1.5 Proteinanalytik
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Organisationseinheit der BAM
- 1 Analytische Chemie; Referenzmaterialien (71)
- 1.5 Proteinanalytik (71)
- 1.9 Chemische und optische Sensorik (10)
- 8 Zerstörungsfreie Prüfung (9)
- 8.1 Sensorik, mess- und prüftechnische Verfahren (9)
- 1.8 Umweltanalytik (7)
- 6 Materialchemie (4)
- 6.3 Strukturanalytik (4)
- 1.7 Organische Spuren- und Lebensmittelanalytik (3)
- 1.1 Anorganische Spurenanalytik (2)
Eingeladener Vortrag
- nein (9)
We introduce a passive smart dust concept as a novel solution for environmental monitoring. Utilizing chemical reagents like colorimetric indicators and other chemosensors, these particles detect varying environmental conditions. We developed paper-based sensors that are both cost-effective and eco-friendly. In practical tests, these sensors, dispersed over a designated area, successfully identified hazardous substances by changing their color when exposed to acids or bases. This color change was remotely detectable using a drone-mounted color camera. The data thus obtained was processed through specialized software, accurately pinpointing areas of contamination. This method proves the efficacy and scalability of passive smart dust technology for real-time, environmentally sustainable remote sensing of hazardous materials
Use/distribution of cost-effective and biocompatible pieces of cellulose paper as passive optical chemosensors and readout by the camera system.
Modification of the paper surface using various chemically sensitive dyes (indicators) provides an optically detectable reaction and conclusions about the target substances and precise location.
Homochirality is an obvious feature of life on Earth. On the other hand, extraterrestrial samples contain largely racemic compounds. The same is true for any common organic synthesis. Therefore, it has been a perplexing puzzle for decades how these racemates could have formed enantiomerically enriched fractions as a basis for the origin of homochiral life forms. Numerous hypotheses have been put forward as to how preferentially homochiral molecules could have formed and accumulated on Earth. In this article, it is shown that homochirality of the abiotic organic pool at the time of formation of the first self-replicating molecules is not necessary and not even probable. It is proposed to abandon the notion of a molecular ensemble and to focus on the level of individual molecules. Although the formation of the first self-replicating, most likely homochiral molecule is a seemingly improbable event, on a closer look, it is almost inevitable that homochiral molecules have formed simply on a statistical basis. In this case, the non-selective leap to homochirality would be one of the first steps in chemical evolution directly out of a racemic “ocean”. Moreover, most studies focus on the chirality of the primordial monomers with respect to an asymmetric carbon atom. However, any polymer with a minimal size that allows folding to a secondary structure, would spontaneously lead to asymmetric higher structures (conformations). Most of the functions of these polymers would be influenced by this inherently asymmetric folding. To summarize, simple and universal mechanisms may have led to homochiral self-replicating systems in the context of chemical evolution. A homochiral monomer pool is deemed unnecessary and probably never existed on primordial Earth.
Antikörper sind in der medizinischen Welt nicht mehr wegzudenken. Ihr Einsatz-Repertoire als Therapeutika für die Behandlung diverser Krankheiten und Infektionen weitet sich von Jahr zu Jahr mehr aus. Dabei basiert ein Großteil der verwendeten Antikörper zu diesem Zeitpunkt auf monoklonalen Antikörpern auf Basis von Hybridomazellen. Doch hier könnten rekombinante Antikörper aufgrund ihrer Vektor-basierten Expression eine vielversprechende und womöglich sogar revolutionierende Alternative bieten. So ergeben sich neue Optimierungsoptionen für Merkmale wie Affinität, Stabilität und Kompatibilität über Anpassungen auf genetischer oder posttranslationaler Ebene. Allerdings ergibt sich kein Anwendungszweck ohne die vorhergehende detaillierte Charakterisierung der Antikörper. Denn nur mit spezifischem Wissen über die diversen Eigenschaften und Wechselwirkungen kann eine effektive und präzise Verwendung in der Diagnostik und Therapeutik gewährleistet werden und auf diese Weise Leben retten.
Aus diesem Grund beschäftigt sich diese Masterarbeit mit der Vertiefung der rekombinanten Antikörper-Thematik. Dazu soll ein Anti-Tetanus-Antikörper erstmals rekombinant in verschiedenen Zelllinien hergestellt und anschließend über umfangreiche analytische Verfahren mit einem Fokus auf die Einflussanalyse der unterschiedlichen posttranslationalen Glykosylierungen charakterisiert werden. Zu diesem Zweck wurden als Grundlage einerseits zwei P1316-Dual-Expressions-Vektoren mit variierenden Insert-Positionen der schweren und leichten Kette durch Gibson-Assembly sowie individuellen pRK5-Plasmide für eine separiert Expression der Antikkörperketten mithilfe Restriktionsfragment-Ligation generiert. In einer Gegenüberstellung der der Expressionsausbeuten stellte sich die Überlegenheit der P1316-Plasmid-Konfiguration heraus, in der das Leichtketten-Insert an der ersten Expressionsstelle positioniert ist. Deshalb wurde dieser Vektor als Ausgangspunkt für die anschließende hochskalierten Herstellung des Anti-Tetanus-Antikörpers in Expi293F-GnTI- und ExpiCHO-S-Zellen verwendet. Ein ELISA bestätigte die Bindungsaktivität der rekombinanten Antikörper am Tetanus-Toxoid unabhängig vom Expressionssystem. Nach einer folgenden Optimierung der für die Aufreinigung verwendeten Protein-A-Affinitätschromatographie, wurde über weitere Charakterisierungsmethoden wie Größenausschlusschromatographie und dynamische Lichtstreuung eine vergleichbare Aggregation aufgrund der sauren Elution aufgezeigt. In diesem Kontext wurde die Notwendigkeit einer Zweistufen-Präparation mit Affinitäts- und Größenausschlusschromatografie für den Erhalt monodisperser Proben verdeutlicht. Zudem wurden die verschiedenen intakten Massen mitsamt ihrer spezifischen Glykan-abhängigen Streumaßen in Relation zum monoklonalen NISTmAB-Referenzantikörper bestimmt. Dabei konnte den Erwartungen nach sowohl die geringere Masse als auch die uniformere Glykosylierung der in Expi293 GnTI- exprimierten Immunoglobuline in Relation zum ExpiCHO-S-Antikörper nachgewiesen werden. Anschließend wurde das Stabilitätsverhalten mittels thermischer Entfaltung und dynamischer Lichtstreuung über einen ausgeweiteten Zeitraum sowie verschiedenen Temperaturen dokumentiert. Grundlegend wurde eine höhere thermische Stabilität der zweiten konstanten Schwerketten-Domäne des aus ExpiCHO-S-Zellen stammenden Antikörpers nachgewiesen. Außerdem konnte eine problemlose Lagerungsfähigkeit beider unterschiedlich glykosylierter Antikörper bei Raumtemperatur für mindestens 4 Wochen festgestellt werden. Des Weiteren wurde die Langzeitlagerung im Rahmen der Auswirkung von Gefrier-Auftau-Zyklen mit gleichbleibenden Methoden sowie zusätzlicher Größenausschlusschromatographie erprobt. Hierbei stellte sich der Erhalt der strukturellen Integrität nach bis zu 15 Zyklen heraus. Allerdings geht die Monodispersität bereits nach wenigen Zyklen verloren, wobei die geringfügiger glykosylierten Expi293F-GnTI-Anti-Tetanus-Antikörper eine stärkere Beeinträchtigung durch multiple Gefrier-Auftau-Zyklen aufwiesen.
