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Optimization of a pipeline for the development of recombinant monoclonal antibodies for diagnostics
Get PDFMolte tecnologie sviluppate in campo diagnostico si basano sull’individuazione di specifici marcatori presenti nei campioni biologici. In questo ambito è ampiamente sfruttata la capacità intrinseca degli anticorpi di riconoscere il loro antigene. Pertanto, è fondamentale implementare i vari processi che permettono il rapido isolamento degli anticorpi e che ne garantiscono un alto livello di produzione.
Grazie al continuo avanzamento delle tecnologie del DNA ricombinante, sono state sviluppate tecnologie in vitro che permettono l'isolamento su larga scala di anticorpi monoclonali.
Il phage display è una tecnologia basata sullo screening di una libreria di anticorpi contro un dato antigene. La libreria di anticorpi è di solito composta da domini variabili delle catene leggere e pesanti di una immunoglobulina combinati casualmente, questi domini sono fusi con una proteina del capside del batteriofago M13. Ogni fago espone sulla sua superficie un singolo peptide, quindi si può facilmente risalire alla sequenza codificante dei frammenti anticorpali.
L'obiettivo principale di questo lavoro è quello di ottimizzare l’intera pipeline che consente lo sviluppo di anticorpi monoclonali ricombinanti interessanti in ambito diagnostico. Questo processo inizia con l'isolamento di cloni in grado di riconoscere l'antigene partendo da una libreria fagica naïve.
Pertanto, questo lavoro è iniziato con la creazione e la caratterizzazione di una libreria fagica naïve.
Una volta costruita la libreria, questa è stata validata mediante NGS, in questo modo è stato possibile avere un'istantanea della sua diversità. La libreria è stata ulteriormente validata mediante il classico sequenziamento di Sanger in modo da poter anche valutare la qualità delle sequenze codificanti dei frammenti anticorpali che la compongono.
Per ottimizzare e validare l'intera pipeline, come antigene “pilota” è stato scelto Interferone γ, una proteina rilevante da un punto di vista diagnostico perché è utilizzata nella diagnosi della tubercolosi. Diverse procedure di biopanning hanno permesso di isolare, complessivamente, 25 scFv diversi. Questi cloni sono stati testati anche su una piattaforma automatizzata progettata per i test immunodiagnostici e questo ne ha permesso una caratterizzazione più approfondita. In questo modo è stato possibile isolare i cloni che mostravano le prestazioni migliori.
Tra questi, sono stati scelti due cloni per essere ulteriormente maturati in modo da migliorare la loro affinità nei confronti dell'antigene. Considerato che il ruolo chiave svolto dalla VH nel riconoscimento dell'antigene è ben noto, è stato deciso di creare due nuove librerie di display fagico composte da scFv tutti recanti la VH dei due cloni parentali e un pannello di VL. La procedura di selezione ha permesso di isolare due cloni che sembrano funzionare meglio rispetto il clone parentale quando testati sulla piattaforma automatizzata.
In campo diagnostico, generalmente, sono utilizzate immunoglobuline nel loro formato nativo. Quindi, una volta selezionati i frammenti anticorpali, questi devono essere clonati in vettori che ne consentano l'espressione in linee cellulari di mammifero. In questo lavoro è stata costruita una serie di vettori che consentono l'espressione delle IgG, IgA, IgM umane e IgG di topo. Tutti i vettori sono stati trasfettati e la produzione di anticorpi è stata buona anche in termini di corretto folding dell'immunoglobulina.
La fase finale di convalida dell'intera pipeline consiste nell'espressione dei tre cloni gli anti-interferone γ come immunoglobuline in formato nativo e nel test della loro funzionalità sulla piattaforma automatizzata come reagenti in un kit diagnostico della tubercolosi commerciale.In diagnostics, many tools rely on the detection of specific markers in biological specimens and widely exploit the ability of antibodies to recognize their antigen. Thus, the implementation of the various processes ranging from antibody isolation to their high rate production is fundamental.
Thanks to the advantages of recombinant DNA technologies, in vitro display methods have been developed for large scale isolation of monoclonal antibodies.
Phage display is a technology based on the screening of an antibody library against a given antigen. The antibody library is usually composed of randomly combined immunoglobulin’s light and heavy variable domains fused with a coat protein of the bacteriophage M13. Each phage exposes on its surface a single peptide, so the coding sequence of the antibody fragments can be easily retrieved.
