR isolates (p-value < 0.05) than non-MDR isolates. No significant association of cytotoxic potential with multidrug resistance or biofilm formation was found (p-value > 0.05). The MDR isolates showing significant cytotoxic effects and strong biofilm formation impose a serious threat for hospitalized patients with weak immune system.
KW  - Pseudomonas aeruginosa
KW  - multidrug resistance
KW  - biofilm
KW  - cytotoxicity
KW  - VideoScan technology
Y1  - 2019
U6  - https://doi.org/10.17179/excli2018-1948
SN  - 1611-2156
VL  - 18
SP  - 79
EP  - 90
PB  - Leibniz Research Centre for Working Environment and Human Factors
CY  - Dortmund
ER  - 
TY  - JOUR
A1  - Roggenbuck, Dirk
A1  - Borghi, Maria Orietta
A1  - Somma, Valentina
A1  - Buettner, Thomas
A1  - Schierack, Peter
A1  - Hanack, Katja
A1  - Grossi, Claudia
A1  - Bodio, Caterina
A1  - Macor, Paolo
A1  - von Landenberg, Philipp
A1  - Boccellato, Francesco
A1  - Mahler, Michael
A1  - Meroni, Pier Luigi
T1  - Antiphospholipid antibodies detected by line immunoassay differentiate among patients with antiphospholipid syndrome, with infections and asymptomatic carriers
JF  - IEEE transactions on geoscience and remote sensing
N2  - Background: Antiphospholipid antibodies (aPL) can be detected in asymptomatic carriers and infectious patients. The aim was to investigate whether a novel line immunoassay (LIA) differentiates between antiphospholipid syndrome (APS) and asymptomatic aPL+ carriers or patients with infectious diseases (infectious diseases controls (IDC)). Methods: Sixty-one patients with APS (56 primary, 22/56 with obstetric events only, and 5 secondary), 146 controls including 24 aPL+ asymptomatic carriers and 73 IDC were tested on a novel hydrophobic solid phase coated with cardiolipin (CL), phosphatic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, beta2-glycoprotein I (beta 2GPI), prothrombin, and annexin V. Samples were also tested by anti-CL and anti-beta 2GPI ELISAs and for lupus anticoagulant activity. Human monoclonal antibodies (humoAbs) against human beta 2GPI or PL alone were tested on the same LIA substrates in the absence or presence of human serum, purified human beta 2GPI or after CL-micelle absorption. Results: Comparison of LIA with the aPL-classification assays revealed good agreement for IgG/IgM a beta 2GPI and aCL. Anti-CL and anti-beta 2GPI IgG/IgM reactivity assessed by LIA was significantly higher in patients with APS versus healthy controls and IDCs, as detected by ELISA. IgG binding to CL and beta 2GPI in the LIA was significantly lower in aPL+ carriers and Venereal Disease Research Laboratory test (VDRL) + samples than in patients with APS. HumoAb against domain 1 recognized beta 2GPI bound to the LIA-matrix and in anionic phospholipid (PL) complexes. Absorption with CL micelles abolished the reactivity of a PL-specific humoAb but did not affect the binding of anti-beta 2GPI humoAbs. Conclusions: The LIA and ELISA have good agreement in detecting aPL in APS, but the LIA differentiates patients with APS from infectious patients and asymptomatic carriers, likely through the exposure of domain 1.
KW  - Antiphospholipid syndrome
KW  - Antiphospholipid antibody
KW  - Phospholipid binding proteins
KW  - Beta2-glycoprotein I
KW  - Line immunoassay
Y1  - 2016
U6  - https://doi.org/10.1186/s13075-016-1018-x
SN  - 1478-6354
SN  - 1478-6362
VL  - 18
PB  - BioMed Central
CY  - London
ER  -