BMB514: Immunological techniques

• have gained a better understanding of theoretical immunology, through "hands-on” experience of immune system
• be able to manufacture, purify and characterise antibodies produced in, for example, rabbit or mouse
• use antibodies for qualitative and quantitative analysis
• be able to develop optimise and validate immunological assays
• be able to determine qualitative parameters such as antigen specificity, epitope specificity, affinity and the involvement of the types of bonds in the reaction between antigen and antibody
• work with cell cultures in the manufacture and characterisation of monoclonal antibodies
• work in accordance to regulations in a sterile laboratory
• assess the results of tests based on the use of immunological reagents.

I. Production of monoclonal antibodies, including immunisation, fusion, selection, cloning and the determination of specificity.

II. Analysis of polyclonal antibodies, purification of IgG, analysis of purity and specificity and the production of mono-specific preparations from poly-specific antisera.

III. Quantitative immunological techniques with special focus on the "sandwich" format in the form of Enzyme-Linked Immunosorbent Assay (ELISA). With regard to ELISA, skills in developing and configuring the assay will be acquired and applied to the quantification of an antigen in various body fluids. The effects of interference by heterophilic anti-Ig antibodies and human anti-animal IgG antibodies will also be examined. In addition, quantitative radio-immunoassay (RIA) and quantitative immunoelectrophoresis and immunodiffusion techniques will be performed.

IV. Analytical immunological techniques. In connection with the production of monoclonal antibodies, students will be instructed about the setup and application of analytical ELISA to identify relevant hybridomas and to monitor cloning. The students are introduced to the use of analytical immunoelectrophoresis techniques to study the specificity of polyclonal antibodies and to monitor chromatographic separations. The students will also be schooled in the use of double immunodiffusion to illustrate the concepts of immunological identity, non-identity and cross-reaction. Western blots will be performed in relation to the identification of specific antigens in a biological fluid and to evaluation of the specificity of the antibodies employed. In the course of the ELISA exercises, knowledge about new technologies to define the relative epitope specificity of monoclonal antibodies will also be acquired.

V. In the course teaches students the use of fluorochrome-labelled antibodies in FACS analysis to identify specific cell types and quantification of receptors on these cells. In addition, students are trained in the use of immunohistochemistry to test antigen localization in tissue sections, using enzyme-labelled antibodies.

VI. Students will be instructed in the immunoprecipitation, isolation and characterization of radioactive-labelled protein mixtures, using specific antibodies in combination with SDS-PAGE and autoradiography. Introduction to immunoagglutination will be acquired through exercises centred on the AB0 blood group system.