Communities worldwide face significant threats from Explosive Remnants of War (ERW), which endanger lives and restrict land usage. From forest fires due to ERWs or in ERW-contaminated areas (e.g., in Jüterbog, Germany) to broader global challenges (e.g., the Ukrainian conflict), the need for efficient detection and removal of these remnants, especially for humanitarian demining, is paramount. Traditional methods, like manual demining, have severe limitations in safety and efficiency. Here, we introduce an innovative solution to these challenges: “Chemosensing Smart Dust.” This technology uses chemoselective dyes that change their fluorescence properties when exposed to explosives like 2,4,6-trinitrotoluene (TNT). Fluorescence-based detection offers superior sensitivity, reduced likelihood of false positives, and enhanced accuracy of explosive detection. Drones, equipped with excitation lasers or LEDs, deploy the Chemosensing Smart Dust over areas of interest and actively detect the fluorescence changes using high-resolution cameras, offering a rapid, safe, and adaptable detection method. Beyond demining, this innovative approach has potential applications in monitoring polluted areas, homeland security, and emergency response.
Beschichtete Zellulose Partikel als kolorimetrische passive Sensoren, die mittels optische Kamerasystem einer Drohne erfasst werden. Somit können Gefahrenstoffe, in ersten Versuchen z. B. Säuren aus sicherer Entfernung, kostengünstig und umweltverträglich detektiert werden.
Quantitative 1H Nuclear Magnetic Resonance (qNMR) of Aromatic Amino Acids for Protein Quantification
(2022)
qNMR is a valuable technique for metrological studies due to the uniformity of its signal response for all chemical species of an isotope of interest, which enables compound-independent calibration. However, protein quantification remained challenging as large molecules produce wide, low-intensity signals that reduce the already low sensitivity. Combining qNMR with the hydrolysis of protein samples into amino acids circumvents many of these issues and facilitates the use of NMR spectroscopy for absolute protein and peptide quantification.In this work, different conditions have been tested for quantifying aromatic amino acids and proteins. First, we examined the pH-based signal shifts in the aromatic region. The preferable pH depends on the selection of the amino acids for quantification and which internal standard substance should be used to avoid peak overlap. Several aromatic compounds, such as terephthalic acid, sulfoisophthalic acid, and benzene tricarboxylic acid, have been applied as internal standards. The quantification of amino acids from an amino acid standard, as well as from a certified reference material (bovine serum albumin), was performed. Using the first two suggested internal standards, recovery was ~ 97 % for histidine, phenylalanine, and tyrosine at a concentration of approximately 1 mM in solution. Acidic hydrolysis of a certified reference material (CRM) of bovine serum albumin (BSA) and subsequent quantification of Phe and Tyr yielded recoveries of 98 ± 2 and 88 ± 4 %, respectively, at a protein concentration of 16 g/L or 250 µM.
Screening against ADAMTS4 reveals a specific peptide, which was turned into an MRI probe. The aneurysm in a mouse modal was visualized via MRI. A differentiation between stable and unstable aneurysm in an early state was performed. Using the probe as tool for an easy and non-invasive rupture assessment is possible.
ABID
(2022)
In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0. This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.
Nonporous corundum powder was functionalized covalently with protein binders to isolate and enrich specific proteins from complex matrices.The common crosslinker glutaraldehyde was compared with a hyperbranched polyglycerol (PG) of around 10 kDa. The latter was oxidized with periodate to generate aldehyde groups that can covalently react with the amines of the surface and the amino groups from the protein via a reductive amination process. The amount of bound protein was quantified via aromatic amino acid analysis (AAAA). As a proof of concept, IgG was extracted with protein A from crude human plasma. The advantages of corundum include the very low price, extremely high physical and chemical stability, pressure resistance, favorable binding kinetics, convenient handling, and flexible application.
High demand for remote sensing of hazardous substances. Possible solution: Use of distributed, low cost, and environmentally safe particles as passive sensors that can be read out remotely
Chemical intelligence on the particle surface can be easily modified Particles enable optically quantifiable response and inference of target substances (also no maintenance or power supply required
Remote sensing of hazardous substances is a key task that can be achieved with the help of remotely operated platforms equipped with specific sensors. A huge variety of methods and used vehicles have been developed for different purposes in recent years. The term smart dust refers to a science fiction novel and develop shortly after into a research proposal at UC Berkley funded by DARPA. Subsequently, the topic gained attraction but was overall considered as to complex for the technologies available at that time. In the launched passive smart dust project, we shift to a simple “chemical intelligent” passive sensor particle on the ground combined a read-out active sensor attached to an Unmanned Aerial Vehicle (UAV). The reactive particle surface can be preadjusted in the lab for exact desired properties regarding certain reactions to hazardous substances.
Moreover, the aimed interaction with the active sensor can be modified. Planed applications allow for different materials e.g., for short time measurement, being ecologically degradable, or weather stable for long time monitoring.
Current trends in materials and life sciences are flanked by the need to push detection limits to single molecules or single cells, enable the characterization of increasingly complex matrices or sophisticated nanostructures, speed up the time of analysis, reduce instrument complexity and costs, and improve the reliability of data. This requires suitable analytical tools such as spectroscopic, separation and imaging techniques, mass spectrometry, and hyphenated techniques as well as sensors and their adaptation to application-specific challenges in the environmental, food, consumer product, health sector, nanotechnology, and bioanalysis. Increasing concerns about health threatening known or emerging pollutants in drinking water, consumer products, and food and about the safety of nanomaterials led to a new awareness of the importance of analytical sciences. Another important driver in this direction is the increasing demand by legislation, particularly in view of the 17 sustainable development goals by the United Nations addressing clean energy, industry, and innovation, sustainable cities, clean water, and responsible consumption and production. In this respect, also the development of analytical methods that enable the characterization of material flows in production processes and support recycling concepts of precious raw materials becomes more and more relevant. In the future, this will provide the basis for greener production in the chemical industry utilizing recycled or sustainable starting materials.
This makes analytical chemistry an essential player in terms of the circular economy helping to increase the sustainability of production processes. In the life sciences sector, products based on proteins, such as therapeutic and diagnostic antibodies, increase in importance. These increasingly biotechnologically produced functional biomolecules pose a high level of complexity of matrix and structural features that can be met only by highly advanced methods for separation, characterization, and detection. In addition, metrological traceability and target definition are still significant challenges for the future, particularly in the life sciences.
