Immunology in the Clinic and Research Lab Flashcards
Describe and explain how antibodies can be used in research and diagnostic techniques
Research Applications:
1) Immunohistochemistry (IHC): This technique is used to detect specific antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues
2) Western Blotting: This technique is used to detect specific proteins in a sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane, where they are probed using antibodies specific to the target protein
3) Immunoprecipitation and Co-immunoprecipitation (IP/Co-IP): These techniques are used to purify and concentrate a protein or complex of proteins from a cell lysate or other complex protein mixture
4) Flow Cytometry: This technique uses fluorescently labeled antibodies to detect specific cell surface markers, which can be used to identify and isolate different cell populations
Diagnostic Applications:
1) Enzyme-Linked Immunosorbent Assay (ELISA): ELISA is a plate-based assay technique used for detecting and quantifying substances such as peptides, proteins, antibodies, and hormones. It uses antibodies to detect the presence and quantity of an antigen
2) Radioimmunoassay (RIA): This is a highly sensitive technique used to measure concentrations of antigens by the use of antibodies
3) Immunofluorescence: This technique uses antibodies labeled with fluorescent dyes to detect specific proteins or other molecules in cells and tissues. This technique is often used in diagnostic tests for autoimmune diseases and in identifying pathogens in tissue samples
4) Rapid Diagnostic Tests (RDTs): RDTs are used to quickly identify specific pathogens or markers of disease in patient samples. Many of these tests, like the rapid strep test or pregnancy tests, use antibodies to detect specific antigens
Describe the fundamental characteristics of enzyme-linked immunosorbent assays
Characteristics of ELISA:
1) Specificity: ELISA is highly specific due to the unique binding between the antibody and the antigen. This means the antibody binds only to a specific antigen, which minimises false-positive results
2) Sensitivity: ELISA is a highly sensitive technique, capable of detecting minute amounts of antigen or antibody (in the range of nanograms to picograms)
3) Versatility: ELISA can be modified to detect either the presence of antigen (direct and sandwich ELISA) or specific antibody (indirect ELISA) in a given sample
4) Quantification: ELISA not only detects the presence of an antigen or antibody but also provides quantitative results, indicating the concentration of the substance in the sample
The Process of ELISA:
1) Coating: The first step in a sandwich ELISA is the coating of the well plate with a capture antibody. This is typically done overnight to allow for adequate binding to the plate
2) Blocking: Once the capture antibody is bound, the remaining sites on the well plate are blocked with a non-specific protein or other blocking agent to prevent non-specific binding
3) Antigen Binding: The sample, which contains the antigen of interest, is added to the wells. The antigen binds specifically to the capture antibody
4) Detection: A detection antibody is added, which binds to a different epitope of the antigen. This antibody is linked to an enzyme, hence the ‘enzyme-linked’ in ELISA
5) Substrate Addition: A substrate is added, which the enzyme will convert to a detectable signal, often a colour change
6) Measurement: The plate is read on a spectrophotometer, which measures the intensity of the colour. The intensity of the colour is directly proportional to the amount of antigen in the sample
Describe the fundamental principles of Western blotting
1) Sample Preparation: This might be cells from a tissue culture, a tissue biopsy, or a sample of bodily fluid
2) Gel Electrophoresis: The proteins in the sample are first separated by size using gel electrophoresis. The negatively charged SDS binds to proteins and gives them a uniform charge-to-mass ratio. When an electric field is applied, these proteins migrate through the gel, with smaller proteins moving faster and therefore farther than larger ones
3) Transfer to a membrane: The proteins are then transferred from the gel to a more robust support medium — a membrane made of nitrocellulose or PVDF (Polyvinylidene difluoride). This is usually achieved using an electric current in a process known as ‘blotting’
4) Blocking Non-Specific Binding Sites: To prevent non-specific binding of antibodies in later steps, the membrane is incubated in a solution of a ‘blocking’ protein which occupies any remaining binding sites on the membrane
5) Incubation with Primary Antibody: This antibody binds specifically to its target protein wherever it is on the membrane
6) Incubation with Secondary Antibody: After washing off unbound primary antibodies, the membrane is incubated with a secondary antibody that recognises the primary antibody. The secondary antibody is usually linked to an enzyme or a fluorescent molecule that will allow detection
