level: Antibodies
Questions and Answers List
Flashcards on the lecture of Antibodies in Biomedical Research
level questions: Antibodies
| Question | Answer |
|---|---|
| what is the difference between linear and conformational epitopes? and why is this important? | linear epitopes are epitopes that are located on the antigen in a linear fashion, while conformational epitopes are spread on the antigen, requiring a fold/3D structure of the antigen when the antibody binds this is important when you're analysing and detecting antibody-antigen reactions. e.g. in ELISA, mostly linear epitopes are used. |
| what is the difference between polyclonal and monoclonal antibodies? | polyclonal antibodies are antibodies that are produced by different B cells and recognise multiple epitopes of a single antigen. they are cheap to produce, but generate mixed populations of antibodies and they are tolerant to small changes in the protein structure. monoclonal antibodies are antibodies that are generated by identical immune cells, which are clones of a single parent cell. this means that they are expensive to produce, but they will only bind to one epitope. monoclonal antibody production generates a single antibody species. they are senstive to protein structure. |
| what is the basic principle of immunoblot and ELISA for detecting the presence of autoantibodies in a sample? | 1. antigens are labelled on a nitrocellulose filter membrane in case of immunoblot and are coated on a 96-well plate in case of ELISA 2. you add your sample that you think contains the autoantibodies. 3. the autoantibody will bind to the antigen. 4. a secondary antibody conjugated with an enzyme is added. 5. substrate is added to visualise the reaction. |
| what are the different types of ELISA? | direct: the use of only a primary antibody conjugated with a substrate. indirect: the use of a primary antibody and a secondary antibody conjugated with a substrate. sandwich: the use of a capture antibody that "captures" the antigen as it is added, as well as a primary antibody and a secondary antibody conjugated with a substrate that bind on top of this to visualise the reaction. competitive: the sample antigen competes with a reference antigen in binding to the capture antibody, which is indicative of the concentration of the sample antigen. |
| what is the principle of cell-based assay? and what is an example? what techniques are considered cell-based assays? | cell-based assays assess the efficacy of compounds in a cellular environment, which aids to understaning of the cell. 1. plasmids that contain the gene of interest are engineerd. 2. the plasmid is transfected into the cells. 3. the corresponding protein is expressed and validated. 4. immunofluorescence is done to check for the presence of the autoantibody. cell-based assays are; flow cytometry, immunocytochemistry, immunofluorescence on cells |
| what is a tissue-based assay? and what is an example? | a tissue-based assay is an experiment that is done on a tissue and gives you insights in tissue characteristics. it is often done for detecting neuronal antibodies. 1. you have a tissue that you want to study. 2. you add a sample that contains autoantibodies. 3. if the antigen is present, the autoantibodies will bind. 4. you add a secondary antibody conjugated with an enzyme. 5. you add a substrate to visualise the reaction. |
| what is immunoprecipitation and how is it done? | immunoprecipitation is the isolation of a specific antigen from a mixture, using the antibody-antigen reaction. 1. add the suitable antibody to the mixture. 2. the antibody binds to the antigen/protein of interest. 3. IgA or IgG is added to make the antibody-antigen complex insoluble. 4. centrifuge to form a pellet so that the supernatant can be removed. |