However, innovative reference materials as required for the health and food sector and the characterization of advanced materials can only be developed when suitable analytical protocols are available. The so-called reproducibility crisis in sciences underlines the importance of improved measures of quality control for all kinds of measurements and material characterization. This calls for thorough method validation concepts, suitable reference materials, and regular interlaboratory comparisons of measurements as well as better training of scientists in analytical sciences.
The important contribution of analytical sciences to these developments is highlighted by a broad collection of research papers, trend articles, and critical reviews from these different application fields. Special emphasis is dedicated to often-overlooked quality assurance and reference materials.
Nonporous corundum powder, known as an abrasive material in the industry, was functionalized covalently with protein binders to isolate and enrich specific proteins from complex matrices. The materials based on corundum were characterized by TEM, ESEM, BET, DLS, and zeta potential measurements. The strong Al-O-P bonds between the corundum surface and amino phosphonic acids are used to introduce functional groups for further conjugations. The common cross-linker glutaraldehyde was compared with a hyperbranched polyglycerol (PG) of around 10 kDa. The latter is oxidized with periodate to generate aldehyde groups that can covalently react with the amines of the surface and the amino groups from the protein via a reductive amination process. The amount of bound protein was quantified via aromatic amino acid analysis (AAAA). This work shows that oxidized polyglycerol can be used as an alternative to glutaraldehyde. With polyglycerol, more of the model protein bovine serum albumin (BSA) could be attached to the surface under the same conditions, and lower nonspecific binding (NSB) was observed. As a proof of concept, IgG was extracted with protein A from crude human plasma. The purity of the product was examined by SDS-PAGE. A binding capacity of 1.8 mg IgG per g of corundum powder was achieved. The advantages of corundum are the very low price, extremely high physical and chemical stability, pressure resistance, favorable binding kinetics, and flexible application.
Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a pilot study, CCQM-P216, was coordinated by the Chinese National Institute of Metrology (NIM), National Research Council of Canada (NRC) and the Bureau International des Poids et Mesures (BIPM). Eleven Metrology Institutes or Designated Institutes and the BIPM participated in the first phase of the pilot study (Part 1). The purpose of this pilot study was to develop measurement capabilities for larger proteins using a recombinant humanized IgG monoclonal antibody against Spike glycoprotein of SARS-CoV-2 (Anti-S IgG mAb) in solution. The first phase of the study was designed to employ established methods that had been previously studies by the CCQM Protein Analysis Working Group, involving the digestion of protein down to the peptide or amino acid level. The global coronavirus pandemic has also led to increased focus on antibody quantitation methods. IgG are among the immunoglobulins produced by the immune system to provide protection against SARS-CoV-2. Anti-SARS-CoV-2 IgG can therefore be detected in samples from affected patients. Antibody tests can show whether a person has been exposed to the SARS-CoV-2, and whether or not they potentially show lasting immunity to the disease. With the constant spread of the virus and the high pressure of re-opening economies, antibody testing plays a critical role in the fight against COVID-19 by helping healthcare professionals to identify individuals who have developed an immune response, either via vaccination or exposure to the virus. Many countries have launched large-scale antibody testing for COVID-19. The development of measurement standards for the antibody detection of SARS-CoV-2 is critically important to deal with the challenges of the COVID-19 pandemic. In this study, the SARS-CoV-2 monoclonal antibody is being used as a model system to build capacity in methods that can be used in antibody quantification. Amino acid reference values with corresponding expanded uncertainty of 36.10 ± 1.55 mg/kg, 38.75 ± 1.45 mg/kg, 18.46 ± 0.78 mg/kg, 16.20 ± 0.67 mg/kg and 30.61 ± 1.30 mg/kg have been established for leucine, valine, phenylalanine, isoleucine and proline, respectively. Agreement between nearly all laboratories was achieved for the amino acid analysis within 2 to 2.5 %, with one participant achieving markedly higher results due to a technical issue found in their procedure; this result was thus excluded from the reference value calculations. The relatively good agreement within a laboratory between different amino acids was not dissimilar to previous results for peptides or small proteins, indicating that factors such as hydrolysis conditions and calibration procedures could be the largest sources of variability. Peptide reference values with corresponding expanded uncertainty of 4.99 ± 0.28 mg/kg and 6.83 ± 0.65 mg/kg have been established for ALPAPIEK and GPSVFPLAPSSK, respectively. Not surprisingly due to prior knowledge from previous studies on peptide quantitation, agreement between laboratories for the peptide-based analysis was slightly poorer at 3 to 5 %, with one laboratory's result excluded for the peptide GPSVFPLAPSSK. Again, this level of agreement was not significantly poorer than that achieved in previous studies with smaller or less complex proteins. To reach the main text of this paper, click on Final Report.
Es wurde im Rahmen dieser Arbeit eine neue Methode der Biokonjugation entwickelt, die es ermöglicht humane Antikörper ortspezifisch mit IgG-bindenden Peptiden zu konjugieren. Als Basis fungierte ein Peptid, welches für den Einsatz gezielt modifiziert wurde. So sollte am C-Terminus ein Biotin eingefügt werden, dass für die spätere Detektion der Biokonjugation genutzt werden kann, während am N-Terminus ein Cross-Linker für die kovalente Bindung zum Antikörper eingefügt wurde. Das Biotin wurde mittels Biotin-Lysin eingebaut. Dies hat den Vorteil, dass die modifizierte Aminosäure direkt in der SPPS genutzt werden kann. Auch der Cross-Linker soll schon während der SPPS in das Peptid integriert werden. Als Cross Linker wurden die zwei heterobifunktionellen Succinimidyl(3-bromoacetamid)propionate und Succinimidyl(4-iodacetyl)aminobenzoat untersucht. Die Aktivierung des Peptides mit dem SBAP-Cross-Linker erfolgte am besten im pH-Bereich zwischen 7,0 und 9,0. Die Modifizierung des Peptides mit dem Iodid-Cross-Linker SIAB unter den gleichen Bedingungen zeigte allerdings keine zufriedenstellenden Ergebnisse. Da das erste Peptid allerdings in den Folgeexperimenten sehr gute Ergebnisse zeigte, musste kein weiterer Linker getestet werden. Zusätzlich zu der Cross-Linker-Wahl sollte der Abstand zwischen dem Cross-Linker und dem Grundgerüst des Peptides auf den Einfluss der Bindung untersucht werden. Dazu wurden drei Kontrollpeptide synthetisiert, die entweder um zwei Aminosäuren zwischen dem ursprünglichen N-Terminus des Peptides und dem SBAP-Linker verlängert wurden, keinen SBAP-Linker beinhalten oder die Verlängerung ohne SBAP-Linker besaßen. Die erfolgreiche Synthese aller vier Peptide wurde mittels MALDI-TOF-MS bestätigt.