7) Detection: If the secondary antibody is enzyme-linked, this involves incubating the membrane with a substrate that the enzyme can convert to a detectable signal (either a colour change or light emission). For fluorescently labelled secondary antibodies, the membrane is simply illuminated with light of the appropriate wavelength
This technique allows researchers to confirm the presence of a specific protein, estimate its molecular weight, and measure relative amounts in different samples
Describe the fundamental principles of an ELISPOT
Enzyme-Linked ImmunoSpot (ELISPOT) is an assay that enables detection and quantification of individual cells secreting specific molecules, most commonly cytokines, but it can also be used for other secreted substances such as antibodies
1) Preparation: The first step is the coating of a high protein-binding capacity plate (often a 96-well plate) with a specific capture antibody. This capture antibody is chosen based on the molecule of interest, for example, an antibody that binds to the cytokine Interferon-gamma (IFN-γ) if you are looking to identify and count IFN-γ-secreting cells
2) Cell Incubation: After the capture antibody has been added and allowed to bind, the plate is washed to remove unbound antibody. Then, the cells of interest are added to the plate and incubated. During this time, if the cells secrete the molecule of interest, it will be captured by the bound antibody on the plate
3) Detection: After the incubation period, the cells are removed from the plate, and a biotinylated detection antibody is added. This antibody is specific for a different epitope on the molecule of interest, allowing it to bind the secreted molecule without disrupting the capture antibody’s binding
4) Visualisation: The plate is then washed to remove unbound detection antibody and a streptavidin-enzyme conjugate is added, which binds to the biotinylated detection antibody. After another washing step to remove unbound streptavidin-enzyme conjugate, a substrate is added that the enzyme can convert into a visible product. The enzyme reaction produces a spot on the membrane at the site of the secreting cell
5) Analysis: Each spot that develops on the plate corresponds to one cell that has secreted the molecule of interest. The spots can be counted using an automated ELISPOT reader system. The number of spots gives a direct measure of the frequency of cells secreting the molecule of interest in the starting cell population
Describe how we quantify the concentration of antibodies in biological samples by nephelometry
Nephelometry is a technique used to measure the concentration of particles - such as antibodies —in a liquid solution based on the scattering of light
1) Basic Principle: When light passes through a solution containing particles, the particles scatter the light in all directions. The scattered light can be detected at a right angle to the incident light. The intensity of the scattered light is directly proportional to the concentration of the particles in the solution
2) Preparation of Samples: In the context of measuring antibodies, the biological sample (such as blood, plasma, or serum) is mixed with specific antigens. If the sample contains antibodies against these antigens, antigen-antibody complexes will form
3) Antigen-Antibody Complex Formation: These antigen-antibody complexes are larger than unbound and scatter light more effectively. This increased scattering can be detected by the nephelometer
4) Light Scattering: A beam of light (usually a laser) is directed through the sample. The antigen-antibody complexes in the sample scatter the light and this scattered light is detected at a right angle to the incident light beam
5) Detection and Quantification: The intensity of the scattered light is measured and compared to a standard curve generated using samples with known antibody concentrations. From this comparison, the concentration of antibodies in the unknown sample can be determined
Difference between ELISA and Western blotting?
ELISA: used when you want to quantify the amount of a specific protein in a sample. It can be highly sensitive and is often used for diagnostic purposes, such as HIV testing. It provides a direct measure of the amount of antigen (if the proper standards are included), but it does not provide any information about the size of the protein or whether it may exist in multiple forms
Western Blotting: used for protein identification and confirmation, as well as for estimating the size of a protein and determining its expression levels. It is particularly useful in research settings for studying changes in protein expression and post-translational modifications. Unlike ELISA, Western blotting can detect proteins that have been modified (e.g., by phosphorylation or cleavage) and can differentiate between proteins that are similar in size
