Master in Industrial Chemistry and Introduction to Chemical Research
Master in Advance Nanoscience and Nanotechnology, Universitat Autònoma de Barcelona.
Diagnostic tests for Ebola virus
In light of the recent emergence of the lethal Ebola virus worldwide, accurate and novel detection methods for the diagnosis of the disease are mandatory.
The objective of this subject is that at the end of the lectures you will be able to design four different approaches for the diagnosis of the virus in different settings in the developed or in the developing world. This four approaches will be described in a written project which will be used for your assessment:
Approach 1. An approach for separating and preconcentrating Ebola virus from complex samples for further isolation of the virus.
Approach 2. An RDT for the detection of the infection in low resource settings in the depeloping world to screen out patients with requires strict surveillance and quarantine.
Approach 3. A test for quantifying the immunological response of the patients under treatment in high income countries.
Approach 4. A confirmatory diagnosis of Ebola hemorrhagic fever in an outbreak setting and assessment of patient viral load as a predictor of outcome.
Instructions For the Project
The main objective is to learn different methods based on Biomolecules (mainly antibodies and DNA based methods) by applying them in the detection of a target. The target selected is the Ebola Virus. Four different analytical challenges will be addressed, as described above. All these approaches will be discussed in the final project. The project will be developed step by step by meeting different milestones, during the lectures. Different deliverables for the 4 approaches will be also performed both in and out the class.
Introductory lesson of different approaches to detect communicable diseases
In this lesson you will find the different approaches in order to obtain a final diagnosis in the case of any infection disease, taking as a model Ebola virus.
Find general information about Ebola virus infection and diseases. Write a summary about the virus that will help us as an introduction for the project.
Find general information about the current diagnosis methods of the Ebola virus. Examples of websites and papers where you can find this information
Milestone 3. THE PROJECT
Describe the problem in an intruduction as well as the current diagnostic of Ebola. Furthermore, describe, step by step, the development of each test procedure as well as the protocol, for the detection of the biomarker related with Ebola Virus, taking into account:
– The production of the bioreagent (for instance, antibodies from the immunization to the purification) or, instead, the availability of commercial reagents.
– the format used for this specific biomarker as well as the platform.
– the solid support and the immobilization strategy for the bioreagent.
– the labelling of the bioreagent
– the signal obtained for the quantification of the biomarker.
Lesson 1. Introduction to bioanalytical chemistry
Lesson 2. Biomolecules and Biorecognition
Amino Acids, Peptides and Proteins. Antibodies. Enzymes. Nucleic Acids. Biorecognition: Enzyme/Substrate. Antigen/antibody. Hybridization. Other affinity interactions in nature. Strept(avidin), Protein A and G. Aptamers. Biomimetic recognition. The importance of water in biorecognition Biological buffers.
Lesson 3. Separation methods of biomolecules
Electrophoresis of proteins and DNA. Gel Electrophoresis (GE). SDS Polyacrylamide Gel Electrophoresis (SDS–PAGE). Southern, Northern and Western blot. Applications of Electrophoresis. Diagnosis of Genetic (Inherited) Disease. Paternity and forensic testing. DNA Sequencing. Diagnosis of infection diseases. Chromatography. Liquid Chromatography for Bioanalysis. Reversed Phase Liquid Chromatography (RP-LC). Ion Exchange Chromatography (IEC). Size Exclusion Chromatography (SEC). Affinity Chromatography. The special case of magnetic bioseparation.
Lesson 4. Production of bioreagents
Synthesis of short peptides and oligonucleotides. Immunological Response. Production of monoclonal and polyclonal antibodies. Antibodies fragmentation. Synthesis the immunogens. Purification of proteins from nature sources or cultures. Purification of antibodies.
Lesson 5. Immunoassays
Classification. Antibody-Antigen Complex Formation. Assay Formats. Homogeneous and heterogeneous immunoassays. Labels in immunoassays: radioisotopes, fluorescence, chemiluminescent, enzymes. Enzyme Immunoassays: ELISA. Immunoassay curve fitting.