Die neue Crosslinking-Methode ist hilfreich, um Immunglobuline des Isotyps G ortspezifisch an ihrer Bindungsstelle mit Protein A oder G zu konjugieren. Die Kopplungen von Protein A und G konnten erfolgreich an Maus- und Human-IgG durchgeführt, sowie die Bedingungen untersucht und optimiert werden. Die Aktivierung von Protein G mit Glutaraldehyd erfolgt am besten bei pH 8 und die anschließende Kopplung mit Maus-IgG1 bei einem pH-Wert von 6. Jedoch wurden mit SIAB und Sulfo-SIAB als Crosslinker im Vergleich zu Glutaraldehyd noch höhere Signale erhalten. Für die Kopplung von SIAB sind 40% DMSO im Reaktionspuffer günstig, währenddessen Sulfo-SIAB gut wasserlöslich ist daher keine Lösungsvermittler benötigt. Es ergab sich ein optimaler pH-Wert von 7,4 um Protein A mit SIAB zu aktivieren und den gleichen pH-Wert um die Kopplung mit Maus-IgG1 durchzuführen. Für die Kopplung von Protein G mit Maus-IgG1 hingegen zeigten die Experimente, dass ein leicht saurer pH-Wert bei pH 6 für den IgG-Kopplungsschritt am günstigsten ist. Während die Inkubationszeit von Maus-IgG1 mit Protein G bei 16 h liegt, muss mit Protein A bis zu 40 h inkubiert werden, um das Kopplungsmaximum zu erreichen. Des Weiteren wurde das Crosslinking von Human-IgG (Herceptin) mit SIAB untersucht. Dabei zeigte sich, wie in der Abbildung 47 zu sehen ist, dass Protein A und G vergleichbar gut an Human-IgG zu koppeln sind. Für das Crosslinking von Protein G mit Maus-IgG1 ist dagegen ein deutlich besserer Umsatz im Vergleich zu Protein A zu erkennen (Abbildung 48). Heterobifunktionale Linker können mit der reaktiveren Gruppe die erste Bindung eingehen, um erst nach Zugabe eines weiteren Reagenzes die zweite Bindung auszubilden und zudem intramolekulare Reaktionen möglichst zu vermeiden.
Das Ziel der Arbeit war die Entwicklung einer magnetischen Anreicherungsmethode für eine Bead-basierte Substanzbibliothek zur Identifizierung von Proteinbindern. Im Ramen des Forschungsbelegs wurde bereits eine Anreicherungsmethode für ein Modellsystem einer Peptidbibliothek entwickelt. Das Modellsystem bestand dabei aus zwei verschiedenen Peptiden. Als Positivkontrolle wurde das FLAG-Peptid gewählt, welches selektiv gegen den Anti-FLAG-Antikörper bindet, und ein weiteres Peptid wurde als Negativkontrolle gewählt. Anhand dieses Modellsystems konnte die zuvor entwickelte Anreicherungsmethode von 20.000 auf 1.000.000 Beads vergrößert werden. Hierbei wurde ein Anreicherungsfaktor von 818 und eine gute Wiederfindungsrate von 82% erreicht.
Im Vergleich zu anderen Proteinen ist die Identifizierung von Antikörpern anhand ihrer Sequenz zum Beispiel mittels "peptide mass fingerprinting" schwierig. Da die Sequenzinformation eines Antikörpers aufgrund der hypersomatischen Mutation während der Affinitätsreifung nicht im Genom eines Organismus gespeichert ist, kann die Aminosäuresequenz nicht auf einfachem Weg der DNA-Sequenzierung gewonnen werden. Das ist nur in seltenen Fällen möglich, wenn dem Endanwender der Zellklon der Antikörper-produzierenden Zelle zugänglich ist. Eine Sequenzierung auf Protein-Ebene ist sehr aufwändig und teuer und wird daher fast nie für die Charakterisierung von analytischen Antikörpern verwendet. Der Mangel an Validierung dieser analytischen Antikörper, die bei Experimenten verwendeten werden, löst aber eine Reihe Probleme aus, die die Wiederholbarkeit dieser Experimente schwierig und in einigen Fällen unmöglich macht. Das sorgt jährlich für verschwendete Forschungsgelder in Milliardenhöhe und hindert den wissenschaftlichen Fortschritt.
Ziel der vorliegenden Arbeit war die Entwicklung einer einfachen und schnellen Methode, die es trotzdem ermöglicht, die Identifikation von Antikörpern sicherzustellen. Dazu wurde eine Methode basierend auf dem "peptide mass fingerprinting" gewählt. Das Problem der unbekannten Aminosäuresequenz der Antikörper wurde gelöst, indem lediglich die Peptidmuster der entstehenden Fingerprint-Spektren zur Identifikation herangezogen wurden. MALDI wurde dabei als Ionisationsmethode für die Massenspektrometrie gewählt, da die resultierenden Spektren im Gegensatz zu ESI-MS einfach auszuwerten sind. Auch kann auf eine vorige Trennung der Peptide mittels LC verzichtet werden, was zusätzlich Analysenzeit spart. Für die Proteinspaltung wurde eine simple saure Hydrolyse mittels Ameisensäure gewählt. Im Vergleich zum herkömmlichen Trypsin-Verdau konnten auf zeitraubende Arbeitsschritte wie Denaturierung, Reduktion und Alkylierung der Antikörper verzichtet werden. Die Hydrolyse mittels Ameisensäure wurde bisher nur auf kleine und mittelgroße Proteine angewendet, sodass im ersten Teil dieser Arbeit mehrere Schritte optimiert wurden bevor zufriedenstellende Fingerprint-Spektren von Antikörpern erhalten wurden.
In dieser Arbeit wurden drei verschiedene Nanomaterialien auf ihre Bindungsfähigkeit zu Proteinen untersucht. Zu Beginn standen dabei die Herstellung stabiler Dispersionen der einzelnen Nanopartikel und die Stabilität der gebildeten Konjugate im Vordergrund. Der Nachweis einer erfolgreichen Konjugatbildung, sprich der Beschichtung von Nanopartikel mit Proteinen, wurde sowohl qualitativ mittels DLS-Messungen als auch über quantitative Protein-Bestimmungen erbracht. Für die Quantifizierung konnten verschiedene Methoden eingesetzt werden. Neben der klassischen Vorgehensweise, welche indirekt über die Quantifizierung von ungebundenem Protein im Überstand erfolgt, konnten ihm Rahmen dieser Arbeit verschiedene direkte Bestimmungsmethoden entwickelt werden. So wurden mittels kolorimetrischer Tests, wie dem BCA-Assay und dem Bradford-Assay, Nanodiamantdispersionen mit Hilfe einer Korrekturwellenlänge vermessen und quantifiziert. Ebenso zum Einsatz kam die Methode der Aminosäureanalytik, welche aufgrund ihrer guten Rückführbarkeit auf Aminosäurestandards Ergebnisse mit hoher Richtigkeit generieren kann und ebenso die Detektion kleiner Proteinmengen möglich macht.
Nach den erfolgten quantitativen Betrachtungen wurden die Protein-beschichteten Nanopartikel auf ihre Anwendbarkeit als Analoga von Virus-like Particles (VLP) bei einer Immunisierung zur Gewinnung von polyklonalen Antikörpern gegen humanes Ceruloplasmin in Kaninchen überprüft. Es konnte mittels ELISA gezeigt werden, dass die Konjugate erfolgreich für die Herstellung von Antikörpern eingesetzt werden können und im zeitlichen Verlauf einer Immunisierung eine Steigerung des Antikörper-Titers zu erreichen ist.