The main objective of this work is to optimize a pipeline for recombinant monoclonal antibody development against antigens of diagnostic interest. This process begins with the isolation from a naïve phage library of clones able to recognize the antigen.
Therefore, this work started with the creation and the characterization of a naïve phage display library.
Once constructed the library, it has been validated with NGS to have an actual snapshot about library diversity and with Sanger sequencing to assess the quality of the coding sequences.
To optimize and validate the whole pipeline, as a “pilot” antigen it has been chosen Interferon γ, a diagnostically relevant protein given its employment in the detection of tuberculosis. Different biopanning procedures allowed to isolate, overall, 25 different scFv. Thanks to a deeper characterization, performed on an automated platform designed for immunodiagnostic testing, it was possible to point out the best performing clones.
Among them, two were chosen to be maturated to further enhance their affinity towards the antigen. Since it is well established the key role played by the VH in antigen recognition, it has been decided to create two new phage display libraries composed of scFv all bearing the VH of the two parental clones and a panel of VLs. The tailored selection procedure allowed to isolate two clones that seemed to perform better than the parental one on the automated platform.
In a diagnostic setting, usually, full-size immunoglobulins are required, so, once selected, the antibody fragments are cloned in vectors allowing the expression in mammalian host cell lines. In this work a set of vectors allowing the expression of the human IgG, IgA, IgM and mouse IgG has been built. All the vectors have been transfected and antibody production was good also in terms of correct folding of the full-size immunoglobulin.
The final step of validation of this whole pipeline consists in the expression of all the three anti-Interferon γ scFv as full-size immunoglobulin and the assay of their functionality on an automated platform as reagents in a commercial tuberculosis diagnostic kit
Usare strumenti empirici per risolvere problemi: Indagine nella scuola secondaria
Get PDFIl saggio presenta un approfondimento dell’indagine Problem Solving e abilità geografiche. Come valutare la competenza nel triennio della scuola secondaria di I grado.
Dopo l’indagine principale è stata realizzata anche una rilevazione, con gli stessi strumenti, nella scuola secondaria di II grado, quindi i risultati delle due popolazioni sono comparabili. L’approfondimento mira a indagare il rapporto fra l’uso di strumenti empirici per misurare le distanze su carta e i risultati nel Problem Solving. Viene presentato il modello di Problem Solving utilizzato da OCSE-PISA anche in relazione alla sua potenziale spendibilità didattica.
L’analisi dei dati conferma la correlazione positiva fra l’uso di strumenti e la performance per gli studenti della secondaria di I grado, senza differenze significative per il genere. Più complessa la situazione degli studenti più grandi: l’uso di alcuni strumenti non risulta incidere sui risultati; esistono differenze di genere circa l’andamento dei punteggi per il numero di strumenti utilizzati.The paper presents a deepening of survey Problem Solving and geographic skills. How to evaluate the competence in the three years of lower secondary school.
A second survey was also conducted, after the main investigation with the same tools, in upper secondary school, then the data of the two populations are comparable. The deepening aims to investigate the relationship between the use of empirical tools to measure distances on maps and performance in Problem Solving. The Problem Solving model used by OECD-PISA is presented, also in relation to its potential educational applications.
The analysis of the data confirms a positive correlation between the use of tools and performance for students of lower secondary school and no gender differences. The results for upper secondary school is more complex: the use of some tools does not affect the performance; there are gender differences in performance by the number of tools used
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Whi5 phosphorylation embedded in the G1 /S network dynamically controls critical cell size and cell fate
Get PDFIn budding yeast, overcoming of a critical size to enter S phase and the mitosis/mating switch - two central cell fate events - take place in the G 1 phase of the cell cycle. Here we present a mathematical model of the basic molecular mechanism controlling the G 1 /S transition, whose major regulatory feature is multisite phosphorylation of nuclear Whi5. Cln3-Cdk1, whose nuclear amount is proportional to cell size, and then Cln1,2-Cdk1, randomly phosphorylate both decoy and functional Whi5 sites. Full phosphorylation of functional sites releases Whi5 inhibitory activity, activating G 1 /S transcription. Simulation analysis shows that this mechanism ensures coherent release of Whi5 inhibitory action and accounts for many experimentally observed properties of mitotically growing or conjugating G 1 cells. Cell cycle progression and transcriptional analyses of a Whi5 phosphomimetic mutant verify the model prediction that coherent transcription of the G 1 /S regulon and ensuing G 1 /S transition requires full phosphorylation of Whi5 functional sites
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