Lesson 6. DNA analysis and amplification
Lesson 7. Rapid and screening methods
RDTs (rapid diagnostic test). Immunochromatography and lateral flow assays. Home pregnancy test. Agglutination techniques. ABO typing for blood group determination. Biosensors. Glucose biosensor. Statistical in screening methods. Cut-off determination. Binary classification test. Problems and exercises (update version)
Lesson 8. Immobilization and labeling of biomolecules.
Solid supports in bioanalytical chemistry. Immobilization strategies of biomolecules in solid supports. Non-specific adsorption. Blocking reagents. Labeling of biomolecules: enzymes, fluorophores, nanoparticles, QDs, biotin. Signal amplification strategies.
Lesson 9. Nanomaterials in Bioscience
Biorecognition with biologically-modified quantum dots, carbon nanotubes, metal and magnetic
nanoparticle: medical diagnosis, nanomedicine and bioanalysis.
– Bioanalytical Chemistry. Susan R. Mikkelsen & Eduardo Cortón. Wiley-interscience. 2004.
– Principles and Techniques of Biochemistry and Molecular Biology. 6 ª ed. Edited by Keith Wilson & John Walker. Cambridge University Press. 2006.
– Bioanalytical Chemistry. Andreas Manz. Nicole Pamme. Dimitri lossifidis. Imperial College Press, 2004..
– Principles and Practice of Bioanalysis. Edited by Richard F. Venn. Taylor & Francis, 2000.
Bibliography. The basic
– David L. Nelson, Michel M. Cox. ‘Lehninger Principios de Bioquímica’. 4ª ed. Ediciones Omega. 2006.
– L. Stryer, Jeremy M. Berg, John L. Tymocxko. Bioquímica. 5 ª ed. Editorial Reverté. 2003.
– Mary K. Campbell, Shawn O. Farrell. Bioquímica. 4 ª ed. Editorial Thomsom. 2004.
– Skoog, Holler i Niemen. «Principios de Análisis Instrumental» 5ª edició. Ed. Mc. Graw Hill. 2001.
– Skoog, West, Holler i Crouch “Fundamentos de Química Analítica” 8ª edició. Ed. Thomsom, 2005.
– Daniel C. Harris «Análisis químico cuantitativo». 2ª ed (5ª en Ingles), Editorial Reverte 2001.
Other suggested bibliography
– ‘Bioquimica. Técnicas y Métodos’. Pilar Roca, Jordi Oliver y Ana Mª Rodríguez. Editorial Hélice. 2003.
– Bryan L. Williams, Keith Wilson. Principios y Técnicas de Bioquímica experimental. Ediciones Omega, 1981.
– J.M. García Segura, J.G. Gavilanes, A. Martínez del Pozo, F. Montero, M. Oñaderra, F. Vivanco. Técnicas instrumentales de análisis en Bioquímica. Editorial Síntesis, 2002.
– D.J. Holme, H. Peck. Analytical Biochemistry. 3rd edition. Prentice Hall, 1998.
– Reiner Wetermeier and Tom Naven. Proteomics in Practice: A Laboratory Manual of Proteome Analysis, Wiley-VCH Verlag-GmbH,. Weinheim, 2002.
– Rodney F. Boyer, Modern Experimental Biochemistry (Third Edition) Benjamin/Cummings, San Francisco, 2000.
– Handbook of Experimental Immunology. Vol. 1. Weir DM, ed. Oxford: Blackwell Scientific Publications, 1986.
– Rodney F. Boyer, Biochemistry Laboratory. Modern Theory and Techniques. Publisher: Benjamin Cummings. 2006.
– Jeremy W Dale and Malcom von Schantz. From Genes to Genomes: Concepts and Applications of DNA Technology. John Wiley & Sons, Ltd. 2002
– Sample Preparation Techniques in Analytical Chemistry. Somenath Mitra, ed. Wiley-Interscience: Hoboken, NJ, 2003.