Vergleichende Darstellung SARS-CoV-2-spezifischer Nanobodys aus unterschiedlichen Wirtsorganismen
(2021)
Aufgrund der anhaltenden COVID-19-Pandemie werden neutralisierende Therapeutika benötigt. Eine Möglichkeit zur Behandlung stellt die Verwendung monoklonaler Anti-SARS-CoV-2-Immun-globuline dar. Ihre Produktion in Säugetierzellen ist jedoch schwer skalierbar, um den weltweiten Bedarf zu decken. VHH-Antikörper, auch Nanobodys genannt, bieten hierfür eine Alternative, da sie eine hohe Temperaturstabilität aufweisen und eine kostengünstige Produktion in prokaryotischen Wirtsorganismen ermöglichen.
In 2019 over 30 000 people were killed or injured by explosions caused by explosives like TNT, PETN, HMX and RDX. Therefore, highly sensitive assays for the detection of TNT are needed. In this study we compared two commercially available TNT antibodies: A1.1.1 and EW75C with a highly optimized indirect competitive ELISA based on a BSA-TNA conjugate. As a result, a precision profile for both antibodies was determined with a LOD of 170 pmol L-1 for the clone A1.1.1 and a LOD of 3,2 nmol L-1 for the clone EW75C. The measurements showed that the clone A1.1.1 is a highly sensitive antibody for the detection of TNT while the clone EW75C does show medium performance at most.
In the cross-reactivity characterization of both antibodies many substances, closely related to the structure of TNT were tested. Both antibodies showed strong cross reactivity with trinitroaniline and trinitrobenzene. For the clone A1.1.1, which is known to originate from immunization of mice with an TNP-glycine-KLH conjugate, this has to be expected. Interestingly the clone EW75C, which was not characterized yet, showed similar behavior. This suggests a TNA-conjugate as immunogen for the EW75C antibody as well. None of both antibodies showed cross-reactions to the high explosives PETN, HMX and RDX. Also, the cross-reactions of nitro musks with the antibodies were investigated. Despite their prohibition, nitro musks are still used in Asia especially and are particularly popular in India. The overall superior clone A1.1.1 showed a significant cross-reactivity to musk ambrette. For practical reasons the influence of musk ambrette on this assay when used in natural environment should be investigated.
In further experiments, the highly sensitive TNT antibody A1.1.1 was digested with papain to obtain monovalent Fab-fragments. Due to its high stability against the digestion, a custom protocol for the IgG1 subclass of mice, to which the clone A1.1.1 belongs, was developed, resulting in a quantitative digestion of the intact antibody to Fab fragments. The success of the digestion was determined with MALDI-TOF-MS and SDS-PAGE. It was shown that this protocol worked for many different antibodies of IgG1 subclass as well.
Protein immobilization for the functionalization of particles is used in various applications, including biosensors, lateral-flow immunoassays (LFIA), bead-based assays, and others. Common methods for the quantification of bound protein are measuring protein in the supernatant before and after coating and calculating the difference. This popular approach has the potential for a significant overestimation of the amount of immobilized protein since layers not directly bound to the surface (soft protein corona) are usually lost during washing and handling. Only the layer directly bound to the surface (hard corona) can be used in subsequent assays. A simplified amino acid analysis method based on acidic hydrolysis and RP-HPLC-FLD of tyrosine and phenylalanine (aromatic amino acid analysis, AAAA) is proposed to directly quantify protein bound to the surface of gold nano- and latex microparticles. The results are compared with indirect methods such as colorimetric protein assays, such as Bradford, bicinchoninic acid (BCA), as well as AAAA of the supernatant. For both particle types, these indirect quantification techniques show a protein overestimation of up to 1700% compared to the direct AAAA measurements. In addition, protein coating on latex particles was performed both passively through adsorption and covalently through EDC/sulfo-NHS chemistry. Our results showed no difference between the immobilization methodologies. This finding suggests that usual protein determination methods are no unambiguous proof of a covalent conjugation on particles or beads.
MALDI-TOF-MS-based identification of monoclonal murine anti-SARS-CoV-2 antibodies within one hour
(2022)
During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used thirty-five monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied onto the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 45 minutes and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 °C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0 (https://gets.shinyapps.io/ABID/). This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.
Development of an affinity-based method for the site-selective synthesis of antibody-drug-conjugates
(2021)
For the site-selective synthesis of ADCs, a variety of obstacles must be overcome. Those include designing bifunctional affinity peptides with reasonably low 𝐾𝑑-values that couple to the mAb in a site-selective manner. These peptides should also include a functional group that links the payload to the mAb under mild conditions without adversely affecting it. The bioconjugation between peptide and antibody and the linker between peptide and payload must be stable and durable to provide safety when used for medical purposes. The usage of metals and organic solvents should be minimized. Within the project, new types of functionalized affinity peptides were designed, and their affinity towards the Fc-fragment was determined.
In der Masterarbeit wurde ein Screening gegen den CCMV mit einer linearen Peptidbibliothek entwickelt, wobei zwei lineare Binder identifiziert wurden. Das resynthetisierte Peptid wurde auf seine Bindungseigenschaften mittels ELISA und MST untersucht. Aufgrund eines Aminosäurefehlers in der selbst durchgeführten Resynthese des Peptids sind die Ergebnisse nicht vollständig übertragbar. Um eine umsetzbare Cyclusgröße für die Peptidbibliothek zu finden, wurden Ringgrößen mit sechs bis acht Aminosäuren untersucht. Da keiner der gewählten Ringgrößen bevorzugt war, wurde die Ringgröße aus acht Aminosäuren für die Peptidbibliothek gewählt. Der Ringschluss wurde durch die Bildung einer Disulfidbrücke erreicht. Dadurch war die Alkylierung der vorhandenen Thiolgruppe in den Abbruchsequenzen notwendig.
Neben den etablierten Alkylierungsreagenzien Iodessigsäure, Iodacetamid und Acrylamid wurden zwei Epoxide mituntersucht. Hierbei konnte nur bei Acrylamid und Propylenoxid eine vollständige Alkylierung beobachtet werden. Eine synthetische Peptidbibliothek aus zehn Aminosäuren pro Kopplungsschritt und einer Peptidlänge von acht Aminosäuren wurde erfolgreich nach der Split-and-Mix-Synthese hergestellt. Neben den kanonischen Aminosäuren wurde die synthetische Aminosäure 3-(3-Pyridyl)-alanin in die Peptidbibliothek mit eingebaut. Peptidsequenzen aus der cyclische Peptidbibliothek konnte mittels MALDI-TOF-MS identifiziert werden. Es konnte außerdem gezeigt werden, dass synthetische Aminosäuren mit proteinogenen Aminosäuren erfolgreich übersetzt werden. Anschließend wurde das entwickelte Screening auf eine cyclische Peptidbibliothek übertragen. Dabei konnte kein Binder identifiziert werden, da zu viele Nebenreaktionen auftraten. Ein alternativer Ringschluss über die Seitenketten von Lysin und Cystein wurden mit ortho-Phthaldialdehyd und 2,4,6-Trichloro-1,3,5-triazin (Cyanurchlorid) untersucht. Beide Bedingungen wiesen keinen erfolgreichen Ringschluss auf. Obwohl der Ringschluss über die Seitenketten von Lysin und Cystein nicht erfolgreich war, sollte ein Austausch von Cystein angestrebt werden, so dass kein freies Cystein in den Abbruchsequenzen vorhanden ist und die Alkylierung überflüssig wäre. Der Ringschluss durch Verwendung anderer Seitenketten bietet einen vielseitigen Ansatz.
Mit dem Testsystem zur Probe
(2021)
Type-I pyrethroids are frequently used for disinfection purposes on airplanes from and to tropical areas. The WHO (Word Health Organization) defines “disinfection” as the procedure whereby health measures are taken to control or kill the insect vectors of human diseases including dengue, yellow fever and malaria.1 Due to this, a new effective and rapid method for pyrethroids analysis directly in the plane is needed.
To detect type-I pyrethroids, gated materials are constructed for the purpose of finely tuning the movement of a cargo from voids of a mesoporous inorganic support to a solution, in response to a predefined stimulus.2 Antibodies are bound to specific functional groups grafted on the support’s surface with the two-fold aim of closing the mesopores and working as biological receptors for the target compounds.2 A specific opening mechanism, activated selectively in presence of pyrethroids as analytes, is able to control the release of an indicator dye previously loaded inside the inorganic support. As only few molecules of pyrethroids are necessary to open a pore and release a large amount of dyes, the system shows an intrinsic signal amplification quantified as a fluorescence emission intensity.3
Different mesoporous silica supports (from nano- and microparticles to platelets and short fibers) were synthetized, characterized and functionalized following different capping strategies. In this contribution, we will compare the temporal response behaviour of the optimized gated materials to verify if the respective delivery systems are properly closed and how fast they can be opened in presence of the analytes. Our results indicate that the trends of the kinetics observed in solution show a better control of the closing/opening mechanism when the epitope region of the antibody (around 10 nm) properly fits the pore size of the carriers.
Because the incorporation of an antibody-gated dye-delivery system with a conventional test-strip-based lateral-flow assay allows for the detection of analytes down to the ppb level in an easy-to-operate manner and an overall assay time of 2–5 min, which is fast for a biochemical test,3 we carried out first model studies for a lateral flow test assay on membranes, using a smartphone setup for read-out.
Cardiovascular diseases are the number one cause of death worldwide and responsible for 3.9 million deaths in Europe and over 1.8 million deaths in the European Union (2017) [1]. Cardiac Troponin I (cTnI) is part of a triple protein complex and the most important biomarker for acute events such as heart attacks. Once a heart attack occurs, the blood stream within the heart is interrupted and further oxygen supply cannot be maintained. As a result, heart cells undergo apoptosis and their proteins will be released into the blood stream. Within the project CardioMet of the European Metrology Program of Research and Innovation (EMPIR) a biosensor for online-monitoring of acute myocardial infarction by detecting cTnI is being developed.
Antibodies are the most used biomolecules in analytical research. Nevertheless, the sequence and structure information of antibodies is often limited, since manufacturers keep them secret or suppliers sell them under different names. This can make it difficult to reproduce even basic experiments performed in publications as the antibodies used might not be identifiable. To overcome these problems, we developed a simple and cheap method for antibody identification by MALDI-TOF-MS fingerprinting. This technique was used to generate a library of antibody fingerprints, which enables the identification and comparison of antibodies in short time.
The trafficking of illegal drugs by criminal networks at borders, harbors, or airports is an increasing issue in public health as these routes ensure the main supply of illegal drugs. The prevention of drug smuggling, including the installation of scanners and other analytical devices to detect ultra-small traces of drugs within a reasonable time frame, remains a challenge. The presented immunosensor is based on a monolithic affinity column with a large excess of immobilized hapten, which traps fluorescently labeled antibodies as long as the analyte cocaine is absent. In the presence of the drug, some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and microfluidic chip-based mixing devices and flow cells. The biosensor achieved limits of detection of 23 pM (7 ppt) of cocaine with a response time of 90 seconds and a total assay time below 3 minutes. With surface wipe sampling, the biosensor was able to detect 300 pg of cocaine. This immunosensor belongs to the most sensitive and fastest detectors for cocaine and offers near-continuous analyte measurement.
Silicon photonic micro-ring resonators (MRR) developed on the silicon-on-insulator (SOI) platform, owing to their high sensitivity and small footprint, show great potential for many chemical and biological sensing applications such as label-free detection in environmental monitoring, biomedical engineering, and food analysis. In this tutorial, we provide the theoretical background and give design guidelines for SOI-based MRR as well as examples of surface functionalization procedures for label-free detection of molecules.
After introducing the advantages and perspectives of MRR, fundamentals of MRR are described in detail, followed by an introduction to the fabrication methods, which are based on a complementary metal-oxide semiconductor (CMOS) technology. Optimization of MRR for chemical and biological sensing is provided, with special emphasis on the optimization of waveguide geometry. At this point, the difference between chemical bulk sensing and label-free surface sensing is explained, and definitions like waveguide sensitivity, ring sensitivity, overall sensitivity as well as the limit of detection (LoD) of MRR are introduced. Further, we show and explain chemical bulk sensing of sodium chloride (NaCl) in water and provide a recipe for label-free surface sensing.
A novel stationary phase for affinity separations is presented. This material is based on sintered borosilicate glass readily available as semi-finished filter plates with defined porosity and surface area. The material shows fast binding kinetics and excellent long-term stability under real application conditions due to lacking macropores and high mechanical rigidity. The glass surface can be easily modified with standard organosilane chemistry to immobilize selective binders or other molecules used for biointeraction. In this paper, the manufacturing of the columns and their respective column holders by 3D printing is shown in detail. The model system protein A/IgG was chosen as an example to examine the properties of such monolithic columns under realistic application conditions. Several specifications, such as (dynamic) IgG capacity, pressure stability, long-term performance, productivity, non-specific binding, and peak shape, are presented. It could be shown that due to the very high separation speed, 250 mg antibody per hour and column can be collected, which surpasses the productivity of most standard columns of the same size. The total IgG capacity of the shown columns is around 4 mg (5.5 mg/mL), which is sufficient for most tasks in research laboratories. The cycle time of an IgG separation can be less than 1 minute. Due to the glass material's excellent pressure resistance, these columns are compatible with standard HPLC systems. This is usually not the case with standard affinity columns, limited to manual use or application in low-pressure systems. The use of a standard HPLC system also improves the ability for automation, which enables the purification of hundreds of cell supernatants in one day. The sharp peak shape of the elution leads to an enrichment effect, which might increase the concentration of IgG by a factor of 3. The final concentration of IgG can be around 7.5 mg/mL without the need for an additional nanofiltration step. The purity of the IgG was > 95% in one step and nearly 99% with a second polishing run.
Vor dem Hintergrund der einzigartigen Modularität, hohen Empfindlichkeit und Selektivität von Antikörper‐gesteuerten Indikatorfreisetzungssystemen (gAID‐Systemen) wurde hier ein Multiplex‐Assay für drei organische Explosivstoffmoleküle (TATP, TNT, PETN) entwickelt, der es erlaubt, die Analyten gleichzeitig in flüssiger Phase mit einem einzelnen Teststreifen und einem Fluoreszenzlesegerät bzw. Smartphone als Detektor in Konzentrationen bis in den unteren ppb‐Bereich in <5 min nachzuweisen. Alle drei Systeme, darunter die hier neu entwickelten Systeme für TNT und PETN, tolerieren zudem nicht nur gepufferte wässrige Modelllösungen, sondern auch komplexere Matrices. Neben einem konventionellen Teststreifen mit einem Kanal erlaubte uns die Anwendung von Wachsdrucktechnologie das Herstellen von mehrkanaligen Streifen mit vergleichbarer analytischer Leistungsfähigkeit, was das enorme Potenzial der modular aufgebauten, hybriden Biosensormaterialien im Hinblick auf eine für den Endanwender maßgeschneiderte Vor‐Ort‐Analytik unterstreicht.
BioPIC - Integration of Biosensors based on Photonic Integrated Circuits by Local-Backside Etching
(2020)
Silicon photonic sensors are promising candidates for lab-on-a-chip solutions with versatile applications and scalable production prospects using complementary metal-oxide semiconductor (CMOS) fabrication methods. However, the widespread use has been hindered because the sensing area adjoins optical and electrical components making packaging and sensor handling challenging. In this work, a local back-side release of the photonic sensor is employed, enabling a separation of the sensing area from the rest of the chip. This approach allows preserving the compatibility of photonic integrated circuits in the front-end of line and metal interconnects in the back-end of line.
The antibody A.1.1.1 was labeled and found to be very sensitive and highly selective for TNT. A novel monolithic affinity column was coated with a Trinitroaniline (TNA)-BSA affinity conjugate and a custom laser induced fluorescence detector were built to allow for continuous and sensitive detection. The affinity column combined with the high sensitivity detector resulted in a limit of detection of approx. 100 pM TNT or 20 ppt TNT for offline detection and was able to detect picogram amounts within three minutes.
The mechanism of this system is based on kinetic competition. This biosensor consists of a monolithic glass column with a vast excess of immobilized hapten, which traps the fluorescently labeled antibody as long as no explosive is present. If the explosive 2,4,6-trinitrotoluene (TNT) is introduced some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein. The fluorescence is detected by highly sensitive laser-induced fluorescence with a conventional CMOS camera. The system achieved limits of detection of approx.1 pM (1 ppt) of the fluorescent label and around 100 pM (20 ppt) of TNT. The total assay time is less than 8 minutes. A cross-reactivity test with 5000 pM solutions of pentaerythritol tetranitrate (PETN), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) showed no cross reactivity.
Gegenwärtig ist das Interesse und der Bedarf von Proteinbindern insbesondere in der Biotechnik und Pharmaforschung sehr groß. Kombinatorische, Partikel-basierte (One-Bead-One-Compound) Peptidbibliotheken sind eine Technik, um selektiv bindende Proteine zu identifizieren. Allerdings beinhaltet das Screening dieser Peptidbibliotheken aufwendige Schritte, wie die Separation, Sequenzierung und Charakterisierung von identifizierten Bindern. In dieser Arbeit wurde ein Chip-System entwickelt, auf dem alle Schritte eines Screenings durchgeführt werden können. Dafür wurde ein Glasobjektträger mit einem magnetisch leitenden, doppelseitigen Klebeband versehen. Die Partikel der Bibliothek wurden durch ein Sieb aufgetragen. Dies führte zu einer geordneten Immobilisierung der Partikel auf dem Chip. Über 30.000 Partikel konnten so auf einem Chip immobilisiert werden. Für die Identifizierung von selektiven Protein-bindenden Peptiden wird die immobilisierte Peptidbibliothek mit einem Fluorophor-markierten Protein inkubiert, bindende Partikel mittels Fluoreszenzscan identifiziert und die Peptidsequenz direkt auf dem Chip mittels Matrix-Assisted-Laser-Desorption/Ionization-(MALDI)-Flugzeit-(TOF)-Massenspektroskopie (MS) bestimmt. Die Durchführung einer Abbruchsequenz-Methode erlaubt die eindeutige Bestimmung der Peptidsequenzen mit einer nahezu 100 % Genauigkeit. Die entwickelte Technologie wurde in einem FLAG-Peptid-Modell validiert. Bei dem Screening wurden neue anti-FLAG-Antikörper-bindende Peptide identifiziert. Anschließend wurden in einem Screening von ca. 30.000 Partikeln IgG-bindende Peptide mit mittleren mikromolaren Dissoziationskonstanten identifiziert. Für die Identifizierung stärkerer Binder wurde eine magnetische Anreicherung entwickelt, die dem Chip-Screening vorgeschaltet werden kann. Hiermit wurden aus ca. 1 Million gescreenter Partikel, Peptide mit Dissoziationskonstanten im niedrigen mikromolaren Bereich identifiziert.
The urgent necessity to carry out reliable and relevant analytical measurements directly at a point-of-need is one of the current drivers for the development of miniaturised analytical systems, quick tests and wearables. Despite their simplicity, this type of tests must guarantee analytical relevance and reliability like laboratory-based analysis, e.g., in terms of sensitivity, selectivity, immunity against false positives and false negatives as well as robustness and repeatability. Keeping in mind the high sensitivity offered by gated indicator-releasing micro- and nanoparticles due to their inherent features of signal amplification, we performed several optimisations to develop a potential biosensor platform for use in rapid tests. Conceptually, these gated materials are closely related to drug delivery systems, consisting of high porous materials usually closed with macromolecular “caps” and loaded with indicator molecules that are released in presence of a target analyte. However, the key difference between the two types of functional materials is that many drug delivery systems should deliver their cargo over a longer period, often many hours, whereas the gated materials prepared for sensing should show fast release kinetics, on the order of <5 min.
With the aim to optimise and adapt gated materials for sensing purposes, we prepared in this work several antibody-gated materials for small-molecule sensing. The materials consisted of porous silica particles containing indicator molecules in the pores and certain hapten molecules grafted to the particle surface close to the pore openings. The pores were then capped with antibodies binding to these haptens, thus inhibiting the escape of the indicators from inside of the pores. In presence of the corresponding analyte, the antibody is displaced from the surface of the material, allowing the escape of the indicators. This allows the detection of the analyte indirectly through an inherent signal amplification. In this work, the insecticide permethrin, a type-I pyrethroid, was selected as target model, because type-I pyrethroids play an important role in airplane disinfection. A first in-depth study of the various chemical tuning options of such antibody gated systems was performed. Different mesoporous silica supports, different functionalisation routes and different loading sequences were assessed. The materials’ performances were evaluated by studying their temporal response behaviour and detection sensitivity, including the tightness of pore closure (through the amount of blank release in absence of analyte) and the release kinetics. Our results indicate that the better the paratope-accommodating Fab region of the antibody “cap” fits into the host material’s pore openings, the better the closing/opening mechanism can be controlled. Because such materials can be used in various different formats from suspension assays[1] via microfluidic chips[2] to test strip-based lateral flow assays,[3] such materials present a powerful analytical particle platform for the sensitive analytics and diagnostics outside of a laboratory, realising sensitivities down to the µg kg–1 range in less analysis times of less than 5 min as we have recently demonstrated.[4]
The urgent necessity to carry out reliable and relevant analytical measurements directly at a point-of-need is one of the current drivers for the development of miniaturised analytical systems, quick tests and wearables. Despite their simplicity, this type of tests must guarantee analytical relevance and reliability like laboratory-based analysis, e.g., in terms of sensitivity, selectivity, immunity against false positives and false negatives as well as robustness and repeatability. Keeping in mind the high sensitivity offered by gated indicator-releasing micro- and nanoparticles due to their inherent features of signal amplification, we performed several optimisations to develop a potential biosensor platform for use in rapid tests. Conceptually, these gated materials are closely related to drug delivery systems, consisting of high porous materials usually closed with macromolecular “caps” and loaded with indicator molecules that are released in presence of a target analyte. However, the key difference between the two types of functional materials is that many drug delivery systems should deliver their cargo over a longer period, often many hours, whereas the gated materials prepared for sensing should show fast release kinetics, on the order of <5 min.
With the aim to optimise and adapt gated materials for sensing purposes, we prepared in this work several antibody-gated materials for small-molecule sensing. The materials consisted of porous silica particles containing indicator molecules in the pores and certain hapten molecules grafted to the particle surface close to the pore openings. The pores were then capped with antibodies binding to these haptens, thus inhibiting the escape of the indicators from inside of the pores. In presence of the corresponding analyte, the antibody is displaced from the surface of the material, allowing the escape of the indicators. This allows the detection of the analyte indirectly through an inherent signal amplification. In this work, the insecticide permethrin, a type-I pyrethroid, was selected as target model, because type-I pyrethroids play an important role in airplane disinfection. A first in-depth study of the various chemical tuning options of such antibody gated systems was performed. Different mesoporous silica supports, different functionalisation routes and different loading sequences were assessed. The materials’ performances were evaluated by studying their temporal response behaviour and detection sensitivity, including the tightness of pore closure (through the amount of blank release in absence of analyte) and the release kinetics. Our results indicate that the better the paratope-accommodating Fab region of the antibody “cap” fits into the host material’s pore openings, the better the closing/opening mechanism can be controlled. Because such materials can be used in various different formats from suspension assays[1] via microfluidic chips[2] to test strip-based lateral flow assays,[3] such materials present a powerful analytical particle platform for the sensitive analytics and diagnostics outside of a laboratory, realising sensitivities down to the µg kg–1 range in less analysis times of less than 5 min as we have recently demonstrated.[4]
The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence a risky venture. We think that it is crucial to improve the screening process to eliminate most of the immanent deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and simultaneous performance of competition experiments. The latter can directly be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones and blank supernatant has been designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the IgG concentration, which is unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration is not possible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media had been used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system, we conclude that this approach should be preferable to most other protocols leading to many of false positives, causing expensive and lengthy confirmation steps to weed out the poor clones.
Thousands of antibodies for diagnostic and other analytical purposes are on the market. However, it is often difficult to identify duplicates, reagent changes, and to assign the correct original publications to an antibody. This slows down scientific progress and might even be a cause of irreproducible research and a waste of resources. Recently, activities were started to suggest the sole use of recombinant antibodies in combination with the open communication of their sequence. In this case, such uncertainties should be eliminated. Unfortunately, this approach seems to be rather a long-term vision since the development and manufacturing of recombinant antibodies
remain quite expensive in the foreseeable future. Also, nearly all commercial antibody suppliers may be reluctant to publish the sequence of their antibodies, since they fear counterfeiting. De-novo sequencing of antibodies is also not feasible today for a reagent user without access to the hybridoma clone. Nevertheless, it seems to be crucial for any scientist to have the opportunity to identify an antibody undoubtedly to guarantee the traceability of any research activity using antibodies from a third party as a tool. For this purpose, we developed a method for the identification of antibodies based on a MALDI-TOF-MS fingerprint. To circumvent lengthy denaturation, reduction, alkylation, and enzymatic digestion steps, the fragmentation was performed with a simple formic acid hydrolysis step. Eighty-nine unknown monoclonal antibodies were used for this study to examine the feasibility of this approach. Although the molecular assignment of peaks was rarely possible, antibodies could be easily recognized in a blinded test, simply from their mass-spectral fingerprint. A general protocol is given, which could be used without any optimization to generate fingerprints for a database. We want to propose that in most scientific projects relying critically on antibody reagents, such a fingerprint should be established to prove and document the identity of the used antibodies and to assign a specific reagent to a datasheet of a commercial supplier, a public database record or an antibody ID.
In this study, we developed a new immunoprecipitation method based on protein-coated nanodiamonds. Performing SDS-PAGE and using the SDS-PAGE buffer as an eluent showed that CER could be successfully enriched from human serum. Based on the copper determination with ICP-MS, the amount of bound CER on the nanodiamonds can be calculated. We could show the fulfilled mass balance of bound CER and CER in the supernatant after incubation with a known amount of CER. For isotope ratio analysis this method can be applied to compare ratios of the total copper content in human serum to copper ratios from CER enrichment.
Peptide-polymer bioconjugates combine oligopeptides with synthetic polymer blocks and can be used for various applications in material sciences. In recent years, bioconjugates were applied as compatibilizers and coatings. Biocombinatorial approaches, such as phage display, have been shown to yield strong binding peptides, which exhibit excellent coating properties as peptide-PEO conjugates. Phage display represents a widely exploited strategy to select peptides or proteins that exhibit highly specific affinity to various substrates. Following a phage display experiment, DNA sequencing of binding phage clones is required in order to get the sequence information of the binding peptides. Traditionally, random clone picking followed by Sanger sequencing was applied. However, this method may not necessarily identify the strongest binding clones. Next-generation sequencing made sequencing of whole phage libraries possible, which highly improved the selection of strong binders. Here, we show that the biocombinatorial method of phage display combined with next generation DNA sequencing of whole phage libraries represents a powerful tool for an application in material chemistry. Phage display is used to find specific target binding peptides for polypropylene surfaces (PP). PP binders are of particular interest because thus far gluing or printing on PP is challenging due to its low surface energy. Scripts for sequence data analysis were developed and promising sequences were synthesized as peptide-PEO conjugates. Fluorescence based adsorption experiments on PP surfaces led to the identification of strong binding sequences and a better understanding of the peptide-surface interactions.
Preactivation crosslinking – An efficient method for the oriented immobilization of antibodies
(2019)
Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.